cyclin-d1 and Breast-Neoplasms

Up to 80% of breast cancers (BCa) are estrogen receptor positive and current treatments target the estrogen receptor (endocrine therapies) and/or CDK4/6 (CDK4/6 inhibitors).. Publications were retrieved from the databases: PubMed, MEDLINE, Embase and Cochrane library. Exclusion criteria were duplication, publication type, non-English language,. While a lack of standardised approach for the detection of. https://www.crd.york.ac.uk/prospero/, identifier CRD42020208179.

Topics: Breast Neoplasms; Cyclin D1; Gene Amplification; Humans; Postmenopause; Prognosis; Receptors, Estrogen

Topics: Breast Neoplasms; Case-Control Studies; Cyclin D1; Female; Genetic Techniques; Genotype; Humans; Meta-Analysis as Topic; Polymorphism, Genetic; Reproducibility of Results; Research Design; Risk

Cyclin D1 (CCND1) polymorphisms, a regulator of the cell cycle progress from G1 to the S phase, may lead to uncontrolled cell proliferation and lack of apoptosis. G870A, a common single-nucleotide polymorphism in CCND1 influences breast cancer risk. However, the association between G870A polymorphism and breast cancer risk is ambiguous so far.. In this case-control study, we analyzed the role of G870A polymorphism with breast cancer risk in Indian women. A meta-analysis of 18 studies was also performed to elucidate this association by increasing statistical power.. In our case-control study, significant risk association of the CCND1 G870A AA genotype with breast cancer in total cohort (odds ratio [OR], 2.98; 95% confidence interval [CI], 1.64-5.42; P value, 4.96e-04) and premenopausal women (OR, 3.31; 95% CI, 1.54-7.08; P value, .003) was found. The results of the meta-analysis showed that AA genotype of the CCND1 G870A polymorphism significantly increases breast cancer risk in total pooled data (AA vs GG+GA: OR = 1.20; 95% CI = 1.03 to 1.39; P value, 0.016*) and Caucasian (AA vs GG+GA: OR = 1.22; 95% CI = 0.99 to 1.51; P value, .056*) but not in Asian population. Further, a significant protective association with breast cancer was also found in the GA vs AA comparison model in pooled data (OR = 0.73; 95% CI = 0.58 to 0.92; P value, .007*) as well as in Caucasian subgroup (OR = 0.62; 95% CI = 0.49 to 0.94; P value, .022*).. CCND1 G870A AA genotype was found associated with breast cancer risk. Future association studies considering the environmental impact on gene expression are required to validate/explore this association.

Topics: Adult; Aged; Breast Neoplasms; Case-Control Studies; Cyclin D1; Female; Humans; India; Middle Aged; Models, Genetic; Polymorphism, Genetic

Cyclin D1 (CCND1) plays an essential role in regulating the progress of the cell cycle from G1 to S phase. There is a common c.870G>A polymorphism in the CCND1 gene. The aim of this study was to investigate the association of CCND1 gene c.870G>A polymorphism with breast cancer risk in a case-control study, which followed by a meta-analysis and an in silico analysis. Three hundred and thirty-five subjects composed of 174 women with breast cancer and 161 healthy controls were included in the case-control study. CCND1 gene c.870G>A genotyping was performed by PCR-RFLP. Meta-analysis was done for 14 studies composed of 7281 cases and 6820 controls. Some bioinformatics tools were applied to investigate the effects of c.870G>A on the mRNA splicing and structure. Our data obtained from case-control study revealed that GA genotype (OR: 1.89, 95%CI: 1.12-3.17, p = 0.017), AA genotype (OR: 1.95, 95%CI: 1.08-3.53, p = 0.027), and A allele (OR: 1.44, 95%CI: 1.06-1.95, p = 0.019) were significantly associated with breast cancer risk. The results of meta-analysis showed a significant association between CCND1 c.870G>A polymorphism and breast cancer risk, especially in Caucasian population. In silico analysis revealed that c.870G>A transition affect CCND1 mRNA splicing and secondary structure.

Topics: Alleles; Breast Neoplasms; Case-Control Studies; Cyclin D1; Female; Genetic Predisposition to Disease; Genotype; Humans; Middle Aged; Polymorphism, Restriction Fragment Length; Polymorphism, Single Nucleotide; Risk Factors

Despite significant advances in early detection and treatment, breast cancer still remains a major cause of morbidity and mortality for women. Our understanding of the molecular heterogeneity of the disease has significantly expanded over the past decade and the role of cell cycle signaling in both breast cancer oncogenesis and anti-estrogen resistance has gained increasing attention. The mammalian cell cycle is driven by a complex interplay between cyclins and their associated cyclin-dependent kinase (CDK) partners, and dysregulation of this process is one of the hallmarks of cancer. Despite this, initial results with broadly acting CDK inhibitors were largely disappointing. However, recent preclinical and phase I/II clinical studies using a novel, oral, reversible CDK4/6 inhibitor, palbociclib (PD-0332991), have validated the role of CDK4/6 as a potential target in estrogen receptor-positive (ER+) breast cancers. This review highlights our current understanding of CDK signaling in both normal and malignant breast tissues, with special attention placed on recent clinical advances in inhibition of CDK4/6 in ER+ disease.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Clinical Trials as Topic; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Estrogen Receptor alpha; Female; Humans; Piperazines; Protein Kinase Inhibitors; Pyridines

Uncontrolled cellular proliferation, mediated by dysregulation of the cell-cycle machinery and activation of cyclin-dependent kinases (CDKs) to promote cell-cycle progression, lies at the heart of cancer as a pathological process. Clinical implementation of first-generation, nonselective CDK inhibitors, designed to inhibit this proliferation, was originally hampered by the high risk of toxicity and lack of efficacy noted with these agents. The emergence of a new generation of selective CDK4/6 inhibitors, including ribociclib, abemaciclib and palbociclib, has enabled tumour types in which CDK4/6 has a pivotal role in the G1-to-S-phase cell-cycle transition to be targeted with improved effectiveness, and fewer adverse effects. Results of pivotal phase III trials investigating palbociclib in patients with advanced-stage oestrogen receptor (ER)-positive breast cancer have demonstrated a substantial improvement in progression-free survival, with a well-tolerated toxicity profile. Mechanisms of acquired resistance to CDK4/6 inhibitors are beginning to emerge that, although unwelcome, might enable rational post-CDK4/6 inhibitor therapeutic strategies to be identified. Extending the use of CDK4/6 inhibitors beyond ER-positive breast cancer is challenging, and will likely require biomarkers that are predictive of a response, and the use of combination therapies in order to optimize CDK4/6 targeting.

Topics: Aminopyridines; Antineoplastic Combined Chemotherapy Protocols; Benzimidazoles; Breast Neoplasms; Cell Cycle; Clinical Trials as Topic; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Female; Forecasting; Humans; Molecular Targeted Therapy; Neoplasms; Piperazines; Purines; Pyridines

Cyclin D1 polymorphism has been reported to be associated with risk of breast cancer, but the published studies have yielded controversial results. This study was undertaken to derive a precise risk estimate for the cyclin D1 polymorphism associated with breast cancer risk. We performed a search of EMBASE, PubMed, and Web of Science. In total, data from 18 publications were pooled and the association was assessed by odds ratios (ORs) with 95 % confidence intervals (CIs). This analysis showed that there was no obvious association between the cyclin D1 polymorphism and breast cancer risk in any of the analyzed genetic model. We found the same negative association in stratified analyses by ethnicity, source of controls, and sample size. Our meta-analysis provides an estimate that the presence of cyclin D1 polymorphism may not confer susceptibility to breast cancer.

Topics: Alleles; Breast Neoplasms; Case-Control Studies; Cyclin D1; Female; Genetic Predisposition to Disease; Genotype; Humans; Odds Ratio; Polymorphism, Genetic; Publication Bias

Progesterone (P4) participates in the regulation of several physiological and pathological processes in mammals through the interaction with its intracellular receptors (PR), which are ligand-dependent transcription factors.Many human cancers depend on P4 for growth and metastasis, especially cancers of reproductive tissues. In women,administration of combined estrogen and progestin hormone replacement therapy for postmenopausal symptoms increases the risk of breast cancer relative to women taking estrogens alone. P4 exerts its actions through various mechanisms classified as classical and non-classical. PR has dual functions as a nuclear transcription factor and as a modulator of cell signaling pathways. Many PR target genes do not contain canonical progesterone response elements (PRE) in their promoter regions and may thus be regulated by PR tethering to other transcription factors and/or rapid signaling,independent of direct PR DNA binding. We review the mechanisms involved in P4 effects on genes implicated in control of cell cycle, proliferation, angiogenesis and metastasis, such as cyclin D1 and epidermal growth factor receptor (EGFR)whose promoters lack PRE sequences, and vascular endothelial growth factor (VEGF) which gene contains PRE in its promoter region. The understanding of the molecular mechanisms involved in the regulation of cyclin D1, EGFR and VEGF expression by P4 will be helpful for the development of new cancer therapies.

Topics: Breast Neoplasms; Cyclin D1; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Progesterone; Receptors, Progesterone; Signal Transduction; Vascular Endothelial Growth Factor A

Cyclin D1 (CCND1), a key regulator of cell cycle progression, is overexpressed in many human cancers, including breast cancer. However, the impact of CCND1 overexpression in these cancers remains unclear and controversial. We conducted a systematic literature search in PubMed and EMBASE with the search terms "cyclin D1", "CCND1", "breast cancer", "prognosis", and potential studies for analysis were selected. Studies with survival data, including progression-free survival (PFS), overall survival (OS) or metastasis-free survival (MFS), were included in this meta-analysis. A total of 33 studies containing 8,537 cases were included. The combined hazard risk (HR) and its 95 % confidence interval (CI) of OS, PFS and MFS were 1.13 (95 % CI 0.87-1.47; P = 0.35), 1.25 (95 % CI 0.95-1.64; P = 0.12), and 1.04 (95 % CI 0.80-1.36; P = 0.76), respectively, for primary breast cancer patients with tumors exhibiting CCND1 overexpression. Interestingly, the impact of CCND1 expression on OS was a 1.67-fold (95 % CI 1.38-2.02; P = 0.00) increased risk for ER-positive breast cancer patients. However, CCND1 overexpression exhibited no association with the PFS or OS of patients who received epirubicin-based neoadjuvant chemotherapy, for which the P values were 0.63 and 0.47, respectively. In summary, CCND1 overexpression impacts the prognosis of ER-positive breast cancer patients, but not patients with unselected primary breast cancer or patients treated with neoadjuvant chemotherapy.

Topics: Breast Neoplasms; Cyclin D1; Female; Gene Expression; Humans; Neoadjuvant Therapy; Prognosis; Publication Bias; Receptors, Estrogen

The higher incidence of breast cancer in developed countries has been tempered by reductions in mortality, largely attributable to mammographic screening programmes and advances in adjuvant therapy. Optimal systemic management requires consideration of clinical, pathological and biological parameters. Oestrogen receptor alpha (ERα), progesterone receptor (PgR) and human epidermal growth factor receptor 2 (HER2) are established biomarkers evaluated at diagnosis, which identify cardinal subtypes of breast cancer. Their prognostic and predictive utility effectively guides systemic treatment with endocrine, anti-HER2 and chemotherapy. Hence, accurate and reliable determination remains of paramount importance. However, the goals of personalized medicine and targeted therapies demand further information regarding residual risk and potential benefit of additional treatments in specific circumstances. The need for biomarkers which are fit for purpose, and the demands placed upon them, is therefore expected to increase. Technological advances, in particular high-throughput global gene expression profiling, have generated multi-gene signatures providing further prognostic and predictive information. The rational integration of routinely evaluated clinico-pathological parameters with key indicators of biological activity, such as proliferation markers, also provides a ready opportunity to improve the information available to guide systemic therapy decisions. The additional value of such information and its proper place in patient management is currently under evaluation in prospective clinical trials. Expanding the utility of biomarkers to lower resource settings requires an emphasis on cost effectiveness, quality assurance and possible international variations in tumor biology; the potential for improved clinical outcomes should be justified against logistical and economic considerations.

Topics: Antigens, Tumor-Associated, Carbohydrate; Antineoplastic Agents; Biomarkers, Tumor; Breast Neoplasms; Carcinoembryonic Antigen; Cell Proliferation; Cost-Benefit Analysis; Cyclin D1; Cyclin E; Developed Countries; Developing Countries; DNA, Neoplasm; Estrogen Receptor alpha; Estrogen Receptor beta; Female; Gene Expression Profiling; Global Health; Health Resources; Humans; Ki-67 Antigen; Molecular Targeted Therapy; Neoplastic Cells, Circulating; Plasminogen Activator Inhibitor 1; Precision Medicine; Predictive Value of Tests; Prognosis; Quality Assurance, Health Care; Receptor, ErbB-2; Receptors, Progesterone; Tissue Polypeptide Antigen; Transcriptome; Urokinase-Type Plasminogen Activator

Cyclin D1 represents a key molecule in the regulation of cell cycle. CCND1 G870A (rs603965) polymorphism has drawn considerable attention as the A allele may generate a variant splice product with possible oncogenic actions. A meta-analysis examining the association between CCND1 G870A polymorphism and breast cancer risk was performed. Separate analyses on Caucasian and Chinese populations were also implemented. Eligible articles were identified for the period up to July 2010. Pooled odds ratios (OR) were appropriately derived from fixed-effects or random-effects models. Sensitivity analysis excluding studies whose genotype frequencies in controls significantly deviated from Hardy-Weinberg Equilibrium (HWE) was performed. Nine case-control studies on Caucasians (7,304 cases and 8,149 controls) and four case-control studies on Chinese (2,607 cases and 3,022 controls) were eligible. At the overall analysis the A allele seemed to be associated with elevated breast cancer risk; the effect seemed to be confined to homozygous carriers (pooled OR = 1.091, 95% CI: 1.008-1.179, P = 0.030, fixed effects) as heterozygous carriers did not exhibit significantly elevated breast cancer risk. No statistically significant associations were demonstrated in Caucasians. On the other hand, Chinese AA carriers exhibited marginally elevated breast cancer risk (pooled OR = 1.144, 95% CI: 0.984-1.329, P = 0.080, fixed effects). Nevertheless, the controls in two out of the four Chinese studies deviated from HWE. In conclusion, this meta-analysis suggests that the A allele of the CCND1 G870A polymorphism may confer additional breast cancer risk when it comes to homozygosity and Chinese populations. The need for additional, methodologically sound studies on Chinese populations seems warranted.

Topics: Breast Neoplasms; Case-Control Studies; Cyclin D1; Female; Genetic Predisposition to Disease; Homozygote; Humans; Models, Genetic; Odds Ratio; Polymorphism, Single Nucleotide; Risk Factors

Cyclin D1 overexpression is found in more than 50% of human breast cancers and causes mammary cancer in transgenic mice. Dysregulation of cyclin D1 gene expression or function contributes to the loss of normal cell cycle control during tumorigenesis. Recent studies have demonstrated that cyclin D1 conducts additional specific functions to regulate gene expression in the context of local chromatin, promote cellular migration and inhibit mitochondrial metabolism. It is anticipated that these additional functions contribute to the pathology associated with dysregulated cyclin D1 abundance. This article discusses evidence that examines the significance of cyclin D1 in breast cancer with emphasis on its role in breast cancer stem cell expansion.

Topics: Adult Stem Cells; Animals; Breast Neoplasms; Cyclin D1; Cyclin-Dependent Kinases; Female; Gene Expression Regulation, Neoplastic; Humans; Mammary Glands, Human; Protein Isoforms

Cyclin D1 (CCND1) plays an essential role in tumor development and progression through regulating the cell transition from G1 to the proliferative S phase. The CCND1 G870A polymorphism has been associated with an increased susceptibility to squamous cell carcinoma of the head and neck, bladder, prostate, and gastric cardiac cancers. There are a number of studies that explored the relationship between CCND1 G870A polymorphism and breast cancer risk, with inconsistent conclusions. In order to better define the predictive value of CCND1 G870A polymorphism in breast cancer, we searched PubMed and EBSCO for relevant publications. A total of 13 studies were indentified, which included 11,235 cases and 12,763 controls. We calculated the summary odds ratios and the corresponding 95% confidence interval. Our meta-analysis showed that carriers of AA genotype have a significantly higher risk in developing breast cancer compared with that of GG genotype (OR = 1.08, 95% CI = 1.01-1.17, p > = 0.03) in overall population. Furthermore, in subgroup analysis, CCND1 G870A polymorphism was associated with a marginally increased risk of breast cancer for Chinese compared to Caucasian populations with an OR = 1.14, 95% CI = 1.00-1.20, p-trend = 0.06 for AA + GA versus GG, if the controls were hospital-based population with an OR = 1.21, 95% CI = 0.99-1.47, p = 0.06 for AA versus GG and if the distributions of genotypes in control groups were consistent with the Hardy-Weinberg equilibrium (HWE) with an OR = 1.08, 95% CI = 1.00-1.15, p = 0.04 for AA versus GA + GG. Our meta-analysis represents the largest study to date indicating that the G870A polymorphism in CCND1 confers an increased risk for breast cancer. Further studies are warranted to explore the preventive measures to detect and manage the breast cancers attributable to the G870A polymorphism.

Topics: Breast; Breast Neoplasms; Case-Control Studies; Cyclin D1; Female; Humans; Polymorphism, Genetic; Prognosis; Risk Factors

Breast cancer progression involves multiple genetic events, which can activate dominant-acting oncogenes and disrupt the function of specific tumor suppressor genes. This article describes several key oncogene and tumor suppressor signaling networks that have been implicated in breast cancer progression. Among the tumor suppressors, the article emphasizes BRCA1/2 and p53 tumor suppressors. In addition to these well characterized tumor suppressors, the article highlights the importance of PTEN tumor suppressor in counteracting PI3K signaling from activated oncogenes such as ErbB2. This article discusses the use of mouse models of human breast that recapitulate the key genetic events involved in the initiation and progression of breast cancer. Finally, the therapeutic potential of targeting these key tumor suppressor and oncogene signaling networks is discussed.

Topics: Animals; Breast Neoplasms; Cyclin D1; Female; Genes, erbB-2; Genes, myc; Genes, Tumor Suppressor; Humans; Mice; Nuclear Proteins; Oncogenes; Transcription Factors

Cyclin D1 (CCND1), a key cell cycle regulatory protein that governs the cell cycle progression from G1 to S phase, can promote cell proliferation or induce growth arrest and apoptosis. Since the identification of a well-characterized functional polymorphism, G870A in exon 4 of CCND1, several molecular epidemiological studies were conducted in recent years to evaluate the association between G870A and breast cancer risk in diverse populations. However, the results remain conflicting rather than conclusive. This meta-analysis on 5,371 cases with breast cancer and 5,336 controls from 7 published case-control studies showed that the variant allele 870A was associated with a significantly increased risk of breast cancer (AA vs. GG: OR = 1.18, 95% CI = 1.06-1.32; AG vs. GG: OR = 1.12, 95% CI = 1.01-1.23; AA/AG vs. GG: OR = 1.14, 95% CI = 1.04-1.25) without any between-study heterogeneity. In the stratified analysis by race, we found that the increased breast cancer risk associated with G870A polymorphism was more evident in Caucasians (OR = 1.14, 95% CI = 1.01-1.28, P = 0.88 for heterogeneity test), but not significant in Asians (OR = 1.10, 95% CI = 0.85-1.42, P = 0.05 for heterogeneity test). The results suggest that CCND1 G870A polymorphism may contribute to breast cancer development, especially in Caucasians. Additional well-designed large studies were required for the validation of this association in different populations.

Topics: Breast Neoplasms; Case-Control Studies; Cyclin D1; Female; Genetic Predisposition to Disease; Genotype; Humans; Polymorphism, Single Nucleotide; Prognosis; White People

Having previously reported the synthesis and anticancer activities of cyclic 5-fluorouracil (5-FU) O,N-acetalic compounds, the decision was made to change 5-FU for uracil (U), with the prospect of finding an antiproliferative agent endowed with a new mechanism of action. The use of a reverse transcription-PCR-based assay decreased cyclin D1 mRNA, suggesting that this cyclic U O,N-acetalic compound exerts its regulatory action on cyclin D1 at the level of transcription. Following the ongoing Anticancer Drug Programme we planned the synthesis of compounds bearing a natural pyrimidine base and also, the oxygen atom at position 1 of the seven-membered cycle was replaced by its isosteric sulfur atom, and its oxidized states. Next, the pyrimidine base was substituted for the purine one, with the objective of increasing both the lipophilicity and the structural diversity of the target molecules. If the previously described compounds were not prodrugs, it would not be necessary to maintain the O,N-acetalic characteristic. Therefore, molecules were designed in which both structural entities (such as the benzoheterocyclic ring and the purine base) were linked by a heteroatom-C-C-N bond. A series of (RS)-9-(2,3-dihydro-1,4-benzoxathiin-3-ylmethyl)-9H-purine derivatives was obtained and the anticancer activity for the most active compounds was correlated with their capability to induce apoptosis. Finally, completing a SAR study, a series of (RS)-6-substituted-7- or 9-(1,2,3,5-tetrahydro-4,1-benzoxazepine-3-yl)-7H- or 9H-purines was prepared. The studies by microarray technology showed that the main molecular targets of some of these compounds are pro-apoptotic genes with protein kinase activity such as GP132, ERN1 or RAC1, which prevent the metastatic progression.

Topics: Acetals; Antineoplastic Agents; Benzene Derivatives; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Drug Design; Fluorouracil; Heterocyclic Compounds; Humans; Inhibitory Concentration 50; Neoplasm Metastasis; Purines; Pyrimidines; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Structure-Activity Relationship

Recent discoveries suggest that several protein kinases are rapidly activated in response to ligand binding to cytoplasmic steroid hormone receptors (SRs), including progesterone receptors (PRs). Thus, PRs act as ligand-activated transcription factor "sensors" for growth factor-initiated signaling pathways in hormonally regulated tissues, such as the breast. Induction of rapid signaling upon progestin binding to PR-B provides a means to ensure that receptors and co-regulators are appropriately phosphorylated as part of optimal transcription complexes. Alternatively, PR-B activated kinase cascades provide additional avenues for progestin-regulated gene expression independent of PR nuclear action. Herein, an overview of progesterone/PR and signaling cross-talk in breast cancer models is provided. Kinases are emerging as key mediators of PR action. Cross-talk between SR and membrane-initiated signaling events suggests a mechanism for coordinate regulation of gene subsets by mitogenic stimuli in hormonally responsive normal tissues, and is suspected to contribute to cancer biology.

Topics: Animals; Breast Neoplasms; Cyclin D1; Cyclin-Dependent Kinase 2; Female; Humans; Mitogen-Activated Protein Kinases; Models, Biological; Phosphorylation; Receptors, Progesterone; Receptors, Steroid; Signal Transduction; Up-Regulation

Progesterone acting through two isoforms of the progesterone receptor (PR), PRA and PRB, regulates proliferation and differentiation in the normal mammary gland in mouse, rat, and human. Progesterone and PR have also been implicated in the etiology and pathogenesis of human breast cancer. The focus of this review is recent advances in understanding the role of the PR isoform-specific functions in the normal breast and in breast cancer. Also discussed is information obtained from rodent studies and their relevance to our understanding of the role of progestins in breast cancer etiology.

Topics: Animals; Breast; Breast Neoplasms; Cyclin D1; Cyclin-Dependent Kinase Inhibitor Proteins; Female; Humans; Mammary Glands, Animal; Mammary Glands, Human; Mice; Phosphorylation; Protein Isoforms; Rats; Receptors, Progesterone; Structure-Activity Relationship

The circadian clock controls a large array of behavioral and physiological systems of fundamental importance to most organisms. Consequently, abnormal functioning of the clock results in severe dysfunctions and pathologies. Although epidemiological studies show a clear correlation between disruption of circadian rhythms and incidence of breast cancer, a molecular interpretation of how clock-related mechanisms may link to tumor development remains elusive. Here we speculate on the molecular pathways that may couple the circadian machinery to breast cancer.

Topics: Acetylation; Animals; ARNTL Transcription Factors; Basic Helix-Loop-Helix Transcription Factors; Breast Neoplasms; Cell Transformation, Neoplastic; Chromatin; Circadian Rhythm; CLOCK Proteins; Cyclin D1; Developed Countries; Disease Susceptibility; Estrogen Receptor alpha; Estrogens; Female; Gene Expression Regulation; Genes, Tumor Suppressor; Histone Acetyltransferases; Histones; Humans; Mammals; Mammary Neoplasms, Experimental; Melatonin; Mice; Models, Biological; Neoplasms, Hormone-Dependent; Nuclear Proteins; Protein Processing, Post-Translational; Risk; Trans-Activators; Transcription Factors

Temsirolimus (CCI779), an intravenous analog of rapamycin, presents immunosuppressive properties and also antiproliferative activity. Its principal target is the mTOR serine/threonin kinase which controls the initiation of the transcription of many ARNm implicated in carcinogenesis. Breast cancers, glioblastoma and renal cell carcinoma were particularly studied with response rates from 10 to 20 %. In haematology, mantle-cell lymphoma is of particular interest because of constitutional activation of cyclin D1 (response rate of 40 %). As a whole these data define temsirolimus as a promising new drug. Current and further developments are based on its association with chemotherapy in a concomitant or sequential way.

Topics: Antineoplastic Agents; Breast Neoplasms; Cyclin D1; Glioblastoma; Humans; Kidney Neoplasms; Lymphoma, Mantle-Cell; Protein Kinase Inhibitors; Protein Kinases; Sirolimus; TOR Serine-Threonine Kinases

Cellular homeostasis in higher organisms is maintained by balancing cell growth, differentiation, and death. Two important systems that transmit extracellular signals into the machinery of the cell nucleus are the signaling pathways that activate nuclear factor kappaB (NF-kappaB) and estrogen receptor (ER). These two transcription factors induce expression of genes that control cell fates, including proliferation and cell death (apoptosis). However, ER has anti-inflammatory effects, whereas activated NF-kappaB initiates and maintains cellular inflammatory responses. Recent investigations elucidated a nonclassical and nongenomic effect of ER: inhibition of NF-kappaB activation and the inflammatory response. In breast cancer, antiestrogen therapy might cause reactivation of NF-kappaB, potentially rerouting a proliferative signal to breast cancer cells and contributing to hormone resistance. Thus, ER ligands that selectively block NF-kappaB activation could provide specific potential therapy for hormone-resistant ER-positive breast cancers.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Breast Neoplasms; Cell Division; Cells, Cultured; Cyclin D1; Estrogens; Female; Gene Expression Regulation; Humans; I-kappa B Proteins; Ligands; Mice; Mice, Knockout; Models, Biological; Neoplasms, Hormone-Dependent; NF-kappa B; Pyrazoles; Receptors, Estrogen; Selective Estrogen Receptor Modulators; Signal Transduction; Transcriptional Activation

Taking a perspective on available evidence that emphasizes relevance to human disease, cyclin D1 is solidly established as an oncogene with an important pathogenetic role in breast cancer and other human tumors. However, the precise cellular mechanisms through which aberrant cyclin D1 expression drives human neoplasia are less well established. Indeed, emerging evidence suggests that cyclin D1 might act, predominantly or at least in part, through pathways that do not involve its widely accepted function as a cell cycle regulator. Although therapeutic exploitation of the role of cyclin D1 as a molecular driver of breast cancer carries great promise, it is also suggested that direct targeting of the cyclin D1 gene or gene products may prove more successful than approaches that rely on arguably incomplete knowledge of the oncogenic mechanisms of cyclin D1.

Topics: Breast Neoplasms; Cyclin D1; Female; Humans; Oncogene Proteins

Estrogen is an important steroid hormone with diverse functions in different parts of the human body. The developmental and physiological role of estrogen is mediated by estrogen receptor alpha (ER-alpha) and newly identified ER-beta. Regulation of expression of various important cellular oncogenes and tumor suppressor genes is a key component of estrogen and ER action. The expression of these genes is crucial in various processes such as cell cycle progression, mammary gland development, growth factor pathways and apoptosis. A very precise and accurate control of these genes is required for normal growth and functioning of cells. Aberrant expression of these genes through elevated expression, gene amplification or mutation may lead to induction and/or progression of different cancers including estrogen-dependent breast cancers. This review briefly describes the role of different genes that are regulated by estrogen in female reproductive tissues and breast cancer.

Topics: BRCA1 Protein; Breast Neoplasms; Cathepsin D; Cyclin D1; Estrogens; Female; Genes, myc; Humans; Membrane Proteins; Presenilin-2; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-fos; Receptors, Estrogen; Receptors, Progesterone; Reproduction

Antioestrogen therapy is a highly effective treatment for patients with oestrogen-receptor (ER)-positive breast cancer, emphasising the central role of oestrogen action in the development and progression of this disease. However, effective antioestrogen treatment is often compromised by acquired endocrine resistance, prompting the need for a greater understanding of the down-stream mediators of oestrogen action that may contribute to this effect. Recent studies have demonstrated a critical link between oestrogen's mitogenic effects and cell cycle progression, particularly at the G1 to S transition where key effectors of oestrogen action are c-Myc and cyclin D1, which converge on the activation of cyclin E-cdk2. These components are rapidly upregulated in response to oestrogen, and can mimic its actions on cell cycle progression, including re-initiating cell proliferation in antioestrogen-arrested cells. Here we review the roles of c-Myc, cyclin D1 and cyclin E in oestrogen action and endocrine resistance, and identify their potential as markers of disease progression and endocrine responsiveness, and as novel therapeutic targets in endocrine-resistant breast cancer.

Topics: Breast Neoplasms; Cyclin D1; Cyclin E; Drug Resistance, Neoplasm; Estrogen Antagonists; Estrogens; Female; Growth Substances; Humans; Proto-Oncogene Proteins c-myc; Receptors, Estrogen; Signal Transduction

Beta-catenin and cyclin D1 have attracted considerable attention due to their proto-oncogenic roles in human cancer. The finding of cyclin D1 as a direct target gene of beta-catenin in colon cancer cells led to the assumption that cyclin D1 upregulation is pivotal to beta-catenin's oncogenicity. Our recent paper shows that this is not the case; cyclin D1 dampens the oncogenicity of activated beta-catenin (MMTV-DN89beta-catenin). The relationships and dependencies of beta-catenin and cyclin D1 point to distinct, essential and sequential roles during alveologenesis. These results support the concept that both beta-catenin's and cyclin D1's actions are more sophisticated than simple acceleration of the cell cycle clock. These proteins are employed at critical junctures involving cell fate decisions that we speculate require specific types of cell cycle to traverse.

Topics: Animals; beta Catenin; Breast Neoplasms; Cell Division; Cell Lineage; Cell Transformation, Neoplastic; Colonic Neoplasms; Cyclin D1; Cytoskeletal Proteins; Female; Humans; Male; Mice; Models, Biological; Signal Transduction; Trans-Activators

Mitogenic growth factor- and integrin-dependent signaling pathways cooperate to control the proliferation of nontransformed cells. As integral mediators of these networks, the Rho family of GTPases play a pivotal role in G1 cell cycle progression, primarily through regulation of cyclin D1 expression, as well as the levels of the cyclin-dependent kinase inhibitors p21cip1 and p27kip1. Such dual control of both the critical positive and negative regulators of G1 progression make the Rho GTPases prime candidates to target the autonomous proliferation which typifies cancer cells. Cyclin D1 has been identified as an important oncogene and the cdk inhibitors as tumor suppressors in human breast carcinogenesis. Evidence pointing to the potential role of Rho-dependent pathways and their interaction with oncogenic Ras in contributing to such cell cycle abnormalities that characterize human breast cancer is also presented.

Topics: Breast Neoplasms; Cell Adhesion; Cyclin D1; Cyclin-Dependent Kinases; Extracellular Matrix; G1 Phase; Humans; Mitogens; rac GTP-Binding Proteins; rho GTP-Binding Proteins; Signal Transduction

The Rho family of GTPases have emerged as key players in regulating a diverse set of biological activities including actin organization, focal complex/adhesion assembly, cell motility, cell polarity, gene transcription and cell-cycle progression. Some Rho GTPases and their signaling components are overexpressed and/or are hyperactive in breast cancer and recent studies have shown a requirement for Rho GTPases in breast cancer cell metastasis in vivo. Herein we describe the contribution of Rho GTPase to the malignant phenotype of breast cancer cells and the role of these pathways as potential targets for breast cancer therapy. Rho GTPases promote cell-cycle progression through cyclin D1, and cyclin D1 in turn reduces cellular adhesion and promotes migration, an example of 'inside-out' signaling by cyclin D1. As cyclin D1 overexpression correlates with metastatic cancer, the 'inside-out' signaling function of cyclin D1 to promote cell migration may represent a useful new therapeutic target.

Topics: Breast Neoplasms; Cell Adhesion; Cell Cycle; Cell Movement; Cyclin D1; Female; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; rho GTP-Binding Proteins; Signal Transduction; Tumor Cells, Cultured

The D-type and E-type cyclins control the G(1) to S phase transition during normal cell cycle progression and are critical components of steroid- and growth factor-induced mitogenesis in breast epithelial cells. Mammary epithelial cell-specific overexpression of these genes leads to mammary carcinoma, while in cyclin D1-deficient mice mammary gland development is arrested prior to lobuloalveolar development. Cyclin D1 null mice are resistant to mammary carcinoma induced by the neu and ras oncogenes, indicating an essential role for cyclin D1 in the development of some mammary cancers. Cyclin D1 and E1 are commonly overexpressed in primary breast cancer, with some evidence of an association with an adverse patient outcome. This observation may result in part from their ability to confer resistance to endocrine therapies. The functional consequences of cyclin E overexpression in breast cancer are likely related to its role in cell cycle progression, whereas that of cyclin D1 may also be a consequence of a more recently defined role in transcriptional regulation.

Topics: Animals; Antineoplastic Agents, Hormonal; Breast Neoplasms; Carcinoma; Cell Cycle; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin D1; Cyclin E; Disease Models, Animal; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Humans; Mammary Neoplasms, Animal; Mice; Oncogene Proteins

In the USA, breast cancer accounts for approximately 30% of all cancers diagnosed in women and is the second leading cause of cancer death in women. An understanding of the molecular genetic events governing breast cancer lead to both prevention and intervention strategies in an attempt to reduce mortality and morbidity from breast cancer. The last three decades of medical research examining the molecular pathogenesis of cancers have provided compelling evidence for the universal disruption of the cell cycle in human tumors. The importance of cell cycle control in human cancer was recognized by the recent award of the Nobel Prize to Drs Nurse and Hartwell for their discovery of the cyclins. More recent studies have demonstrated a critical interface between hormonal signaling and the cell cycle. In parallel, epidemiological studies have identified as being associated with breast cancer important dietary and environmental components that regulate hormonal signaling. This review describes the intersection of these two fields of study, which together imply a role for dietary prevention and intervention in human breast cancer perhaps through altering cell cycle components.

Topics: Androgens; Breast Neoplasms; Cell Cycle; Cyclin D1; Cyclin-Dependent Kinases; Diet; Estrogens; Fatty Acids, Unsaturated; Female; Genistein; Humans; PPAR gamma; Receptors, Androgen; Receptors, Estrogen; Vitamin A

Chromosome locus 11q13 is frequently amplified in a number of human cancers including carcinoma of the breast where up to 15% carry this chromosomal abnormality. Originally 11q13 amplification was thought to involve a single amplicon spanning many megabases, but more recent data have identified four core regions within 11q13 that can be amplified independently or together in different combinations. Although the region harbors several genes with known or suspected oncogenic potential, the complex structure of the amplicons and the fact that 11q13 is gene-rich have made definitive identification of specific genes that contribute to the genesis and progression of breast cancer a difficult and continuing process. To date CCND1, encoding the cell cycle regulatory gene cyclin D1, and EMS1, encoding the filamentous actin binding protein and c-Src substrate cortactin, are the favored candidates responsible for the emergence of two of the four amplification cores.

Topics: Breast Neoplasms; Chromosomes, Human, Pair 11; Cortactin; Cyclin D1; Female; Gene Amplification; Humans; Microfilament Proteins; Neoplasm Proteins

The central involvement of estrogen in the development of the mammary gland and in the genesis of breast cancer has lent impetus to studies of the links between estrogen action and the cell cycle machinery. Recent studies of the estrogenic regulation of molecules with known roles in the control of G1/S phase progression have resulted in significant advances in understanding these links. Estrogens independently regulate the expression and function of c-Myc and cyclin D1 and the induction of either c-Myc or cyclin D1 is sufficient to recapitulate the effects of estrogen on cell cycle progression. These pathways converge at the activation of cyclin E-Cdk2 complexes. The active cyclin E-Cdk2 complexes are depleted of the cyclin dependent kinase (CDK) inhibitor p21(WAF1/CIP1) because of estrogen-mediated inhibition of nascent p21(WAF1/CIP1). Insulin and estrogen synergistically stimulate cell cycle progression, and the ability of estrogen to antagonize an insulin-induced increase in p21(WAF1/CIP1) gene expression appears to underlie this effect. Antiestrogen treatment of MCF-7 cells leads to an acute decrease of c-Myc expression, a subsequent decline in cyclin D1, and ultimately arrest of cells in a state with features characteristic of quiescence. An antisense-mediated decrease in c-Myc expression results in decreased cyclin D1 expression and inhibition of DNA synthesis, mimicking the effects of antiestrogen treatment and emphasizing the importance of c-Myc as an estrogen/antiestrogen target. These data identify c-Myc, cyclin D1, p21(WAF1/CIP1) and cyclin E-Cdk2 as central components of estrogen regulation of cell cycle progression and hence as potential downstream targets that contribute to the role of estrogen in oncogenesis.

Topics: Animals; Breast Neoplasms; CDC2-CDC28 Kinases; Cell Cycle; Cell Cycle Proteins; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; Estrogen Receptor Modulators; Estrogens; Female; Humans; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-myc

The overexpression of estrogen receptor alpha (ERalpha) is frequently observed in the early stage of breast cancer. We previously reported that the specific promoter of the ERalpha gene is responsible for this enhanced transcription of the gene, and identified the cis-acting elements which play an important role in its transcription. Furthermore, methylation of the ERalpha gene promoters also contribute to the regulation of gene transcription. Elucidation of these mechanisms of ERalpha gene expression may provide useful information for the early detection and chemoprevention of breast cancer. On the other hand, the expression of ERbeta has been reported in breast cancer. We have also assessed the significance and function of ERbeta and its variant types in breast cancer, and suggest that ERbeta and ERbetacx specifically suppress the function of ERalpha through different mechanisms. ERbeta isoforms may be important functional modulators of the estrogen-signaling pathway in breast cancer cells, and might affect the clinical outcome of patients. Moreover, to address the role of these ERs on the estrogen-dependent growth of breast cancer cells and to develop a diagnostic tool, we have analyzed the gene expression profiles of estrogen-responsive genes using cDNA microarray. Based on these results, the expression of several candidate genes in breast cancer tissues were analyzed by real-time RT-PCR and by immunohistochemical techniques, in order to discover new predictive factors for the endocrine therapy of patients with breast cancer. These studies could provide new clues for the elucidation of the estrogen-dependent mechanisms of cancer and the clinical benefits for patients.

Topics: Breast Neoplasms; Cyclin D1; Estrogen Receptor alpha; Estrogen Receptor beta; Female; Humans; Receptors, Estrogen; Receptors, Progesterone

Beta-catenin regulates cell-cell adhesion and transduces signals from many pathways to regulate the transcriptional activities of Tcf/Lef DNA binding factors. Gene ablation and transgenic expression studies strongly support the concept that beta-catenin together with Lef/Tcf factors act as a switch to determine cell fate and promote cell survival and proliferation at several stages during mammary gland development. Mice expressing the negative regulator of Wnt/beta-catenin signaling (K14-Dkk) fail to form mammary buds, and those lacking Lef-1 show an early arrest in this process at stage E13.5. Stabilized deltaN89beta-catenin initiates precocious alveologenesis during pubertal development, and negative regulators of endogenous beta-catenin signaling suppress normal alveologenesis during pregnancy. Stabilized beta-catenin induces hyperplasia and mammary tumors in mice. Each of the beta-catenin-induced phenotypes is accompanied by upregulation of the target genes cyclin D1 and c-myc. Cyclin D1, however, is dispensable for tumor formation and the initiation of alveologenesis but is essential for later alveolar expansion.

Topics: Animals; beta Catenin; Breast Neoplasms; Cadherins; Cell Adhesion; Cell Division; Cell Survival; Cyclin D1; Cytoskeletal Proteins; DNA-Binding Proteins; Female; Humans; Lymphoid Enhancer-Binding Factor 1; Mammary Glands, Animal; Mammary Glands, Human; Mammary Neoplasms, Animal; Mice; Models, Biological; Mucin-1; Phenotype; Proto-Oncogene Proteins c-myc; Signal Transduction; Trans-Activators; Transcription Factors; Up-Regulation

Nuclear factor of kappaB (NF-kappaB) is a group of sequence-specific transcription factors that is best known as a key regulator of the inflammatory and innate immune responses. Recent studies of genetically engineered mice have clearly indicated that NF-kappaB is also required for proper organogenesis of several epithelial tissues, including the mammary gland. Mice have shown severe lactation deficiency when NF-kappaB activation is specifically blocked in the mammary gland. In addition, there are strong suggestions that NF-kappaB may play an important role in the etiology of breast cancer. Elevated NF-kappaB DNA-binding activity is detected in both mammary carcinoma cell lines and primary human breast cancer tissues.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cyclin D1; Female; Humans; I-kappa B Kinase; Mammary Glands, Animal; Mammary Glands, Human; Mammary Neoplasms, Animal; Mice; Models, Biological; NF-kappa B; Phosphorylation; Pregnancy; Protein Serine-Threonine Kinases; Signal Transduction

Topics: Animals; Breast Neoplasms; Cell Transformation, Neoplastic; Cyclin D1; Gene Expression Regulation, Neoplastic; Genes, bcl-1; Genes, erbB-2; Genes, ras; Humans; Mammary Neoplasms, Experimental; Mice; Proto-Oncogenes

The molecular basis of sensitivity to therapeutic radiation and chemotherapy is a complex product of cellular and tissue responses. Certain genetic factors can be highlighted as being of special importance in the response of breast cancers to treatment. The breast cancer susceptibility genes, BRCA1 and BRCA2, determine the phenotype of the tumor, with BRCA1- or BRCA2-deficient tumors showing marked sensitivity to ionizing radiation and drugs that produce double-strand breaks. However, the extent to which loss of BRCA1 or BRCA2 function occurs in sporadic cancer has not yet been determined. The ATM protein plays a significant role in determining the response to therapy, but how frequently the function of ATM is disrupted in breast cancer is debated. Although the p53 protein is a major determinant of the response to ionizing radiation and cytotoxic drugs, there is no consistency in how p53 affects the survival of cells, because an impairment of DNA repair is offset by reduced apoptosis. Growth factors that sustain the proliferation of breast cancer cells may impact the response to therapy by inhibiting apoptosis. Loss of cell-cycle checkpoint responses may result in increased sensitivity, particularly if the checkpoint controls the G2 transition. Overexpression of cyclin D, which shortens the duration of the G1 transition, is associated with mild radiation resistance, perhaps by inhibiting apoptosis. Overall, there is much more to be understood in the complex response of breast cancers to therapy, and many other proteins play important roles in the response to treatment. The focus of our investigation is on those genetic alterations in tumors that affect the response to therapy, which will ultimately allow strategies to achieve therapeutic gain.

Topics: Ataxia Telangiectasia; Ataxia Telangiectasia Mutated Proteins; Breast Neoplasms; Cell Cycle Proteins; Cyclin D1; DNA-Binding Proteins; Drug Tolerance; Female; Genes, BRCA1; Genes, BRCA2; Genes, erbB-2; Humans; Protein Serine-Threonine Kinases; Radiation Tolerance; Tumor Suppressor Proteins

Breast cancer is one of the most common cancer forms affecting many women. The disease nevertheless has widely varying behavior and therefore patient outcome, and an important undertaking is to define and understand the molecular mechanisms behind these actions. Defects in the G1/S transition in the cell cycle affect both tumor proliferation and the fidelity of check points responsible for chromosomal integrity and DNA damage response and has lately been shown to represent one of a rather limited set of key aberrations in the transformation process. Many cell cycle regulatory proteins are either oncogenes or suppressor genes or are closely associated to the transformation process. The types of aberrations in the G1/S transition seem to be different in various cancers but are nevertheless often linked to clinical behaviors. In this review the role of multiparameter analyses of cell cycle regulatory proteins in breast cancer will be outlined with special attention to pattern analyses as well as the definition of two contrasting pathways in tumorigenesis defined by either cyclin D1 or cyclin F overexpression.

Topics: Breast Neoplasms; Cell Cycle; Cyclin D1; Cyclin E; Cyclin-Dependent Kinases; G1 Phase; Humans; S Phase

Topics: Apoptosis; Breast Neoplasms; Carcinoma in Situ; Carcinoma, Ductal, Breast; Cyclin D1; Female; G1 Phase; Humans; Loss of Heterozygosity; Precancerous Conditions; Response Elements; Transcriptional Activation; Transfection

p27 is an inhibitor of cyclin dependent kinase involved in the regulation of the cell cycle. In this commentary we discuss the current knowledge on p27 in breast cancer and its significance in predicting the outcome. p27 protein levels are high in most cases of breast carcinomas, are correlated with the levels of cyclin D1 and estrogen receptor, and could be a useful predictor of survival, because they are low in aggressive carcinomas. Immunodetection of p27 in breast tumors could be useful in the assessment of prognosis, especially in those cases in which the commonly used parameters are insufficient, and might ultimately influence the therapy of this disease.

Topics: Breast Neoplasms; Cell Cycle Proteins; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Enzyme Inhibitors; Female; Humans; Immunohistochemistry; Lymph Nodes; Microtubule-Associated Proteins; Middle Aged; Multivariate Analysis; Prognosis; Proportional Hazards Models; Survival Analysis; Tumor Suppressor Proteins

In MCF-7 breast cancer cells, the insulin-like growth factor 1 receptor (IGF-1R) and the oestrogen receptor (ER) are coexpressed and the two signalling systems are engaged in a crosstalk that results in synergistic growth. However, coupling between the signalling cascades is poorly understood. Oestradiol enhances IGF-1R signalling by inducing the expression of insulin receptor substrate 1 (IRS-1), a substrate of the IGF-1R. Oestradiol induced expression of IRS-1 results in enhanced tyrosine phosphorylation of IRS-1 after IGF-1 stimulation, followed by enhanced mitogen activated protein kinase, phosphoinositide 3' kinase, and Akt activation. Oestradiol can also potentiate the effect of IGF-1 on the expression of cyclin D1 and cyclin E, and on the phosphorylation of the retinoblastoma protein (RB). These effects are greatly diminished in SX13 cells, which exhibit a 50% reduction in IGF-1R expression but few functional IGF-1Rs at the surface. Oestradiol and IGF-1 regulate the expression of two cyclin dependent kinase inhibitors, p21 and p27, differently. Whereas IGF-1 increases p21 expression and reduces p27 expression, oestradiol has no effect on p21. In summary, in MCF-7 cells, oestrogen potentiates the effect of IGF-1 on IGF-1R signalling and its effects on certain cell cycle components.

Topics: Breast Neoplasms; Cell Division; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Estradiol; Female; Humans; Insulin-Like Growth Factor I; Microfilament Proteins; Mitogen-Activated Protein Kinases; Muscle Proteins; Phosphatidylinositol 3-Kinases; Phosphorylation; Receptor, IGF Type 1; Retinoblastoma Protein; Signal Transduction; Tumor Cells, Cultured; Tyrosine

A number of growth factors, growth factor receptors and cell cycle regulatory proteins have been implicated in the genesis of mammary carcinomas both in animal models as well as in human breast tumour samples. Studies on the development of the mammary gland has revealed that several of the proto-oncogenes, or their closely related gene-family members, have a function in the normal growth and differentiation of the gland. In this review the role of fibroblast growth factor signalling and the critical requirement for the cell cycle regulator, cyclin D1 is discussed with respect to their normal function in mammary gland development and abnormal role in mammary carcinogenesis.

Topics: Animals; Breast; Breast Neoplasms; Cyclin D1; Female; Fibroblast Growth Factors; Humans; Mammary Glands, Animal; Mammary Neoplasms, Animal; Mice; Mice, Transgenic; Pregnancy; Pregnancy, Animal; Signal Transduction

Topics: Biomarkers, Tumor; Breast Neoplasms; CDC2 Protein Kinase; Cell Cycle Proteins; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Female; Humans; Ki-67 Antigen; Microtubule-Associated Proteins; Prognosis; Proliferating Cell Nuclear Antigen; Tumor Suppressor Proteins

Topics: Biomarkers, Tumor; Breast Neoplasms; Cyclin D1; Female; Humans; Prognosis; Telomerase

Topics: Age of Onset; Anticarcinogenic Agents; BRCA1 Protein; BRCA2 Protein; Breast Neoplasms; Breast Neoplasms, Male; Carcinoma; Case Management; Cyclin D1; DNA Mutational Analysis; Estrogen Antagonists; Female; Gene Frequency; Genes, BRCA1; Genes, erbB-2; Genetic Counseling; Genetic Testing; Humans; Male; Mammography; Mastectomy; Neoplasm Proteins; Neoplasms, Multiple Primary; Neoplastic Syndromes, Hereditary; Oncogenes; Ovarian Neoplasms; Ovariectomy; Phenotype; Raloxifene Hydrochloride; Retrospective Studies; Risk; Tamoxifen; Transcription Factors

Topics: Breast Neoplasms; Cell Cycle; Cell Cycle Proteins; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase Inhibitor p27; Genes, p53; Genes, Retinoblastoma; Humans; Microtubule-Associated Proteins; Mutation; Tumor Suppressor Proteins

A central issue in the study of neoplastic transformation is to understand how proto-oncogene products deregulate normal processes of cell growth and differentiation: an intrinsic aspect of this is to probe the sequence of events leading to altered expression of proto-oncogenes. In the past few years, studies aimed at understanding the regulation and function of protein synthesis initiation factors, eIF4E initially, culminated in the unexpected finding that a moderate overexpression of this factor results in dramatic phenotypic changes, including rapid proliferation and malignant transformation. Conversely, the tumorigenic properties of cancer cells can be strongly inhibited by antisense-RNA against eIF4E, or overexpression of the inhibitory proteins: 4E-BPs. Furthermore, eIF4E is elevated in carcinomas of the breast, head and neck (HNSCC) and prostate, but not in typical benign lesions. This is a strong indication that elevated eIF4E expression may mark a critical transition in cancer progression. Establishing a greater protein synthesis output may be a necessary step for cancer cells in order to sustain their rapid proliferation. However, analysis of cells transformed by eIF4E revealed that the synthesis of only a few proteins was greatly enhanced, while synthesis of most was minimally increased. One possible explanation is that eIF4E causes these effects by specifically increasing the translational efficiency of several oncogene transcripts, leading to overexpression of their products. The feasibility of this hypothesis was confirmed experimentally with the identification of several important products that are specifically upregulated in eIF4E-overexpressing cells. These include: c-Myc, cyclin DI and ODC, which control cycle progression and tumorigenesis; basic fibroblast growth factor (FGF-2) and vascular endothelial growth factor (VEGF), which are powerful promoters of cell growth and angiogenesis. A deeper understanding of the mRNAs that are strongly dependent on excess eIF4E/F for efficient translation will eventually result in fuller understanding of the fundamental role of translational control in different pathophysiological conditions, including malignancy.

Topics: Animals; Apoptosis; Breast Neoplasms; Carcinoma; Cell Division; Cyclin D1; Disease Progression; Endothelial Growth Factors; Eukaryotic Initiation Factor-4E; Fibroblast Growth Factor 2; Gene Expression Regulation, Neoplastic; Genes, myc; Head and Neck Neoplasms; Humans; Lymphokines; Metalloendopeptidases; Neoplasm Proteins; Neoplasms; Peptide Initiation Factors; Proto-Oncogene Mas; RNA, Messenger; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Topics: Animals; Breast; Breast Neoplasms; Cell Line; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; DNA-Binding Proteins; Oncogene Proteins v-myb; Proto-Oncogene Proteins; Receptors, Estrogen; Transcription Factors; Transfection

Extensive studies of BRCA1- and BRCA2-associated breast tumours have been carried out in the few years since the identification of these familial breast cancer predisposing genes. The morphological studies suggest that BRCA1 tumours differ from BRCA2 tumours and from sporadic breast cancers. Recent progress in immunohistochemistry and molecular biology techniques has enabled in-depth investigation of molecular pathology of these tumours. Studies to date have investigated issues such as steroid hormone receptor expression, mutation status of tumour suppressor genes TP53 and c-erbB2, and expression profiles of cell cycle proteins p21, p27 and cyclin D1. Despite relative paucity of data, strong evidence of unique biological characteristics of BRCA1-associated breast cancer is accumulating. BRCA1-associated tumours appear to show an increased frequency of TP53 mutations, frequent p53 protein stabilization and absence of imunoreactivity for steroid hormone receptors. Further studies of larger number of samples of both BRCA1- and BRCA2-associated tumours are necessary to clarify and confirm these observations.

Topics: Breast Neoplasms; Cathepsin D; Cell Cycle Proteins; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclins; Female; Genes, erbB-2; Genes, p53; Humans; Immunohistochemistry; Microtubule-Associated Proteins; Receptors, Estrogen; Tumor Suppressor Proteins

The cyclin D1/CCND1 oncogene (PRAD1) is amplified in 15% of primary human breast cancers and overexpressed in 30-50% of breast cancers, suggesting that mechanisms in addition to DNA amplification may lead to deregulated expression of this gene in breast cancer. Cyclin D1 overexpression at a higher frequency than gene amplification is also seen in a variety of other tumors. Cyclin D1 overexpression without amplification could result from a trans-acting regulatory disturbance or could be a consequence of a clonal regulatory mutation in one allele of the gene. We have, therefore, examined whether the overexpression of cyclin D1 mRNA is derived from one parental allele or both alleles in tumor cell lines with or without amplification of the cyclin D1 gene. Eight tumor cell lines, MCF-7, SK-BR-3, ZR-75-1, U-2-OS, SK-LMS-1, DLD1, HCT15, and HT29, out of 20 tumor cells initially examined were found to be heterozygous at the polymorphic NciI site within exon 4 of the cyclin D1 gene. Polymerase chain reaction and NciI digestion (PCR-RFLP) analysis of genomic DNA demonstrated DNA amplification of one allele in the ZR-75-1 cells and HT29 cells and no such imbalance in cyclin D1 gene copy number in the other cells, consistent with Southern blot analyses. Reverse-transcription polymerase chain reaction analysis and NciI digestion (RT-PCR-RFLP) of total cDNA revealed that the overexpressed cyclin D1 mRNA is preferentially derived from the amplified allele in the ZR-75-1 and HT29 cells. In contrast, the other tumor cells overexpressed cyclin D1 mRNA equally from both alleles. This finding strongly suggests that, in breast, sarcoma, and in colon cancer cells with cyclin D1 overexpression and normal gene copy number, elevated levels of cyclin D1 mRNA result from a trans-acting influence on both alleles rather than a clonal somatic mutation or rearrangement in or near a single cyclin D1 gene.

Topics: Alleles; Breast; Breast Neoplasms; Cell Line, Transformed; Cyclin D1; Epithelial Cells; Gene Expression Regulation, Neoplastic; Humans; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Tumor Cells, Cultured

Topics: Breast Neoplasms; Cell Cycle Proteins; Cell Movement; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Female; Growth Substances; Humans; Phenotype; Point Mutation

Cyclin D1 protein plays an important part in regulating the progress of the cell during the G1 phase of the cell cycle. The cyclin D1 gene, CCND1, is amplified in approximately 20% of mammary carcinomas, and the protein is over-expressed in approximately 50% of cases. This has led to intensive study to ascertain whether cyclin D1 is a biological marker in breast cancer; however, the clinical work has produced unexpected results. Work in cell lines and in transgenic mice indicate that CCND1 is a weak oncogene and it was expected that, like c-erbB-2, over-expression of cyclin D1 protein would be associated with a poor prognosis. Early immunohistochemical prognostic studies produced equivocal results but we, and others, have recently shown that strong staining for cyclin D1 is more likely to be seen in well differentiated, estrogen receptor positive carcinomas. Furthermore, we have found that over-expression of cyclin D1 is actually associated with a good outcome, both in terms of prognosis and response to endocrine treatment. Cyclin D1 is frequently over-expressed in ductal carcinoma in situ but not in benign breast disease, including atypical ductal hyperplasia; hence its expression appears to be closely linked with carcinogenesis. In order to help explain the apparent beneficial effects of cyclin D1 over-expression, a number of closely associated cell cycle proteins have also been evaluated, including the cyclin dependent kinase inhibitor p27, which blocks the activating effects of cyclin D1. Initial reports show that high levels of p27 are associated with a good prognosis and we have shown a positive association between p27 and cyclin D1 expression. These clinical results of cyclin D1 are an example of how information obtained from basic cell biology studies needs to be complemented by clinical studies to ascertain the true worth of a prognostic marker.

Topics: Animals; Biomarkers, Tumor; Breast Neoplasms; Cell Cycle Proteins; Cell Line; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Female; Humans; Mice; Mice, Knockout; Microtubule-Associated Proteins; Prognosis; Receptors, Estrogen; Tamoxifen; Tumor Suppressor Proteins

Cyclins are regulatory subunits for cyclin dependent kinases in the coordination of the cell cycle. Cyclins can also serve non-cell cycle functions, such as the transactivation of estrogen receptor by cyclin D. Evidence for the participation of the G1 cyclins D and E in breast cancer is summarized, including transgenic and knockout mice, transfections, and expression patterns in cohort studies. Overexpression of cyclin D has been reported in ductal carcinoma in situ (DCIS), and similar overexpression of cyclin E is suggested. Strategies to reduce cyclin expression are discussed as potential prevention efforts.

Topics: Animals; Breast Neoplasms; Cyclin D1; Cyclin E; Cyclins; Female; Humans; Mice; Precancerous Conditions; Retinoids; Tamoxifen

The control of the cell-cycle-associated protein cyclin D1 and its variable behaviour in normal and transformed cells is described and contrasted with its activity in vivo. The role of cyclin D1 as a prognostic and predictive marker is examined in clinical breast cancer. High levels of the protein are seen in well differentiated, oestrogen receptor positive tumours, which respond well to tamoxifen treatment for metastatic disease.

Topics: Animals; Antineoplastic Agents, Hormonal; Biomarkers, Tumor; Breast Neoplasms; Cyclin D1; Cyclins; Female; Humans; Mice; Neoplasm Proteins; Oncogene Proteins; Prognosis; Tamoxifen; Treatment Outcome

The deregulation of cyclin D1 (BCL-1, PRAD1, CCND1) protein, normally synthesized in the G1 phase of the cell cycle, has been implicated in the pathogenesis of some malignant neoplasms, including invasive mammary carcinomas. We used rabbit polyclonal antibody 19 to detect cyclin D1 in 55 infiltrating ductal carcinomas and compared the findings to six important clinicopathologic parameters and cyclin D1 gene amplification. Nuclear immunoreactivity of variable intensity for cyclin D1 was present in 35% of the neoplasms, whereas immunoreactivity of normal mammary epithelial nuclei was absent. No significant correlations were observed between immunoreactivity and patient age, axillary lymph node status, estrogen receptors, progesterone receptors, histologic grade, or any of its three components. There was a correlation between cyclin D1 immunostaining and tumor size (P = 0.013). Fourteen of 15 tumors 2 cm or less were negative, whereas 7 of 12 neoplasms larger than 4 cm were immunopositive. Fifteen percent of the invasive carcinomas had cyclin D1 gene amplification. Of these eight tumors, six showed cyclin D1 immunoreactivity (P = 0.017). In this study, cyclin D1 was detected immunohistochemically in approximately one-third of infiltrating ductal carcinomas; approximately one-third of these had detectable cyclin D1 gene amplification. These results further implicate cyclin D1 in breast tumorigenesis and are additional evidence for the role of cell cycle regulatory proteins in invasive mammary carcinoma.

Topics: Adult; Aged; Breast Neoplasms; Carcinoma, Ductal, Breast; Cyclin D1; Cyclins; Gene Amplification; Humans; Immunohistochemistry; Middle Aged; Oncogene Proteins

In this paper we describe how research on the mouse mammary tumor virus model of breast cancer resulted in the identification of an amplified region of DNA on human chromosome 11 band q13. This amplification occurs in approximately 15% of primary breast cancers. Several candidate oncogenes map within the amplicon but by analysing expression of these genes a strong case can be made for a role for cyclin D1 in tumorigenesis. Immunohistochemical staining indicates that cyclin D1 is expressed at elevated levels in around 40% of breast cancers, including those with the 11q13 amplification. The potential function of cyclin D1 as a regulator of early cell division cycle events would be consistent with a role in neoplasia.

Topics: Blotting, Southern; Breast Neoplasms; Chromosomes, Human, Pair 11; Cyclin D1; Cyclins; Gene Amplification; Humans; Oncogene Proteins

One in six primary human breast cancers has DNA amplification centered on the cyclin D1 gene (CCND1) on chromosome 11q13. This genetic abnormality is preferentially associated with estrogen-receptor positive tumors and may define a sub-class of patients with an adverse prognosis. Although CCND1 has the credentials of a cellular oncogene, being a target for chromosomal translocation and retroviral integration, the 11q13 amplicon encompasses several other markers and CCND1 is not the only candidate for the key gene on the amplified DNA. To assess their relative importance, we have constructed a physical map of the amplified DNA and compared the extent and frequency of amplification across the region. Since it is likely that the gene providing the selective force for amplification will be expressed at elevated levels, we have also examined expression of both RNA and protein. By these criteria, cyclin D1 remains the strongest candidate for the key oncogene on the amplicon and we are currently investigating the functional consequences of its over-expression.

Topics: Breast Neoplasms; Chromosome Mapping; Chromosomes, Human, Pair 11; Cyclin D1; Cyclins; DNA, Neoplasm; Genetic Linkage; Genetic Markers; Humans; Oncogene Proteins; Receptors, Estrogen; Translocation, Genetic

The D-type cyclins are among the candidate 'G1 cyclins' in higher eukaryotes that may regulate G1-S-phase progression. The human cyclin D1 gene, also known as PRAD1 (and previously as D11S287), is a putative proto-oncogene strongly implicated in several types of human tumors, including parathyroid adenomas, B-cell neoplasms (as the 'BCL-1 oncogene'), and breast and squamous cell cancers. The mechanism by which deregulated production of cyclin D1/PRAD1, and perhaps other D-type cyclins, contributes to tumor development is only beginning to be deciphered.

Topics: Adenoma; Breast Neoplasms; Carcinoma, Squamous Cell; Cell Cycle; Cell Division; Cell Transformation, Neoplastic; Cyclin D1; Cyclins; Gene Expression Regulation, Neoplastic; Humans; Leukemia, B-Cell; Lymphoma, B-Cell; Multigene Family; Oncogene Proteins; Parathyroid Neoplasms; Proto-Oncogene Mas; Proto-Oncogenes

Cyclin D/CDK4/6 is critical in controlling the G1 to S checkpoint. CCND, the gene encoding cyclin D, is known to be amplified in a variety of solid tumors. Palbociclib is an oral CDK4/6 inhibitor, approved in advanced breast cancer in combination with endocrine therapy. We explored the efficacy of palbociclib in patients with nonbreast solid tumors containing an amplification in CCND1, 2, or 3.. Patients with tumors containing a CCND1, 2, or 3 amplification and expression of the retinoblastoma protein were assigned to subprotocol Z1B and received palbociclib 125 mg once daily for 21 days of a 28-day cycle. Tumor response was assessed every two cycles.. Forty patients were assigned to subprotocol Z1B; 4 patients had outside assays identifying the CCND1, 2, or 3 amplification and were not confirmed centrally; 3 were ineligible and 2 were not treated (1 untreated patient was also ineligible), leaving 32 evaluable patients for this analysis. There were no partial responses; 12 patients (37.5%) had stable disease as best response. There were seven deaths on study, all during cycle 1 and attributable to disease progression. Median progression-free survival was 1.8 months. The most common toxicities were leukopenia (n = 21, 55%) and neutropenia (n = 19, 50%); neutropenia was the most common grade 3/4 event (n = 12, 32%).. Palbociclib was not effective at treating nonbreast solid tumors with a CCND1, 2, or 3 amplification in this cohort. These data do not support further investigation of single-agent palbociclib in tumors with CCND1, 2, or 3 amplification.

Topics: Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cyclin D1; Female; Humans; Neutropenia; Piperazines; Pyridines

Ribociclib has received approval in the pre/peri- and postmenopausal disease settings on the basis of the MONALEESA trials. MONALEESA-2 demonstrated that ribociclib plus letrozole significantly improved progression-free survival compared with placebo plus letrozole as first-line therapy in postmenopausal patients with HR-positive, HER2-negative advanced breast cancer. Subsequently, ongoing trials reported significant progression-free survival improvements with ribociclib in combination with either fulvestrant in postmenopausal patients with advanced breast cancer who were either treatment naive or received ≤1 line of prior endocrine therapy in the advanced disease setting (MONALEESA-3) or tamoxifen/nonsteroidal aromatase inhibitor with ovarian function suppression in pre/perimenopausal women (MONALEESA-7). This review summarizes the MONALEESA clinical program. ClinicalTrials.gov identifiers: NCT01958021 (MONALEESA-2), NCT02422615 (MONALEESA-3), NCT02278120 (MONALEESA-7).

Topics: Adolescent; Adult; Aminopyridines; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Aromatase Inhibitors; Breast Neoplasms; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Estrogens; Female; Fulvestrant; Humans; Letrozole; Middle Aged; Postmenopause; Progression-Free Survival; Purines; Receptor, ErbB-2; Tamoxifen; Young Adult

Topics: Adult; Aged; Antineoplastic Agents, Immunological; Biomarkers, Tumor; Breast Neoplasms; Chemotherapy, Adjuvant; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Female; Gene Expression Regulation, Neoplastic; Humans; Middle Aged; Neoplasm Grading; Neoplasm Metastasis; Neoplasm Staging; Prognosis; Receptor, ErbB-2; Trastuzumab; Treatment Outcome; Young Adult

The retinoblastoma (RB) gene is a tumor-suppressor gene that plays a central role in regulating the cell cycle. Inactivation of this gene is involved in breast cancer.. A total of 827 patients with breast cancer treated with taxane-based adjuvant chemotherapy were included in the study. Protein expression of RB, phosphorylated RB (pRB), p16, cyclin D1 and p53 was evaluated by immunohistochemistry.. Neither of the retinoblastoma markers (RB and pRB) reached statistical significance in terms of their association with disease-free or overall survival. Nevertheless, when clustering analysis was performed, patients with tumors featuring low levels of p16, cyclin D1 and p53 with concomitantly high levels of pRB had reduced risk for relapse (Wald's p=0.015).. The p53-mediated sensitivity of breast cancer cells to chemotherapeutic agents appears to be driven mostly by pRB. Using agents that enhance RB phosphorylation might possibly increase the chemosensitivity of breast cancer cells.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents, Phytogenic; Breast Neoplasms; Chemotherapy, Adjuvant; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Female; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Middle Aged; Paclitaxel; Phosphorylation; Prognosis; Retinoblastoma Protein; Tumor Suppressor Protein p53; Young Adult

To evaluate the prognostic role of elaborate molecular clusters encompassing cyclin D1, cyclin E1, p21, p27 and p53 in the context of various breast cancer subtypes.. Cyclin E1, cyclin D1, p53, p21 and p27 were evaluated with immunohistochemistry in 1077 formalin-fixed paraffin-embedded tissues from breast cancer patients who had been treated within clinical trials. Jaccard distances were computed for the markers and the resulted matrix was used for conducting unsupervised hierarchical clustering, in order to identify distinct groups correlating with prognosis.. Luminal B and triple-negative (TNBC) tumors presented with the highest and lowest levels of cyclin D1 expression, respectively. By contrast, TNBC frequently expressed Cyclin E1, whereas ER-positive tumors did not. Absence of Cyclin D1 predicted for worse OS, while absence of Cyclin E1 for poorer DFS. The expression patterns of all examined proteins yielded 3 distinct clusters; (1) Cyclin D1 and/or E1 positive with moderate p21 expression; (2) Cyclin D1 and/or E1, and p27 positive, p53 protein negative; and, (3) Cyclin D1 or E1 positive, p53 positive, p21 and p27 negative or moderately positive. The 5-year DFS rates for clusters 1, 2 and 3 were 70.0%, 79.1%, 67.4% and OS 88.4%, 90.4%, 78.9%, respectively.. It seems that the expression of cell cycle regulators in the absence of p53 protein is associated with favorable prognosis in operable breast cancer.

Topics: Adult; Aged; Antineoplastic Agents; Breast; Breast Neoplasms; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Female; Humans; Immunohistochemistry; Middle Aged; Oncogene Proteins; Prognosis; Survival Analysis; Triple Negative Breast Neoplasms; Tumor Suppressor Protein p53; Young Adult

Palbociclib (PD-0332991) is an oral, small-molecule inhibitor of cyclin-dependent kinases (CDKs) 4 and 6 with preclinical evidence of growth-inhibitory activity in oestrogen receptor-positive breast cancer cells and synergy with anti-oestrogens. We aimed to assess the safety and efficacy of palbociclib in combination with letrozole as first-line treatment of patients with advanced, oestrogen receptor-positive, HER2-negative breast cancer.. In this open-label, randomised phase 2 study, postmenopausal women with advanced oestrogen receptor-positive and HER2-negative breast cancer who had not received any systemic treatment for their advanced disease were eligible to participate. Patients were enrolled in two separate cohorts that accrued sequentially: in cohort 1, patients were enrolled on the basis of their oestrogen receptor-positive and HER2-negative biomarker status alone, whereas in cohort 2 they were also required to have cancers with amplification of cyclin D1 (CCND1), loss of p16 (INK4A or CDKN2A), or both. In both cohorts, patients were randomly assigned 1:1 via an interactive web-based randomisation system, stratified by disease site and disease-free interval, to receive continuous oral letrozole 2.5 mg daily or continuous oral letrozole 2.5 mg daily plus oral palbociclib 125 mg, given once daily for 3 weeks followed by 1 week off over 28-day cycles. The primary endpoint was investigator-assessed progression-free survival in the intention-to-treat population. Accrual to cohort 2 was stopped after an unplanned interim analysis of cohort 1 and the statistical analysis plan for the primary endpoint was amended to a combined analysis of cohorts 1 and 2 (instead of cohort 2 alone). The study is ongoing but closed to accrual; these are the results of the final analysis of progression-free survival. The study is registered with the ClinicalTrials.gov, number NCT00721409.. Between Dec 22, 2009, and May 12, 2012, we randomly assigned 165 patients, 84 to palbociclib plus letrozole and 81 to letrozole alone. At the time of the final analysis for progression-free survival (median follow-up 29.6 months [95% CI 27.9-36.0] for the palbociclib plus letrozole group and 27.9 months [25.5-31.1] for the letrozole group), 41 progression-free survival events had occurred in the palbociclib plus letrozole group and 59 in the letrozole group. Median progression-free survival was 10.2 months (95% CI 5.7-12.6) for the letrozole group and 20.2 months (13.8-27.5) for the palbociclib plus letrozole group (HR 0.488, 95% CI 0.319-0.748; one-sided p=0.0004). In cohort 1 (n=66), median progression-free survival was 5.7 months (2.6-10.5) for the letrozole group and 26.1 months (11.2-not estimable) for the palbociclib plus letrozole group (HR 0.299, 0.156-0.572; one-sided p<0.0001); in cohort 2 (n=99), median progression-free survival was 11.1 months (7.1-16.4) for the letrozole group and 18.1 months (13.1-27.5) for the palbociclib plus letrozole group (HR 0.508, 0.303-0.853; one-sided p=0.0046). Grade 3-4 neutropenia was reported in 45 (54%) of 83 patients in the palbociclib plus letrozole group versus one (1%) of 77 patients in the letrozole group, leucopenia in 16 (19%) versus none, and fatigue in four (4%) versus one (1%). Serious adverse events that occurred in more than one patient in the palbociclib plus letrozole group were pulmonary embolism (three [4%] patients), back pain (two [2%]), and diarrhoea (two [2%]). No cases of febrile neutropenia or neutropenia-related infections were reported during the study. 11 (13%) patients in the palbociclib plus letrozole group and two (2%) in the letrozole group discontinued the study because of adverse events.. The addition of palbociclib to letrozole in this phase 2 study significantly improved progression-free survival in women with advanced oestrogen receptor-positive and HER2-negative breast cancer. A phase 3 trial is currently underway.. Pfizer.

Topics: Administration, Oral; Aged; Antineoplastic Combined Chemotherapy Protocols; Aromatase Inhibitors; Biomarkers, Tumor; Breast Neoplasms; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinase Inhibitor p16; Disease-Free Survival; Drug Administration Schedule; Europe; Female; Humans; Intention to Treat Analysis; Letrozole; Middle Aged; Molecular Targeted Therapy; Nitriles; North America; Piperazines; Postmenopause; Proportional Hazards Models; Protein Kinase Inhibitors; Pyridines; Receptor, ErbB-2; Receptors, Estrogen; Republic of Korea; South Africa; Time Factors; Treatment Outcome; Triazoles

Cholesterol lowering statins have been demonstrated to exert anti-tumoral effects on breast cancer by decreasing proliferation as measured by Ki67. The biological mechanisms behind the anti-proliferative effects remain elusive. The aim of this study was to investigate potential statin-induced effects on the central cell cycle regulators cyclin D1 and p27.. This phase II window-of-opportunity trial (Trial registration: ClinicalTrials.gov NCT00816244 , NIH) included 50 patients with primary invasive breast cancer. High-dose atorvastatin (80 mg/day) was prescribed to patients for two weeks prior to surgery. Paired paraffin embedded pre- and post-statin treatment tumor samples were analyzed using immunohistochemistry for the expression of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and the cell cycle regulators cyclin D1 and p27. Corresponding frozen tumor sample pairs were analyzed for expression of the genes coding for cyclin D1 and p27, CCND1 and CDKN1B, respectively.. Forty-two patients completed all study parts, and immunohistochemical evaluation of ER and PR was achievable in 30 tumor pairs, HER2 in 29 tumor pairs, cyclin D1 in 30 tumor pairs and p27 in 33 tumor pairs. The expression of ER, PR and HER2 did not change significantly following atorvastatin treatment. Cyclin D1 expression in terms of nuclear intensity was significantly decreased (P = 0.008) after statin treatment in paired tumor samples. The protein expression of the tumor suppressor p27, evaluated either as the fraction of stained tumor cells or as cytoplasmic intensity, increased significantly (P = 0.03 and P = 0.02, respectively). At the transcriptional level, no significant differences in mRNA expression were detected for cyclin D1 (CCND1) and p27 (CDKN1B). However, CCND1 expression was lower in tumors responding to atorvastatin treatment with a decrease in proliferation although not significantly (P = 0.08).. We have previously reported statin-induced anti-proliferative effects in breast cancer. This study suggests that cell cycle regulatory effects may contribute to these anti-proliferative effects via cyclin D1 and p27.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Breast Neoplasms; Cell Cycle; Cell Proliferation; Cholesterol; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Female; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Immunohistochemistry; Ki-67 Antigen; Middle Aged; Phosphorylation; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone; RNA, Messenger

Observational data show that nonsteroidal anti-inflammatory drug (NSAID) use is associated with a lower rate of breast cancer. We evaluated the effect of etodolac, an FDA-approved NSAID reported to inhibit cyclooxygenase (COX) enzymes and the retinoid X receptor alpha (RXR), on rationally identified potential biomarkers in breast cancer. Patients with resectable breast cancer planned for initial management with surgical resection were enrolled and took 400 mg of etodolac twice daily prior to surgery. Protein and gene expression levels for genes related to COX-2 and RXRα were evaluated in tumor samples from before and after etodolac exposure. Thirty subjects received etodolac and 17 subjects were assayed as contemporaneous or opportunistic controls. After etodolac exposure mean cyclin D1 protein levels, assayed by immunohistochemistry, decreased (P = 0.03). Notably, pre- versus post cyclin D1 gene expression change went from positive to negative with greater duration of etodolac exposure (r = -0.64, P = 0.01). Additionally, etodolac exposure was associated with a significant increase in COX-2 gene expression levels (fold change: 3.25 [95% CI: 1.9, 5.55]) and a trend toward increased β-catenin expression (fold change: 2.03 [95% CI: 0.93, 4.47]). In resectable breast cancer relatively brief exposure to the NSAID etodolac was associated with reduced cyclin D1 protein levels. Effect was also observed on cyclin D1 gene expression with decreasing levels with longer durations of drug exposure. Increased COX-2 gene expression was seen, possibly due to compensatory feedback. These data highlight the utility of even small clinical trials with access to biospecimens for pharmacodynamic studies.

Topics: Administration, Oral; Aged; beta Catenin; Biomarkers, Tumor; Breast Neoplasms; Cyclin D1; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Etodolac; Female; Gene Expression; Humans; Middle Aged; Preoperative Period; Retinoid X Receptor alpha

Several trials have confirmed that the pathological complete response (pCR) rates after neoadjuvant chemotherapy (NAC) are significantly lower in HER2-positive/ER-positive patients than in HER2-positive/ER-negative patients. To understand this phenomenon, we investigated the association between NAC resistance and CCND1, which is frequently overexpressed in ER-positive tumors. Pretreatment formalin-fixed tumor tissues were collected from 75 HER2-positive patients receiving NAC comprised anthracyclines, taxanes, and trastuzumab. Seventeen gene transcripts along with PIK3CA mutations were detected using MassARRAY (Sequenom, San Diego, CA). The gene expression levels were dichotomized according to the median values. The immunohistochemical expression of ER, PTEN, BCL-2, and cyclin D1 was scored. The relationship between the variables was assessed using the Spearman correlation. A logistic regression analysis was performed to detect predictors of pCR, which was defined as no invasive tumor in the breast or axilla. Forty-seven percent of the cases were ER-positive and 52 % (40/63 % in ER-positive/ER-negative) achieved a pCR. Among the ER-positive patients, the CCND1 gene expression level was 2.1 times higher than that in ER-negative patients and was significantly correlated with the expression of cyclin D1 protein. In a univariate analysis, a pCR was associated with high mRNA levels of ESR1, PGR, LMTK3, HER2, IGF1R, INPP4B, PDL-1, BCL-2, and CCND1 (P ≤ 0.05). In contrast, none of these genes were significantly correlated with a pCR among the ER-negative tumors and only EGFR was significantly correlated with a pCR. PIK3CA mutations or PTEN loss were not associated with a pCR in either group. After excluding ESR1 (r = 0.58), PGR (r = 0.64), and IGF1R (r = 0.59), the expressions of which were correlated with CCND1, a multivariate analysis revealed that CCND1 [P = 0.043; OR, 0.16] and HER2 [P = 0.012; OR, 11.2] retained its predictive value for pCR among ER-positive patients, but not among ER-negative patients. A High Level of CCND1 gene expression is a poor predictor of a pCR and provides a rationale for evaluating CDK4/6 inhibitors in HER2-positive/ER-positive breast cancer patients.

Topics: Adult; Aged; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Breast Neoplasms; Class I Phosphatidylinositol 3-Kinases; Cyclin D1; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Humans; Middle Aged; Mutation; Neoadjuvant Therapy; Phosphatidylinositol 3-Kinases; Predictive Value of Tests; Receptor, ErbB-2; Trastuzumab; Treatment Outcome

Limonene is a bioactive food component found in citrus peel oil that has shown chemopreventive and chemotherapeutic activities in preclinical studies. We conducted an open-label pilot clinical study to determine the human breast tissue disposition of limonene and its associated bioactivity. We recruited 43 women with newly diagnosed operable breast cancer electing to undergo surgical excision to take 2 grams of limonene daily for two to six weeks before surgery. Blood and breast tissue were collected to determine drug/metabolite concentrations and limonene-induced changes in systemic and tissue biomarkers of breast cancer risk or carcinogenesis. Limonene was found to preferentially concentrate in the breast tissue, reaching high tissue concentration (mean = 41.3 μg/g tissue), whereas the major active circulating metabolite, perillic acid, did not concentrate in the breast tissue. Limonene intervention resulted in a 22% reduction in cyclin D1 expression (P = 0.002) in tumor tissue but minimal changes in tissue Ki67 and cleaved caspase-3 expression. No significant changes in serum leptin, adiponectin, TGF-β1, insulin-like growth factor binding protein-3 (IGFBP-3), and interleukin-6 (IL-6) levels were observed following limonene intervention. There was a small but statistically significant postintervention increase in insulin-like growth factor I (IGF-I) levels. We conclude that limonene distributed extensively to human breast tissue and reduced breast tumor cyclin D1 expression that may lead to cell-cycle arrest and reduced cell proliferation. Furthermore, placebo-controlled clinical trials and translational research are warranted to establish limonene's role for breast cancer prevention or treatment.

Topics: Anticarcinogenic Agents; Biomarkers, Tumor; Breast; Breast Neoplasms; Caspase 3; Citrus; Cyclin D1; Cyclohexenes; Female; Follow-Up Studies; Humans; Immunoenzyme Techniques; Insulin-Like Growth Factor Binding Protein 3; Insulin-Like Growth Factor I; Interleukin-6; Limonene; Middle Aged; Monoterpenes; Neoplasm Staging; Pilot Projects; Prognosis; Terpenes; Tissue Distribution; Transforming Growth Factor beta1

Most women with estrogen receptor expressing breast cancers receiving anti-estrogens such as tamoxifen may not need or benefit from them. Besides the estrogen receptor, there are no predictive biomarkers to help select breast cancer patients for tamoxifen treatment. CCND1 (cyclin D1) gene amplification is a putative candidate tamoxifen predictive biomarker. The RSF1 (remodeling and spacing factor 1) gene is frequently co-amplified with CCND1 on chromosome 11q. We validated the predictive value of these biomarkers in the MA.12 randomized study of adjuvant tamoxifen vs. placebo in high-risk premenopausal early breast cancer. Premenopausal women with node-positive/high-risk node-negative early breast cancer received standard adjuvant chemotherapy and then were randomized to tamoxifen (20 mg/day) or placebo for 5 yrs. Overall survival (OS) and relapse-free survival (RFS) were evaluated. Fluorescent in-situ hybridization was performed on a tissue microarray of 495 breast tumors (74% of patients) to measure CCND1 and RSF1 copy number. A multivariate Cox model to obtain hazard ratios (HR) adjusting for clinico-pathologic factors was used to assess the effect of these biomarkers on Os and RFS. 672 women were followed for a median of 8.4 years. We were able to measure the DNA copy number of CCND1 in 442 patients and RSF1 in 413 patients. CCND1 gene amplification was observed in 8.7% and RSF1 in 6.8% of these patients, preferentially in estrogen receptor-positive breast cancers. No statistically significant interaction with treatment was observed for either CCND1 or RSF1 amplification, although patients with high RSF1 copy number did not show benefit from adjuvant tamoxifen (HR = 1.11, interaction p = 0.09). Unlike CCND1 amplification, RSF1 amplification may predict for outcome in high-risk premenopausal breast cancer patients treated with adjuvant tamoxifen.

Topics: Antineoplastic Agents, Hormonal; Breast Neoplasms; Cyclin D1; Humans; In Situ Hybridization, Fluorescence; Nuclear Proteins; Premenopause; Tamoxifen; Trans-Activators; Treatment Outcome

Gene amplification of CCND1 is observed in a subgroup of breast cancers with poor prognosis, whereas overexpression of the protein cyclin D1 has been linked to both worse and better clinical outcome. CCND1 amplification and protein overexpression have also been associated with resistance to treatment with tamoxifen or even to a potentially detrimental effect of tamoxifen.. To clarify these challenging and partly contrasting treatment predictive and prognostic links for cyclin D1 we analysed a large cohort of postmenopausal breast cancer patients randomised to receive either adjuvant anastrozole or tamoxifen, as part of the Arimidex, Tamoxifen, Alone or in Combination (ATAC) trial. The CCND1 amplification status and protein expression of cyclin D1 were assessed by chromogenic in situ hybridisation and immunohistochemistry, respectively, in 1,155 postmenopausal, oestrogen-receptor-positive breast cancer patients included in the TransATAC substudy.. Amplification of CCND1 was observed in 8.7% of the tumours and was associated with increased risk of disease recurrence (hazard ratio = 1.61; 95% confidence interval, 1.08 to 2.41) after adjustment for other clinicopathological parameters. In contrast, nuclear expression of cyclin D1 protein was associated with decreased recurrence rate (hazard ratio = 0.6; 95% confidence interval, 0.39 to 0.92). The intensity of nuclear or cytoplasmic expression was not of prognostic value. There was no significant interaction between cyclin D1 status and treatment efficacy, ruling out any major detrimental effect of tamoxifen in CCND1-amplified postmenopausal breast cancer.. In summary, CCND1 amplification and low nuclear expression of cyclin D1 predicted poor clinical outcome in postmenopausal breast cancer patients treated with either anastrozole or tamoxifen.. Current Controlled Trials ISRCTN18233230.

Topics: Anastrozole; Antineoplastic Agents, Hormonal; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cyclin D1; Female; Follow-Up Studies; Gene Amplification; Gene Expression Regulation, Neoplastic; Humans; Ki-67 Antigen; Middle Aged; Neoplasm Recurrence, Local; Nitriles; Postmenopause; Predictive Value of Tests; Prognosis; Receptors, Estrogen; Tamoxifen; Treatment Outcome; Triazoles

Endocrine therapy in patients with breast cancer can be limited by the problem of resistance. Preclinical studies suggest that complete blockade of the estrogen receptor (ER) combined with inhibition of the epidermal growth factor receptor can overcome endocrine resistance. We tested this hypothesis in a phase II neoadjuvant trial of anastrozole and fulvestrant combined with gefitinib in postmenopausal women with newly diagnosed ER-positive breast cancer. After a baseline tumor core biopsy, patients were randomized to receive anastrozole and fulvestrant or anastrozole, fulvestrant, and gefitinib (AFG) for 3 weeks. After a second biopsy at 3 weeks, all patients received AFG for 4 months and surgery was done if the tumor was operable. The primary endpoint was best clinical response by RECIST criteria and secondary endpoints were toxicity and change in biomarkers. The study closed after 15 patients were enrolled because of slow accrual. Median patient age was 67 years and median clinical tumor size was 7 cm. Four patients had metastatic disease present. Three patients withdrew before response was assessed. In the remaining 12 patients, there were two complete clinical responses (17%), three partial responses (25%), five had stable disease (41%), and two (17%) had progressive disease. Most common adverse events were rash in four patients, diarrhea in four, joint symptoms in three, and abnormal liver function tests in three. There were no grade 4 toxicities and all toxicities were reversible. At 3 weeks, cell proliferation as measured by Ki-67 was significantly reduced in the AFG group (P value = 0.01), with a parallel reduction in the expression of the Cyclin D1 (P value = 0.02). RNA microarray data showed a corresponding decrease in the expression of cell cycle genes. These results suggest that AFG was an effective neoadjuvant therapy and consistently reduced proliferation in ER-positive tumors.

Topics: Anastrozole; Antineoplastic Agents, Hormonal; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Breast Neoplasms, Male; Cell Cycle; Cyclin D1; Estradiol; Female; Fulvestrant; Gefitinib; Humans; Ki-67 Antigen; Male; Mitogen-Activated Protein Kinases; Neoadjuvant Therapy; Nitriles; Oncogene Protein v-akt; Proto-Oncogene Proteins; Quinazolines; Receptors, Estrogen; Receptors, Progesterone; Triazoles

Cross-talk between the estrogen receptor (ER) and the phosphoinositide-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathways is a mechanism of resistance to endocrine therapy, and blockade of both pathways enhances antitumor activity in preclinical models. This study explored whether sensitivity to letrozole was enhanced with the oral mTOR inhibitor, everolimus (RAD001).. Two hundred seventy postmenopausal women with operable ER-positive breast cancer were randomly assigned to receive 4 months of neoadjuvant treatment with letrozole (2.5 mg/day) and either everolimus (10 mg/day) or placebo. The primary end point was clinical response by palpation. Mandatory biopsies were obtained at baseline and after 2 weeks of treatment (ie, day 15). Samples were assessed for PI3K mutation status (PIK3CA) and for pharmacodynamic changes of Ki67, phospho-S6, cyclin D1, and progesterone receptor (PgR) by immunohistochemistry.. Response rate by clinical palpation in the everolimus arm was higher than that with letrozole alone (ie, placebo; 68.1% v 59.1%), which was statistically significant at the preplanned, one-sided, alpha = 0.1 level (P = .062). Marked reductions in progesterone receptor and cyclin D1 expression occurred in both treatment arms, and dramatic downregulation of phospho-S6 occurred only in the everolimus arm. An antiproliferative response, as defined by a reduction in Ki67 expression to natural logarithm of percentage positive Ki67 of less than 1 at day 15, occurred in 52 (57%) of 91 patients in the everolimus arm and in 25 (30%) of 82 patients in the placebo arm (P < .01). The safety profile was consistent with historical results of everolimus monotherapy; grades 3 to 4 adverse events occurred in 22.6% of patients who received everolimus and in 3.8% of patients who received placebo.. Everolimus significantly increased letrozole efficacy in neoadjuvant therapy of patients with ER-positive breast cancer.

Topics: Adult; Aged; Aged, 80 and over; Antibiotics, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Aromatase Inhibitors; Biomarkers, Tumor; Biopsy; Breast Neoplasms; Cell Proliferation; Chemotherapy, Adjuvant; Class I Phosphatidylinositol 3-Kinases; Cyclin D1; Double-Blind Method; Europe; Everolimus; Female; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Ki-67 Antigen; Letrozole; Middle Aged; Mutation; Neoadjuvant Therapy; Nitriles; Palpation; Phosphatidylinositol 3-Kinases; Phosphorylation; Postmenopause; Receptors, Estrogen; Receptors, Progesterone; Ribosomal Protein S6 Kinases; Sirolimus; Time Factors; Treatment Outcome; Triazoles; United States

The objective of our study was to determine the clinical relevance of cyclin D1 expression in hormone receptor-positive breast cancer patients who were treated with tamoxifen-based therapy.. We assessed expression of cyclin D1 in surgical specimens of breast carcinoma by means of immunohistochemistry. Patients had been enrolled in either Austrian Breast and Colorectal Cancer Study Group (ABCSG) Trial 05 or ABCSG Trial 06 and received tamoxifen as part of their adjuvant treatment. Overall survival and relapse-free survival were analyzed with Cox models adjusted for clinical and pathologic factors.. Cyclin D1 was expressed in 140 of 253 (55%) tumors of ABCSG Trial 05 and in 569 of 948 (60%) tumors of ABCSG Trial 06. Expression of cyclin D1 was associated with poor outcome in both cohorts. Overall survival was significantly shorter in patients with cyclin D1-positive tumors compared with patients with cyclin D1-negative tumors [adjusted hazard ratio (HR) for death (ABCSG Trial 05), 2.47; 95% confidence interval (95% CI), 1.08-5.63; P = 0.03; adjusted HR for death (ABCSG Trial 06), 1.78; 95% CI, 1.36-2.34; P < 0.0001]. Relapse-free survival was also shorter in patients with cyclin D1-positive tumors than in patients with cyclin D1-negative tumors [adjusted HR for relapse (ABCSG Trial 05), 2.73; 95% CI, 1.50-4.96; P = 0.001; adjusted HR for relapse (ABCSG Trial 06), 1.52; 95% CI, 1.14-2.04; P = 0.005].. Cyclin D1 expression is an independent poor prognostic factor in women with early-stage, hormone receptor-positive breast cancer who received adjuvant tamoxifen-based therapy.

Topics: Adult; Aged; Aged, 80 and over; Aminoglutethimide; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Chemotherapy, Adjuvant; Cyclin D1; Cyclophosphamide; Disease-Free Survival; Female; Fluorouracil; Follow-Up Studies; Goserelin; Humans; Methotrexate; Middle Aged; Prognosis; Receptors, Cytoplasmic and Nuclear; Recurrence; Tamoxifen; Treatment Outcome

Hypoxia in breast cancer is associated with poor prognosis and down-regulation of the estrogen receptor. Carbonic anhydrase IX (CA IX) is a hypoxia-inducible gene that has been associated with poor outcome in many epithelial cancers. Previous studies of CA IX in breast cancer have been carried out on mixed cohorts of premenopausal and postmenopausal patients with locally advanced disease and varying treatment regimens. We examined the potential prognostic and predictive role of CA IX in premenopausal breast cancer patients.. Using tissue microarrays, we analyzed CA IX expression in 400 stage II breast cancers from premenopausal women. The patients had previously participated in a randomized control trial comparing 2 years of tamoxifen to no systemic adjuvant treatment. Median follow-up was 13.9 years.. CA IX expression correlated positively with tumor size, grade, hypoxia-inducible factor 1alpha, Ki-67, cyclin E, and cyclin A2 expression. CA IX expression correlated negatively with cyclin D1, estrogen receptor, and progesterone receptor. CA IX expression was associated with a reduced relapse-free survival (P=0.032), overall survival (P=0.022), and breast cancer-specific survival (P=0.005). Multivariate analysis revealed that CA IX was an independent prognostic marker in untreated patients with one to three positive lymph nodes (hazard ratio, 3.2; 95% confidence interval, 1.15-9.13; P=0.027).. CA IX is marker of poor prognosis in premenopausal breast cancer patients and it is an independent predictor of survival in patients with one to three positive lymph nodes. As all these patients received locoregional radiation therapy, CA IX may be associated with resistance to radiotherapy.

Topics: Adult; Antigens, Neoplasm; Antineoplastic Agents, Hormonal; Biomarkers, Tumor; Blotting, Western; Breast Neoplasms; Carbonic Anhydrase IX; Carbonic Anhydrases; Chemotherapy, Adjuvant; Cyclin A; Cyclin A2; Cyclin D1; Cyclin E; Female; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Immunohistochemistry; Ki-67 Antigen; Lymphatic Metastasis; Mastectomy, Segmental; Middle Aged; Premenopause; Prognosis; Radiation Tolerance; Radiotherapy; Receptors, Estrogen; Receptors, Progesterone; Survival Analysis; Tamoxifen

Antioestrogen treatment by tamoxifen is a well-established adjuvant therapy for oestrogen receptor-alpha (ERalpha) positive breast cancer. Despite ERalpha expression some tumours do not respond to tamoxifen and we therefore delineated the potential link between the cell cycle regulator and ERalpha co-factor, cyclin D1, and tamoxifen response in a material of 167 postmenopausal breast cancers arranged in a tissue array. The patients had been randomised to 2 years of tamoxifen treatment or no treatment and the median follow-up time was 18 years. Interestingly in the 55 strongly ERalpha positive samples with moderate or low cyclin D1 levels, patients responded to tamoxifen treatment whereas the 46 patients with highly ERalpha positive and cyclin D1 overexpressing tumours did not show any difference in survival between tamoxifen and no treatment. Survival in untreated patients with cyclin D1 high tumours was slightly better than for patients with cyclin D1 low/moderate tumours. However, there was a clearly increased risk of death in the cyclin D1 high group compared to an age-matched control population. Our results suggest that cyclin D1 overexpression predicts for tamoxifen treatment resistance in breast cancer, which is line with recent experimental data using breast cancer cell lines and overexpression systems.

Topics: Aged; Antineoplastic Agents, Hormonal; Breast Neoplasms; Case-Control Studies; Chemotherapy, Adjuvant; Cyclin D1; Female; Follow-Up Studies; Humans; Immunohistochemistry; Middle Aged; Postmenopause; Prognosis; Risk Factors; Survival Analysis; Tamoxifen; Up-Regulation

Locally advanced and/or inflammatory breast cancer (LABC) is a heterogeneous disease. Molecular markers may help to understand this heterogeneity. This paper reports the results of a study assessing the potential prognostic or predictive value of HER-2, p53, cyclinD1, MIB1, ER and PgR expression by immunohistochemistry from patients included in an EORTC-NCIC-SAKK trial.. A total of 448 patients with a cytological or histological diagnosis of LABC were randomised into a trial comparing two anthracycline-based neoadjuvant regimens. Chemotherapy was followed by standard locoregional therapy. Survival was comparable in both arms. We collected and analysed centrally paraffin-embedded tumour specimens from 187 (72.5%) of 258 patients that had a histological diagnosis.. Of the patients included in this molecular marker study 114 relapsed and 91 died. In the multivariate analysis p53 positivity was associated with a shorter progression-free survival [hazard ratio (HR) = 1.96; 95% CI 1.33-2.91; P = 0.0008) and a shorter overall survival (HR = 1.98; 95% CI 1.28-3.06; P = 0.002). PgR positivity predicted for a longer overall survival (HR = 0.54; 95% CI 0.35-0.83; P = 0.0045).. p53 was an independent factor predicting for survival. In order to clarify whether p53 is a pure prognostic and/or a predictive factor, a phase III trial is being conducted (EORTC 10994/BIG 00-01 study) using functional assay in yeast from frozen tumour samples.

Topics: Administration, Oral; Adult; Aged; Anthracyclines; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Breast Neoplasms; Cyclin D1; Cyclophosphamide; Epirubicin; Female; Fluorouracil; Gene Expression Regulation, Neoplastic; Granulocyte Colony-Stimulating Factor; Humans; Immunohistochemistry; Infusions, Intravenous; Ki-67 Antigen; Middle Aged; Neoadjuvant Therapy; Predictive Value of Tests; Prognosis; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone; Survival Analysis; Treatment Outcome; Tumor Suppressor Protein p53

Gossypol has demonstrated in vitro effects on cell cycle regulation and anti-tumor activity against mammary carcinoma cell lines. This Phase I/II study assesses both the effect of gossypol on cell cycle regulatory proteins in vivo and the clinical effect. Twenty women with refractory metastatic breast cancer received oral gossypol at daily doses between 30 and 50 mg per day. Gossypol plasma levels were measured (n = 8) and the modulation of the retinoblastoma (Rb) gene protein and Cyclin D1 was assessed by serial biopsies (n = 4). Grade I-II toxicities with gossypol treatment included nausea in 30% of patients, fatigue 15%, emesis 15%, altered taste sensation 15% and diarrhea in 10% of patients. Two of the three patients receiving 50 mg/day experienced dose limiting dermatologic toxicity (grade III). One patient had a minor response and two patients had stable disease with > 50% decline in serial assessments of the serum tumor markers. Immunohistochemical analysis of cyclin D1 and Rb expression in serial biopsies of four patients revealed both a concurrent decrease in cyclin D1 expression and an increase in nuclear Rb expression in three patients. The maximal tolerated dose (MTD) of gossypol was 40 mg/day. Gossypol appears to affect the expression of Rb protein and cyclin D1 in breast cancer metastases at doses achievable, yet had negligible antitumor activity against anthracycline and taxane refractory metastatic breast cancer. The cell cycle regulatory effects of gossypol suggest a potential role for gossypol as a modulating agent in conjunction with other cell cycle specific compounds.

Topics: Administration, Oral; Adult; Aged; Breast Neoplasms; Cell Cycle; Cyclin D1; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Fatigue; Female; Gossypol; Humans; Immunohistochemistry; Middle Aged; Nausea; Retinoblastoma Protein; Taste Disorders; Treatment Outcome

CDK4/6 inhibitors are the standard treatment in advanced HR+/HER2- breast cancer patients. Nevertheless, the resistance to CDK4/6 inhibitors is inevitable and the strategies to overcome resistance are of great interest. Here, we show that the palbociclib-resistant breast cancer cells expressed significantly higher levels of Cyclin D1 and CDK4 proteins because of upregulated protein synthesis. Silencing Cyclin D1 or CDK4 led to cell cycle arrest while silencing Cyclin E1 or CDK2 restored the sensitivity to palbociclib. Furthermore, PI3K/mTOR pathway was hyper-activated in palbociclib-resistant cells, leading to more phosphorylated 4E-BP1 and higher levels of Cyclin D1 and CDK4 translation. Targeting PI3K/mTOR pathway with a specific PI3Kα inhibitor (BYL719) or an mTOR inhibitor (everolimus) reduced the protein levels of Cyclin D1 and CDK4, and restored the sensitivity to palbociclib. The tumor samples expressed significantly higher levels of Cyclin D1, CDK4, p-AKT and p-4E-BP1 after progression on palbociclib treatment. In conclusion, our findings suggest that overexpressed Cyclin D1 and CDK4 proteins lead to the resistance to CDK4/6 inhibitor and PI3K/mTOR inhibitors are able to restore the sensitivity to CDK4/6 inhibitors, which provides the biomarker and rationale for the combinational use of CDK4/6 inhibitors and PI3K/mTOR inhibitors after CDK4/6 inhibitor resistance in breast cancer.

Topics: Breast Neoplasms; Cyclin D1; Cyclin-Dependent Kinase 4; Female; Humans; MTOR Inhibitors; Phosphatidylinositol 3-Kinases; Protein Kinase Inhibitors; TOR Serine-Threonine Kinases

The prevalence rate of breast carcinoma (BC) among multiple ethnic populations required more explanations to understand the pathogenesis mechanisms for the development of this type of cancer. The principal purpose of this work is to validate the correlation of the CCND1 (c.723G > A; rs9344) variant with an increased risk of breast carcinoma.. This retrospective case-controlled study was designed appertaining to 200 women including 100 BC patients and 100 unrelated cancer-free controls. The amplification of genomic DNA was genotyped utilizing the PCR-RFLP technique.. The frequencies of the CCND1 (c.723G > A; rs9344) variant revealed a significant association with increased risk of breast carcinoma under different genetic models including allelic (OR = 2.84, P-value < 0.001), recessive (OR = 4.83, P-value < 0.001), and dominant (OR = 3.19, P-value < 0.001) models.. Our findings concluded that the genetic biomarker of the CCND1 (c.723G > A; rs9344) variant is correlated with an elevated risk of breast carcinoma among Egyptian women.

Topics: Breast Neoplasms; Case-Control Studies; Cyclin D1; Female; Genetic Predisposition to Disease; Genotype; Humans; Polymorphism, Single Nucleotide; Retrospective Studies

Breast cancer is one of the most common cancers worldwide and the discovery of new cytotoxic agents is needed. Enaminones are regarded to be a significant structural motif that is found in a variety of pharmacologically active compounds however the number of studies investigating the anticancer activities of N-propargylic β-enaminones (NPEs) is limited. Herein we investigated the potential cytotoxic and apoptotic effects of 23 different NPEs (1-23) on human breast cancer cells. Cytotoxicity was evaluated via MTT assay. Apoptotic cell death and cell cycle distributions were investigated by flow cytometry. CM-H2DCFDA dye was used to evaluate cellular ROS levels. Expression levels of Bcl-2, Bax, p21, and Cyclin D1 were measured by quantitative real-time PCR. ADME properties were calculated using the ADMET 2.0 tool. NPEs 4, 9, 16, and 21 showed selective cytotoxic activity against breast cancer cells with SI values >2. NPEs induced apoptosis and caused significant changes in Bcl-2 and Bax mRNA levels. The cell cycle was arrested at the G0/G1 phase and levels of p21 and Cyclin D1 were upregulated in both breast cancer cells. ROS levels were significantly increased by NPEs, suggesting that the cytotoxic and apoptotic effects of NPEs were mediated by ROS. ADME analysis revealed that NPEs showed favorable distributions in both breast cancer cell lines, meaning good lipophilicity values, low unfractionated values, and high bioavailability. Therefore, these potential anticancer compounds should be further validated by in vivo studies for their appropriate function in human health with a safety profile, and a comprehensive drug interaction study should be performed.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Humans; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species

Columnar cell lesions (CCLs) are recognised breast cancer precursor lesions. Intraductal papillomas are usually lined by benign (polyclonal) cells. Although papillomas with monoclonal lesions (atypical ductal hyperplasia (ADH)/ductal carcinoma in situ (DCIS)) have been described, CCLs have not been described in papillomas.. We present two papillary breast lesions lined by a single layer of luminal cells resembling atypical CCL/flat epithelial atypia (FEA). We compared these two lesions with 13 benign intraductal papillomas, and 2 papillomas with ADH/DCIS grade 1 features as controls were immunohistochemically stained for the oestrogen receptor alpha (oestrogen receptor) and progesterone receptors (PR), cytokeratin 5 (CK5) and cyclin D1.. Oestrogen receptor/PR expression was variable, with areas with ≥85% hormone receptor positivity in both morphologically normal papillomas and papillomas with ADH. In ADH areas, CK5 expression was seen in ≤5% of cells while cyclin D1 expression was high (>60%). The two papillary lesions with FEA were 100% oestrogen receptor and 90% cyclin D1 positive, and low on PR/CK5. There was only one morphologically normal papilloma with similar areas of low CK5 (5%) and high cyclin D1 expression; in all other morphologically benign papillomas CK5 expression varied between 10% and 50% and cyclin D1 expression was ≤50%. The papillary lesion with FEA that could be tested showed 16q losses, the hallmark genetic change in low nuclear grade breast neoplasias, in contrast to nine morphologically benign papillomas that could be tested.. We present two papillomatous breast lesions with atypical CCL morphology and 16q loss, for which we propose the term papillary FEA.

Topics: Breast; Breast Neoplasms; Carcinoma in Situ; Carcinoma, Intraductal, Noninfiltrating; Cyclin D1; Female; Humans; Hyperplasia; Papilloma; Papilloma, Intraductal; Receptors, Estrogen

Cyclin D1 overexpression may contribute to development of various cancers, including breast cancer, and thus may serve as a key cancer diagnostic marker and therapeutic target. In our previous study, we generated a cyclin D1-specific single-chain variable fragment antibody (ADκ) from a human semi-synthetic single-chain variable fragment library. ADκ specifically interacted with recombinant and endogenous cyclin D1 proteins through an unknown molecular basis to inhibit HepG2 cell growth and proliferation.. Here, using phage display and in silico protein structure modeling methods combined with cyclin D1 mutational analysis, key residues that bind to ADκ were identified. Notably, residue K112 within the cyclin box was required for cyclin D1-ADκ binding. In order to elucidate the molecular mechanism underlying ADκ anti-tumor effects, a cyclin D1-specific nuclear localization signal-containing intrabody (NLS-ADκ) was constructed. When expressed within cells, NLS-ADκ interacted specifically with cyclin D1 to significantly inhibit cell proliferation, induce G1-phase arrest, and trigger apoptosis of MCF-7 and MDA-MB-231 breast cancer cells. Moreover, the NLS-ADκ-cyclin D1 interaction blocked binding of cyclin D1 to CDK4 and inhibited RB protein phosphorylation, resulting in altered expression of downstream cell proliferation-related target genes.. We identified amino acid residues in cyclin D1 that may play key roles in the ADκ-cyclin D1 interaction. A nuclear localization antibody against cyclin D1 (NLS-ADκ) was constructed and successfully expressed in breast cancer cells. NLS-ADκ exerted tumor suppressor effects via blocking the binding of CDK4 to cyclin D1 and inhibiting phosphorylation of RB. The results presented here demonstrate anti-tumor potential of intrabody-based cyclin D1-targeted breast cancer therapy.

Topics: Breast Neoplasms; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Female; G1 Phase; Humans; Phosphorylation

Cyclin D1 (CCND1), a mediator of cell cycle control, has a G870A polymorphism which results in the formation of two splicing variants: full-length CCND1 (CCND1a) and C-terminally truncated CCND1 species (CCND1b). However, the role of CCND1a and CCND1b variants in cancer chemoresistance remains unknown. Therefore, this study aimed to explore the molecular mechanism of alternative splicing of CCND1 in breast cancer (BC) chemoresistance. To address the contribution of G870A polymorphism to the production of CCND1 variants in BC chemoresistance, we sequenced the G870A polymorphism and analysed the expressions of CCND1a and CCND1b in MCF-7 and MCF-7/ADM cells. In comparison with MCF-7 cells, MCF-7/ADM cells with the A allele could enhance alternative splicing with the increase of SC-35, upregulate the ratio of CCND1b/a at both mRNA and protein levels, and activate the CDK4/CyclinD1-pRB-E2F1 pathway. Furthermore, CCND1b expression and the downstream signalling pathway were analysed through Western blotting and cell cycle in MCF-7/ADM cells with knockdown of CCND1b. Knockdown of CCND1b downregulated the ratio of CCND1b/a, demoted cell proliferation, decelerated cell cycle progression, inhibited the CDK4/CyclinD1-pRB-E2F1 pathway and thereby decreased the chemoresistance of MCF-7/ADM cells. Finally, CCND1 G870A polymorphism, the alternative splicing of CCDN1 was detected through Sequenom Mass ARRAY platform, Sanger sequencing, semi-quantitative RT-PCR, Western blotting and immunohistochemistry in clinical BC specimens. The increase of the ratio of CCND1b/a caused by G870A polymorphism was involved in BC chemoresistance. Thus, these findings revealed that CCND1b/a ratio caused by the polymorphism is involved in BC chemoresistance via CDK4/CyclinD1-pRB-E2F1 pathway.

Topics: Alternative Splicing; Breast Neoplasms; Cyclin D1; Cyclin-Dependent Kinase 4; Drug Resistance, Neoplasm; E2F1 Transcription Factor; Female; Humans; Polymorphism, Genetic; Retinoblastoma Protein

Patients undergoing neoadjuvant chemotherapy (NC) for invasive breast cancer (IBC) need indicators to track their progress during treatment. The goal of this research is to learn how cyclin D1 works in conjunction with taxane and non-taxane therapy for people with IBC.. There were 31 examples divided into two groups, based on: those using a different type of NC (taxane- or non-taxane-based), and NC administration time (before or after). Tumor grade, age, PR, ER, Ki-67, HER2, and Cyclin D1 expression were among the factors considered. Using immunohistochemical labeling, we were able to categorize cyclin D1 levels according to a threshold value, and we supplemented this with data we found in our databases. To analyze the data, we used a modified linear model.. The expression of Cyclin D1 decreased after NC delivery (p=0.086). Cyclin D1 expression was reduced in the taxane group (p=0.792). The non-taxane group also saw no differences in outcomes (p = 0.065). There was a larger decrease in Cyclin D1 expression in the non-taxane group compared to the taxane group, but the difference was not statistically significant (p=0.200).. Cyclin D1 expression, even if the differences are not statistically significant, may be a prognostic indicator of NC reaction in IBC. The involvement of Cyclin D1 in NC warrants more research with bigger IBC sample sizes.

Topics: Breast Neoplasms; Cyclin D1; Female; Humans; Neoadjuvant Therapy; Prognosis; Receptor, ErbB-2

In metastatic breast cancer, HER2-activating mutations frequently co-occur with mutations in PIK3CA, TP53, or CDH1. Of these co-occurring mutations, HER2 and PIK3CA are the most commonly comutated gene pair, with approximately 40% of HER2-mutated breast cancers also having activating mutations in PIK3CA. To study the effects of co-occurring HER2 and PIK3CA mutations, we generated genetically engineered mice with the HER2V777L; PIK3CAH1047R transgenes (HP mice) and studied the resulting breast cancers both in vivo as well as ex vivo using cancer organoids. HP breast cancers showed accelerated tumor formation in vivo and increased invasion and migration in in vitro assays. HP breast cancer cells were resistant to the pan-HER tyrosine kinase inhibitor, neratinib, but were effectively treated with neratinib plus the HER2-targeted antibody-drug conjugate trastuzumab deruxtecan. Proteomic and RNA-seq analysis of HP breast cancers identified increased gene expression of cyclin D1 and p21WAF1/Cip1 and changes in cell-cycle markers. Combining neratinib with CDK4/6 inhibitors was another effective strategy for treating HP breast cancers, with neratinib plus palbociclib showing a statistically significant reduction in development of mouse HP tumors as compared to either drug alone. The efficacy of both the neratinib plus trastuzumab deruxtecan and neratinib plus palbociclib combinations was validated using a human breast cancer patient-derived xenograft with very similar HER2 and PIK3CA mutations to the HP mice. Further, these two drug combinations effectively treated spontaneous lung metastasis in syngeneic mice transplanted with HP breast cancer organoids. This study provides valuable preclinical data to support the ongoing phase 1 clinical trials of these drug combinations in breast cancer.. In HER2-mutated breast cancer, PIK3CA mutation activates p21-CDK4/6-cyclin D1 signaling to drive resistance to HER2-targeted therapies, which can be overcome using CDK4/6 inhibitors.

Topics: Animals; Breast Neoplasms; Cell Transformation, Neoplastic; Class I Phosphatidylinositol 3-Kinases; Cyclin D1; Cyclin-Dependent Kinase 4; Drug Resistance, Neoplasm; Female; Humans; Mice; Mutation; Proteomics; Receptor, ErbB-2

Estrogen receptor (ER) is a transcription factor that affects the expression of some genes involved in the progression and development of breast cancer (BC). Hesperetin (Hst) is a flavonoid that inhibits the proliferation of BC cells. In this study, we investigated the effect of Hst on the cell viability of MCF-7 cells and the gene expression of the ERα, ERβ, IL-6, Ps2, and Cyclin D1.. In this study, cell viability was determined by MTT assay. The cells were seeded in RPMI-1640 medium and then exposed to different concentrations of Hst (0, 25, 50, 100, 200, and 400 µM) for 24 h, and IC50 was calculated. Real-time PCR was used to assess the expression of ERα, ERβ, pS2, Cyclin D1, and IL-6 mRNA. MCF-7 cells were seeded in RPMI-1640 medium and then exposed to different concentrations of Hst (0, 25, 50, 100, and 200 µM) for 24 h. Real-time PCR was carried out using a Step One Real-Time PCR System (ABI, USA) and Amplicon SYBR Green reagents.. The MTT assay revealed increased cytotoxicity with higher concentrations of Hst, and the IC. The results of our study demonstrate that Hst has the ability to induce cell death in MCF-7 cells. Furthermore, it was observed that Hst reduces the expression of the ER gene and enhances its activity, which can affect the downstream pathways of the ER.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Estrogen Receptor alpha; Estrogen Receptor beta; Female; Gene Expression; Humans; Interleukin-6; Receptors, Estrogen

Chemotherapy failure is often caused by drug resistance, for which no effective treatment strategy has been established. Many studies have been undertaken with the aim of overcoming drug resistance using natural products. Arctigenin (ATG), a natural product, has been investigated for its anti-cancer effects in HER2-overexpressing, ER-positive, and triple-negative breast cancer cells. We investigated the efficacy of ATG against self-established doxorubicin (DOX)-resistant breast cancer cells (MCF-DR and MDA-DR cells) derived from MCF-7 and MDA-MB-231 cells, respectively. ATG was found to increase DOX intracellular levels by downregulating multidrug Resistance 1 (MDR1) mRNA expression in DOX-resistant cells. In addition, combined treatment with DOX and ATG (DOX/ATG) reduced the viability of and colony formation by DOX-resistant cells. DOX/ATG also significantly induced G2/M cell cycle arrest by suppressing the Cyclin D1/CDK4/RB pathways and suppressed the expressions of MDR1 and Cyclin D1 by inhibiting the Mitogen-activated protein kinase (MAPK)/Activating protein-1 (AP-1) signaling pathways. Furthermore, DOX/ATG induced DNA damage and attenuated the expressions of RAD51 and Ku80. However, PARP1 (Poly [ADP-ribose] polymerase1) cleavage and AIF (Apoptosis-inducing factor) induced apoptosis did not occur despite DNA damage-induced cell death. Rather, flow cytometry showed that DOX/ATG caused necrosis. In summary, DOX/ATG increased intracellular DOX levels by inhibiting MDR1 and inducing G2/M arrest by inhibiting the Cyclin D1/CDK4/RB pathways and causing necrosis by damaging DNA. Our results suggest that ATG might be used as an adjuvant to enhance the efficacy of DOX in DOX-resistant breast cancer.

Topics: Apoptosis; Breast Neoplasms; Cell Cycle Checkpoints; Cell Line, Tumor; Cyclin D1; Doxorubicin; Drug Resistance, Neoplasm; Female; G2 Phase Cell Cycle Checkpoints; Humans; Necrosis

To study of the expression of epithelial-mesenchymal transition markers in various molecular subtypes of cancer and in benign breast diseases with different risks of malignant transformation.. An immunohistochemical study of the expression of E-cadherin, collagen II, integrin 1β, cyclin D1 was carried out in breast tissue samples: 58 with invasive breast carcinoma of no special type and 17 with benign breast diseases with a high and low risk of malignant transformation.. Patients with a triple negative molecular subtype are characterized by lower expression of collagen II compared with individuals with luminal A and B+ subtypes. A distinctive feature of patients with the HER2+ subtype was increased expression of cyclin D1 compared with the luminal A subtype, and in patients with the luminal B+ subtype, the expression of integrin 1β is higher than in the luminal B- and HER2+ subtypes. The expression of cyclin D1 in patients of both groups with benign diseases was lower than in all groups with invasive carcinoma, but at the same time, in patients with a low risk of malignant transformation, the expression of cyclin D1 was higher than in those with an increased risk.. The study revealed the relationship between the expression of markers and molecular subtypes of breast tumors, in addition, some patients with benign diseases were found who need further more careful monitoring and may be at risk for the development of malignancy.. Изучение экспрессии маркеров эпителиально-мезенхимального перехода в различных молекулярных подтипах рака и в доброкачественных заболеваниях молочной железы с разным риском злокачественной трансформации.. Проведено иммуногистохимическое исследование экспрессии E-кадгерина, коллагена II, интегрина 1β, циклина D1 в образцах ткани молочной железы: 58 с инвазивной карциномой неспецифического типа и 17 с доброкачественными заболеваниями с высоким и низким риском злокачественной трансформации.. Для пациенток с тройным негативным молекулярным подтипом характерна более низкая экспрессия коллагена II по сравнению с лицами при люминальном A и B+ подтипах. Отличительной чертой пациенток с HER2+ подтипом была повышенная экспрессия циклина D1 по сравнению с люминальным A подтипом, а у пациенток с люминальным B+ подтипом экспрессия интегрина 1β выше, чем при люминальном B- и HER2+ подтипах. Экспрессия циклина D1 у пациенток обеих групп с доброкачественными заболеваниями была ниже, чем у всех групп с инвазивной карциномой, но при этом у лиц с низким риском злокачественной трансформации экспрессия циклина D1 выше, чем у таковых с повышенным риском.. Исследование позволило выявить взаимосвязь экспрессии маркеров с молекулярными подтипами опухолей молочной железы, кроме того, были обнаружены некоторые пациентки с доброкачественными заболеваниями, которые нуждаются в дальнейшем более тщательном контроле и могут составлять группу риска по развитию малигнизации.

Topics: Biomarkers, Tumor; Breast Neoplasms; Cyclin D1; Epithelial-Mesenchymal Transition; Female; Humans; Integrins; Prognosis; Receptor, ErbB-2

Breast cancer (BC) is the most prevalent cancer in women, with increasing incidence and death rates in recent years. Disruptions of different signaling pathways partially cause breast cancer. Hence, different genes through particular pathways are involved in BC tumorigenesis.. In this study, we evaluated the expression level of GLIS2 and CCND1 genes in 50 patients. Also, in-silico analyses were used to enrich related signaling pathways involving the mentioned genes.. The results showed an increased expression level of Cyclin D1 and decreased expression level of GLIS2 in BC patients. Moreover, a relationship between aberrant expression levels of GLIS2 and CCND1 and BC development was determined.. These observations could help uncover new therapeutic targets for treating patients with BC in the progressive stage.

Topics: Breast Neoplasms; Cyclin D1; Female; Gene Amplification; Humans

We evaluated the immunoexpression of potential markers involved in the HER2 pathway in invasive breast carcinoma with HER2 amplification treated with trastuzumab.. Samples of ninety patients diagnosed and treated at two public Brazilian hospitals with overexpressed invasive carcinoma between 2009 and 2018 were included. Several markers (Bcl-2, CDK4, cyclin D1, EGFR, IGF1, IGF-1R, MDM2, MUC4, p16, p21, p27, p53, PTEN, RA, TNFα, and VEGF) were immune analyzed in the tumor by immunohistochemistry and then correlated with clinicopathological variables.. Tumor sample expression results determined potential markers of good prognosis with statistically significant values: cyclin D1 with a nuclear grade, and recurrence; IGF-1 with tumor size, and death; p16 with a response after treatment; PTEN with a response after treatment, and death. Markers of poor prognosis: p53 with histological, and nuclear grade; IGF-1R with a compromised lymph node. The treatment resistance rate after trastuzumab was 40%; the overall survival was 4.13 years (95% CI 5.1-12.5) and the disease-free survival was 3.6 years (95% CI 5.1-13.1).. The tumor samples profile demonstrated that cyclin D1, IGF-1, p16, and PTEN presented the potential for a good prognosis and p53 and IGF-1R for worse.

Topics: Biomarkers, Tumor; Breast Neoplasms; Cyclin D1; Female; Humans; Insulin-Like Growth Factor I; Prognosis; Receptor, ErbB-2; Trastuzumab; Tumor Suppressor Protein p53

Fighting breast tumors mandates finding different agents devoid of chemotherapy side effects. Repurposing existing drugs, such as statins, presents a promising avenue for the development of novel cancer therapeutics. Based on the different effects of statin members, this study aims to evaluate the effect of two of the most promising lipophilic statins, Simvastatin and Pitavastatin, and their combination with a conventional chemotherapeutic regimen of doxorubicin and cyclophosphamide on breast cancer cells. MDA-MB-231 and MCF7 cell lines were used to analyze the effects of Pitavastatin and simvastatin in combination with doxorubicin/cyclophosphamide. Cell viability and cell cycle were analyzed and certain apoptosis-related genes such as Bax, Bcl2, and caspase-3, besides cyclin D1 were analyzed using qPCR. The viability of breast cancer cells decreased significantly after treatment with a doxorubicin/cyclophosphamide combination in the presence of Pitavastatin or simvastatin compared with dual doxorubicin/cyclophosphamide with a higher effect in MDA-MB-231 cells than MCF7. In MDA-MB-231, The triple combination of Pitavastatin or simvastatin with doxorubicin/cyclophosphamide resulted in an increase in the expression levels of apoptotic markers than treatment with doxorubicin/cyclophosphamide combination (Bax (p-value = 0.09& 0.02, respectively), Bax/Bcl2 ratio (p-value = 0.0002& <0.0001, respectively)). However, the increase in caspase3 wasn't significant (p-value = 0.45& 0.09, respectively). Moreover, the expression of cyclin D1 decreased (p-value = 0.0002& <0.0001, respectively) and the cell cycle was arrested in the G1 phase. Combination of Pitavastatin or simvastatin with doxorubicin/ cyclophosphamide may induce apoptosis in breast cancer cells via upregulation of the Bax/Bcl2 pathway, potentially providing a promising new therapeutic strategy for breast cancer.

Topics: Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Cell Line, Tumor; Cyclin D1; Cyclophosphamide; Doxorubicin; Female; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Proto-Oncogene Proteins c-bcl-2; Simvastatin

The anticancer effects of ruxolitinib and calcitriol against breast cancer were reported previously. However, the effect of ruxolitinib and calcitriol combination treatment on various molecular subtypes of breast cancer remains unexplored. In this study, we used MCF-7, SKBR3, and MDA-MB-468 cells to investigate the effect of ruxolitinib and calcitriol combination treatment on cell proliferation, apoptosis, cell cycle, and cell signaling markers, in vitro and in vivo. Our results revealed the synergistic anticancer effect of ruxolitinib and calcitriol combination treatment in SKBR3 and MDA-MB-468 cells, but not in MCF-7 cells in vitro, via cell proliferation inhibition, apoptosis induction, cell cycle arrest, and the alteration of cell signaling protein expression, including cell cycle-related (cyclin D1, CDK1, CDK4, p21, and p27), apoptosis-related (c-caspase and c-PARP), and cell proliferation-related (c-Myc, p-p53, and p-JAK2) proteins. Furthermore, in the MDA-MB-468 xenograft mouse model, we demonstrated the synergistic antitumor effect of ruxolitinib and calcitriol combination treatment, including the alteration of c-PARP, cyclin D1, and c-Myc expression, without significant drug toxicity. The combination exhibited a synergistic effect in HER2-enriched and triple-negative breast cancer subtypes. In conclusion, our results suggest different effects of the combination treatment of ruxolitinib and calcitriol depending on the molecular subtype of breast cancer.

Topics: Animals; Apoptosis; Breast Neoplasms; Calcitriol; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Humans; Mice; Nitriles; Poly(ADP-ribose) Polymerase Inhibitors; Pyrazoles; Pyrimidines; Triple Negative Breast Neoplasms

The most prevalent malignancy among women is breast cancer. Phytochemicals and their derivatives are rapidly being recognized as possible cancer complementary therapies because they can modify signaling pathways that lead to cell cycle control or directly alter cell cycle regulatory molecules. The phytochemicals' poor bioavailability and short half-life make them unsuitable as anticancer drugs. Applying PLGA-PEG NPs improves their solubility and tolerance while also reducing drug adverse effects. According to the findings, combining anti-tumor phytochemicals can be more effective in regulating several signaling pathways linked to tumor cell development. The point of the study was to compare the anti-proliferative impacts of combined artemisinin and metformin on cell cycle arrest and expression of cyclin D1 and apoptotic genes (bcl-2, Bax, survivin, caspase-7, and caspase-3), and also hTERT genes in breast cancer cells. T-47D breast cancer cells were treated with different concentrations of metformin (MET) and artemisinin (ART) co-loaded in PLGA-PEG NPs and free form. The MTT test was applied to assess drug cytotoxicity in T47D cells. The cell cycle distribution was investigated using flow cytometry and the expression levels of cyclin D1, hTERT, Bax, bcl-2, caspase-3, and caspase-7, and survivin genes were then determined using real-time PCR. The findings of the MTT test and flow cytometry revealed that each state was cytotoxic to T47D cells in a time and dose-dependent pattern. Compared to various state of drugs (free and nano state, pure and combination state) Met-Art-PLGA/PEG NPs demonstrated the strongest anti-proliferative impact and considerably inhibited the development of T-47D cells; also, treatment with nano-formulated forms of Met-Art combination resulted in substantial downregulation of hTERT, Bcl-2, cyclin D1, survivin, and upregulation of caspase-3, caspase-7, and Bax, in the cells, as compared to the free forms, as indicated by real-time PCR findings. The findings suggested that combining an ART/MET-loaded PLGA-PEG NP-based therapy for breast cancer could significantly improve treatment effectiveness.

Topics: Alkylmercury Compounds; Antineoplastic Agents; Apoptosis; Artemisinins; bcl-2-Associated X Protein; Benzalkonium Compounds; Benzoflavones; Breast Neoplasms; Carbanilides; Caspase 3; Caspase 7; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Ethylmercury Compounds; Female; Heterocyclic Compounds; Humans; Metformin; Methacholine Compounds; Nanoparticles; Oximes; Plasmalogens; Sulfonylurea Compounds; Survivin; Trimethyltin Compounds

The management of metastatic estrogen receptor (ER) positive HER2 negative breast cancer (ER+) has improved; however, therapeutic resistance and disease progression emerges in majority of cases. Using unbiased approaches, as expected PI3K and MTOR inhibitors emerge as potent inhibitors to delay proliferation of ER+ models harboring PIK3CA mutations. However, the cytostatic efficacy of these drugs is hindered due to marginal impact on the expression of cyclin D1. Different combination approaches involving the inhibition of ER pathway or cell cycle result in durable growth arrest via RB activation and subsequent inhibition of CDK2 activity. However, cell cycle alterations due to RB loss or ectopic CDK4/cyclin D1 activation yields resistance to these cytostatic combination treatments. To define means to counter resistance to targeted therapies imparted with RB loss; complementary drug screens were performed with RB-deleted isogenic cell lines. In this setting, RB loss renders ER+ breast cancer models more vulnerable to drugs that target DNA replication and mitosis. Pairwise combinations using these classes of drugs defines greater selectivity for RB deficiency. The combination of AURK and WEE1 inhibitors, yields synergistic cell death selectively in RB-deleted ER+ breast cancer cells via apoptosis and yields profound disease control in vivo. Through unbiased efforts the XIAP/CIAP inhibitor birinapant was identified as a novel RB-selective agent. Birinapant further enhances the cytotoxic effect of chemotherapies and targeted therapies used in the treatment of ER+ breast cancer models selectively in the RB-deficient setting. Using organoid culture and xenograft models, we demonstrate the highly selective use of birinapant based combinations for the treatment of RB-deficient tumors. Together, these data illustrate the critical role of RB-pathway in response to many agents used to treat ER+ breast cancer, whilst informing new therapeutic approaches that could be deployed against resistant disease.

Topics: Breast Neoplasms; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Cytostatic Agents; Drug Resistance, Neoplasm; Female; Humans; Receptors, Estrogen; Retinoblastoma Protein

Topics: Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Lobular; Cyclin D1; Cyclooxygenase 2; Female; Humans; Insulin Receptor Substrate Proteins

Claudin 6 (CLDN6) is an important component of tight junctions. Through the PDZ binding motif, CLDN6 binds to a variety of signaling proteins that contain the PDZ domain to regulate different signaling pathways, and plays an important role in the occurrence and development of tumors. Our previous work showed that CLDN6 was expressed at low levels in breast cancer cells, and overexpression of CLDN6 inhibited breast cancer cell proliferation, migration and invasion. However, the mechanism of how CLDN6 works remains unclear. In this study, we aimed to explore the mechanism by which CLDN6 inhibits breast cancer cell malignant behavior. As a result, overexpression of CLDN6 inhibited the proliferation of breast cancer cells along with the downregulation of cyclin D1, which plays an important role in regulating cell proliferation. After overexpression of Sp1 in CLDN6-overexpressing cells, the expression of cyclin D1 was upregulated. On the other hand, CLDN6 inhibited breast cancer cell migration and invasion along with the downregulation of IL-8, CXCR2 and FAK. When treated with IL-8, the migration and invasion ability were promoted along with the upregulation of CXCR2 and p-FAK, and the cytoskeleton was rearranged in CLDN6-overexpressing cells. Furthermore, when treated with the ERK signaling activator PMA, the proliferation, migration and invasion abilities were promoted along with the upregulation of Sp1, cyclin D1 and IL-8 in CLDN6-overexpressin cells. In conclusion, CLDN6 suppressed ERK/Sp1/cyclin D1 and ERK/IL-8 signaling to inhibit proliferation, migration and invasion in breast cancer cells. The mechanism may provide experimental evidence for the treatment of breast cancer targeting CLDN6.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Claudins; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Interleukin-8

Cyclin D1 has been shown to participate in the pathogenesis of breast cancer. This cell cycle-related protein has direct or indirect interactions with long non-coding RNAs (lncRNAs). In the present two-step study, we first identified CCND1-related lncRNAs through an in silico approach. Then, we measured expression of CCND1 mRNA and five lncRNAs in paired breast cancer samples and their matched non-cancerous samples obtained from adjacent tissues. HOTTIP expression was significantly higher in breast cancer tissues compared with adjacent tissues (expression ratio (95% CI)= 4.63 (1.56-13.76), P value= 0.0070). Similarly, CBR3-AS1 was up-regulated in cancerous tissues compared with control tissues (expression ration (95% CI)= 3.26 (1.35-7.86), P value= 0.0122). Expression of HOTTIP was higher in estrogen receptor (ER) negative samples compared with ER positive ones (-4.35 ± 1.33 versus -4.63 ± 0.62, P value=0.002). CBR3-AS1 could differentiate between these two sets of samples with AUC±SD, sensitivity, specificity and P values of 0.7 ± 0.05, 0.9, 0.49 and 0.003, respectively. These values were 0.68 ± 0.04, 0.87, 0.34 and 0.04 for HOTTIP. Although we could not find difference in expression of CCND1 between these two sets of samples, we reported up-regulation of two CCND1-related lncRNAs in breast cancer samples. These lncRNAs are putative markers for breast cancer.

Topics: Breast Neoplasms; Cell Cycle Proteins; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; RNA, Long Noncoding; Up-Regulation

To translate a clinical research finding into daily clinical practice requires well-controlled clinical trials. We have demonstrated the usage of absolute quantitation of Ki67 and cyclinD1 protein levels to improve prognosis of Luminal-like patients based on overall survival (OS) analysis of a cohort of 155 breast cancer specimens (cohort 1). However, this finding is considered the D level of evidence (LOE) to require subsequent validation before it may be used in daily clinical practice. To set the stage for future clinical trials, our findings were validated through OS analysis of an independent cohort (cohort 2) of 173 Luminal-like patients.. Both Ki67 and cyclinD1 levels were measured absolutely and quantitatively using the Quantitative Dot Blot (QDB) method in cohort 2. The proposed cutoffs for both biomarkers from cohort 1 were re-evaluated in cohort 2 and in the merged cohort of 1 and 2, respectively, through univariate, multivariate and Kaplan-Meier survival analysis.. The proposed cutoffs of 2.31 nmol/g for Ki67 and 0.44 μmol/g for cyclinD1 were validated as effective cutoffs in cohort 2 and the merged cohort through OS analysis. The combined use of both biomarkers allowed us to identify patients with both biomarker levels below the cutoffs (59.3%) with10-year survival probability (SP) of 89%, in comparison to those above the cutoffs (8.3%) with 8 year SP of 28% through OS analysis in the merged cohort.. This study validated our findings that absolute quantitation of Ki67 and cyclinD1 allows effective subtyping of luminal-like patients. It sets the stage for prospective or prospective-retrospective clinical studies.

Topics: Biomarkers, Tumor; Breast Neoplasms; Cyclin D1; Female; Humans; Ki-67 Antigen; Prognosis; Prospective Studies; Retrospective Studies

Abnormalities in the cyclin D1-CDK4/6 complex have been implicated in breast cancer proliferation and resistance to treatment. Recently, new drugs have been developed to target CDK4/6. Meanwhile, liquid biopsy has received great interest in oncology. In this study, we analyzed cyclin D1 gene (CCND1) copy number variation (CNV) in circulating tumor DNA (ctDNA) from luminal B breast cancer patients.. This study included 31 patients with luminal B breast cancer who underwent resection. We analyzed CCND1 CNV in ctDNA by digital droplet PCR.. Of the 31 luminal B breast cancers, CCND1 CNV was positive in 5 cases. Patients with CCND1 CNV positivity had significantly shorter recurrence-free survival than patients with negative CCND1 CNV.. CCND1 CNV in ctDNA was associated with poor prognosis in patients with luminal B breast cancer. This biomarker could be a useful prognostic factor.

Topics: Breast Neoplasms; Circulating Tumor DNA; Cyclin D1; DNA Copy Number Variations; Female; Gene Amplification; Genes, bcl-1; Humans; Prognosis; Receptors, Estrogen

Breast cancer represents the most frequent cancer and cause of death in women worldwide and in Tunisia. Cyclin D1 is a gene of cell cycle regulation. It represents a potential oncogene in invasive breast cancer; however; the results are conflicting. We performed a retrospective study aiming to analyze the prognostic impact of cyclin D1 expression in patients with invasive breast carcinoma of no special type and its relation with clinical-pathological features.. One hundred cases of invasive breast carcinoma of no special type diagnosed between 2009 and 2011 were included in this study. Immunohistochemical (IHC) staining was performed for cyclin D1 in all cases. Results were analyzed statistically.. Cyclin D1 positivity was seen in 74 cases (74%), of which 32 cases (32%) showed strong immunoreactivity. Cyclin D1 staining was statistically significantly associated with estrogen receptor (ER) and progesterone receptor (PR) positivity (. These results confirm that cyclin D1 overexpression can be employed as a beneficial prognostic marker and suggest that anti-cyclin D1 therapy may be efficient, especially for ER positive tumors.

Topics: Biomarkers, Tumor; Breast Neoplasms; Cyclin D1; Disease-Free Survival; Female; Humans; Prognosis; Receptors, Estrogen; Retrospective Studies

Overexpression of human epidermal growth factor receptor 2 (HER2) in various cancers is correlated with poor patient survival. Trastuzumab, a recombinant humanized monoclonal antibody against HER2, has been considered to be a first-line therapy for HER2-positive breast cancer patients, but its usefulness is limited by the development of resistance. In this study, we established resistant cells by long-term treatment with trastuzumab. These cells showed higher proliferation, invasion, and migration abilities than the wild-type cells. Mammaglobin 1 (MGB1), cyclin D1, E1, A2, and phosphorylated NF-κB (p-p65) were upregulated in resistant cells. These proteins regulate cell proliferation, migration, and invasion of resistant cells. Depletion of MGB1 decreased cyclin and p-p65 expression. Cyclin D1 and A2, but not E1 expression, were affected by p-p65 downregulation. In summary, our results indicate that MGB1 expression is increased in breast cancer cells that have gained resistance to trastuzumab, and suggest that MGB1 promotes aggressiveness through cyclin and NF-κB regulation.

Topics: Breast Neoplasms; Cell Line, Tumor; Cyclin D1; Female; Humans; Mammaglobin A; NF-kappa B; Trastuzumab

Cyclin D1 (CCND1) is a critical regulator of cell proliferation and its overexpression has been linked to the development and progression of several malignancies. CCND1 overexpression is recognized as a major mechanism of therapy resistance in several cancers; tumors that rely on CCND1 overexpression to evade cancer therapy are extremely sensitive to its ablation. Therefore, targeting CCND1 is a promising strategy for preventing tumor progression and combating therapy resistance in cancer patients. Although CCND1 itself is not a druggable target, it can be targeted indirectly by inhibiting its regulators. CCND1 steady-state levels are tightly regulated by ubiquitin-mediated degradation, and defects in CCND1 ubiquitination are associated with increased CCND1 protein levels in cancer. Here, we uncover a novel function of ubiquitin-specific protease 27X (USP27X), a deubiquitinating enzyme (DUB), in regulating CCND1 degradation in cancer. USP27X binds to and stabilizes CCND1 in a catalytically dependent manner by negatively regulating its ubiquitination. USP27X expression levels correlate with the levels of CCND1 in several HER2 therapy-resistant breast cancer cell lines, and its ablation leads to a severe reduction of CCND1 protein levels, inhibition of tumor growth, and resensitization to targeted therapy. Together, the results presented in our study are the first to expose USP27X as a major CCND1 deubiquitinase and provide a mechanistic explanation for how this DUB fosters tumor growth.. As a deubiquitinating enzyme, USP27X is a druggable target. Our study illuminates new avenues for therapeutic intervention in CCND1-driven cancers.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Humans; Proteolysis; Ubiquitin-Specific Proteases

Bilirubin, as an essential constituent of cellular signaling pathways, may have a role in cell growth and apoptosis in breast cancer, although the biochemical relevance is still unclear. The purpose of the present study is to recognize the mechanism underlying bilirubin-induced apoptosis in breast cancer cell lines.. To detect the cell viability, MTT assay was carried out. Apoptosis was assessed by flow cytometry analysis and caspase activities were determined by colorimetric method. The expression of AhR, cyclin D1, cyclin A, p53, p27, Bcl-2, and Bax were examined using real-time PCR. The cell viability has been reduced by bilirubin in a dose-dependent manner and an intrinsic apoptotic response has been occurred that was evidenced by the elevation of caspase-3 and - 9 activities. Bilirubin induced cell arrest in cell-cycle progression, which was associated with the induction of AhR expression, down-regulation of cyclin D1, cyclin A, and upregulation of p53 and p27 expression. Following bilirubin treatment, Bcl-2 was decreased and Bax protein was increased in both cell lines.. To discuss, bilirubin, as a naturally occurring antiproliferative molecule, mediates growth inhibition by induction of cell cycle arrest and apoptosis in MCF-7 and MDA-MB-468 breast cancer cells. It is associated with the suppression of cyclin A, D1, and Bcl-2; induction of p53, p27, and Bax together with the activation of caspase-3 and - 9.

Topics: Apoptosis; bcl-2-Associated X Protein; Bilirubin; Breast Neoplasms; Caspase 3; Cell Line, Tumor; Cyclin A; Cyclin D1; Female; G1 Phase Cell Cycle Checkpoints; Humans; Proto-Oncogene Proteins c-bcl-2; Tumor Suppressor Protein p53

The incidence of breast cancer continues to rise despite decades of laboratory, epidemiological and clinical research. Breast cancer is still the leading cause of cancer death in women. Cyclin D1 is one of the most important oncoproteins associated with cancer cell proliferation and is overexpressed in more than 50% of cases. Curcumin and chrysin are plant-derived components that are believed to assist in inhibiting the viability of breast cancer cells. These agents are involved in cancer cells' growth and reducing cyclin D1 expression. In this study, the hypothesis of combining curcumin and chrysin is applied to analyze the potential synergistic effect in inhibiting cancer cell proliferation and down-regulation of cyclin D1. Furthermore, applying PLGA-PEG NPs could improve the bioavailability of free curcumin and chrysin components and at the same time increases the anti-cancer potential of this compound.. PLGA-PEG NPs were synthesized via the ring-opening polymerization technique and characterized with FT-IR and FE-SEM for chemical structure and morphological characteristics, respectively. Next, curcumin and chrysin were loaded in PLGA-PEG NPs and MTT assay was performed to assess the cytotoxic effect of these agents. T-47D cells were treated with appropriate concentrations of these agents and cyclin D1 expression level was evaluated by real-time PCR.. The obtained results from FT-IR and FE-SEM techniques illustrated that curcumin and chrysin were efficiently encapsulated into PLGA-PEG NPs. Curcumin, chrysin, and curcumin-chrysin in free and nano-encapsulated forms exhibited an anti-cancer effect on T-47D cells in a time- and dose-dependent manner, especially in a combination of free and encapsulated forms demonstrated synergistic anti-cancer effects. Compared to free form, Nano-curcumin, Nano-chrysin, and Nano-combination remarkably down-regulated cyclin D1 gene expression. (p-value < 0.05).. Our results revealed that the curcumin-chrysin combination has a synergistic effect and the encapsulated form of this nano-component has more inhibition on cyclin D1 expression.
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Topics: Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Curcumin; Cyclin D1; Female; Humans; MCF-7 Cells; Nanoparticles; Polyethylene Glycols; Spectroscopy, Fourier Transform Infrared

This study mainly aimed to explore the influences of Calcium Voltage-Gated Channel Subunit Alpha1 B (CACNA1B) on the development of breast cancer and the related mechanism.. The information of patients with breast cancer from TCGA database was used for analyses of CACNA1B expression and its prognostic value. Loss- and gain- of functions of CACNA1B were conducted in MCF7 and Bcap-37 cells, respectively. CCK-8, colony formation and transwell assays were applied for evaluating the cell viability and motility. Western blot was used for protein expression detection.. We revealed that highly expressed CACNA1B in breast cancer tissues was related to poor prognosis according to the data gained from TCGA database. The outcomes of functional assays showed that depletion of CACNA1B restrained MCF7 cell growth, invasion and migration and high-expression of CACNA1B fortified the growth, invasion and migration in Bcap-37 cells. Finally, we manifested that silencing CACNA1B obviously raised the protein expression level of E-cadherin and reduced the protein levels of Cyclin D1, N-cadherin and Snail in MCF7 cells, whilst, over-expression of CACNA1B reduced the level of E-cadherin and increased the expression of Cyclin D1, N-cadherin and Snail in Bcap-37 cells.. These results identified CACNA1B as a forwarder of the growth, invasion and migration in breast cancer cells.

Topics: Breast Neoplasms; Calcium Channels, N-Type; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells

This study aimed to explore the mutational characteristics and significance of cell cycle-related genes (CCGs) in hormone-receptor positive, human epidermal growth factor receptor 2 negative breast cancer (HR+/HER2- BC).. A total of 1668 HR+/HER2- BC patients from the Guangdong Provincial People's Hospital (GDPH) cohort (n = 321) and METABRIC cohort (n = 1347) were included. Tumor samples from HR+/HER2- BC patients were collected for a next-generation sequencing assay in GDPH cohort, including 15 key CCGs. The association between CCGs alterations and overall survival were identified via the Cox regression analysis. The functional roles of the CCGs were explored via the Metascape database.. Based on multivariate Cox regression analysis, a set of five key CCGs (CDK4, CCND1, CDKN1A, CDKN1C, and CHEK2) were identified as independent prognostic variables in HR+/HER2- BC patients. Besides, the five-CCGs-based risk score was used to effectively classify patients into the low-risk and high-risk groups (P < 0.0001). The potential functional pathways of the CCGs included cell cycle, cyclin D associated events in G1, and regulation of G1/S transition of mitotic cell cycle.. We performed the integrated analysis of the CCGs in HR+/HER2- BC patients. It has the potential to guide individualized precision oncology therapeutic schemes in HR+/HER2- BC patients.

Topics: Adult; Aged; Aged, 80 and over; Breast Neoplasms; Checkpoint Kinase 2; Cohort Studies; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p57; Female; High-Throughput Nucleotide Sequencing; Humans; Middle Aged; Mutation; Prognosis; Receptors, Estrogen; Receptors, Progesterone; Young Adult

In breast cancer, tumor-associated macrophages with activated phenotypes promote tumor invasion and metastasis. The more aggressive mesenchymal-like breast cancer cells have a selective advantage, skewing macrophages toward the more immunosuppressive subtype. However, the mechanism underlying this shift is poorly understood. Cyclin D1b is a highly oncogenic variant of cyclin D1. Our previous study showed that non-metastatic epithelial-like breast cancer cells were highly metastatic

Topics: Animals; Breast Neoplasms; Cyclin D1; Female; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Metastasis; Phenotype; Tumor Microenvironment; Tumor-Associated Macrophages

Using chip array assays, we identified differentially expressed genes via a comparison between luminal A breast cancer subtype and normal mammary ductal cells from healthy donors. In silico analysis confirmed by western blot and immunohistochemistry revealed that C-JUN and C-FOS transcription factors are activated in luminal A patients as potential upstream regulators of these differentially expressed genes. Using a chip-on-chip assay, we identified potential C-JUN and C-FOS targets. Among these genes, the NRIP1 gene was revealed to be targeted by C-JUN and C-FOS. This was confirmed after identification and validation with transfection assays specific binding of C-JUN and C-FOS at consensus binding sites. NRIP1 is not only upregulated in luminal A patients and cell lines but also regulates breast cancer-related genes, including PR, ESR1 and CCND1. These results were confirmed by NRIP1 siRNA knockdown and chip array assays, thus highlighting the putative role of NRIP1 in PGR, ESR1 and CCND1 transcriptional regulation and suggesting that NRIP1 could play an important role in breast cancer ductal cell initiation.

Topics: Adult; Aged; Breast Neoplasms; Carcinoma, Ductal, Breast; Cyclin D1; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Humans; Intracellular Signaling Peptides and Proteins; JNK Mitogen-Activated Protein Kinases; MCF-7 Cells; Middle Aged; Nuclear Proteins; Nuclear Receptor Interacting Protein 1; Proto-Oncogene Proteins c-fos; Transcriptome

Endocrine therapy is a mainstay for the treatment of hormone receptor-positive breast cancer (BC); however, only a fraction of patients experience a pronounced response to antagonists of estrogen signaling. There is a need to identify predictors for efficacy of this treatment.. This study included 138 patients with newly diagnosed metastatic BC, who received upfront endocrine therapy. Archival biopsy specimens were tested for CCND1 and FGFR1 gene amplification and mRNA expression by PCR-based methods.. CCND1 and FGFR1 amplification was detected in 24 (17.9%) and 28 (20.9%) of 134 evaluable cases, respectively; 9 carcinomas had concurrent alterations of these two genes. Presence of amplification in at least one locus was more common in tumors of higher grade (p = 0.018) and was associated with higher Ki-67 proliferation index (p = 0.036). CCND1 gene amplification was associated with shorter progression-free survival (PFS) in patients receiving aromatase inhibitors (AI) [16.0 months vs. 32.4 months, HR = 3.16 (95% CI 1.26-7.93), p = 0.014]. FGFR1 status did not significantly affect PFS of AI-treated women; however, objective response to AI was observed less frequently in FGFR1-amplified BC as compared to cases with normal FGFR1 copy number [2/15 (13.3%) vs. 22/46 (47.8%), p = 0.031]. Meanwhile, CCND1/FGFR1 gene status did not influence the outcome of tamoxifen-treated patients.. Presence of CCND1 and/or FGFR1 amplification is associated with worse outcomes of AI therapy in patients with metastatic BC.

Topics: Adult; Aged; Aged, 80 and over; Aromatase Inhibitors; Breast Neoplasms; Cell Proliferation; Cyclin D1; Drug Resistance, Neoplasm; Female; Gene Amplification; Humans; Kaplan-Meier Estimate; Ki-67 Antigen; Middle Aged; Progression-Free Survival; Receptor, ErbB-2; Receptor, Fibroblast Growth Factor, Type 1; Receptors, Estrogen; Receptors, Progesterone; RNA, Messenger; Tamoxifen; Treatment Outcome

Topics: Animals; Apoptosis; Biomarkers, Tumor; Breast Neoplasms; Cell Movement; Cell Proliferation; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Phosphorylation; Prognosis; RNA Stability; RNA-Binding Proteins; Tumor Cells, Cultured; Ubiquitin-Protein Ligases; Xenograft Model Antitumor Assays; Zebrafish

Breast cancer is one of the most frequent cancers in women and the globally leading cause of cancer-related deaths. Bioinformatics and experimental analyses found that miR-937-5p may play a proto-oncogenic role in breast cancer; however, the specific effects and the molecular mechanism need further investigation. GSEA-KEGG and GSEA-GO suggested that miR-937-5p might be related to cell cycle and DNA replication. The experimental data indicated that miR-937-5p inhibition significantly repressed the proliferation of breast carcinoma cells and elicited S-phase cell cycle arrest. Meanwhile, the protein levels of proliferating marker ki-67 and cell cycle regulators Cyclin A2, Cyclin B1, CDK1, and Cyclin D1 were also decreased by miR-937-5p inhibition. miR-937-5p could directly bind to and negatively regulate SOX17. SOX17 overexpression also significantly repressed the proliferation of breast carcinoma cells and elicited S-phase cell cycle arrest and decreased ki-67, β-catenin, c-Myc, Cyclin A2, Cyclin B1, Cyclin D1, and CDK1 protein contents. More importantly, the effects of miR-937-5p were reversed by SOX17.

Topics: 3' Untranslated Regions; Antagomirs; Base Sequence; Breast Neoplasms; CDC2 Protein Kinase; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin A2; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; S Phase Cell Cycle Checkpoints; Sequence Alignment; SOXF Transcription Factors; Wnt Signaling Pathway

Circular RNA (circRNA) is abnormally expressed in cancers and has been linked to cancer progression, including breast cancer (BC). However, the role and mechanism of circ-UBR1 in BC progression remains to be further studied.. Quantitative real-time PCR (qRT-PCR) was conducted to analyze the expression of circ-UBR1, miR-1299 and Cyclin D1 (CCND1). Cell counting kit 8 (CCK8) assay was used to measure cell viability. Cell apoptosis and cell cycle distribution were analyzed by flow cytometry. Then, the migration and invasion of cells were determined by transwell assay. Moreover, BC tumor xenograft model was built to evaluate the function of circ-UBR1 silencing on BC tumor volume and weight. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were applied to illuminate the interaction between miR-1299 and circ-UBR1 or CCND1. In addition, relative CCND1 protein expression was assessed using western blot (WB) analysis.. Our results revealed that circ-UBR1 was upregulated in BC, and its silencing could inhibit BC cell proliferation, metastasis, and promote apoptosis in vitro, as well as restrain BC tumor growth in vivo. Meanwhile, we found that circ-UBR1 could sponge miR-1299, and miR-1299 inhibitor could reverse the effect of circ-UBR1 knockdown on BC cell progression. Furthermore, CCND1 was a target of miR-1299, and CCND1 overexpression could reverse the effect of miR-1299 mimic on BC cell progression. Also, the downregulation of circ-UBR1 could inhibit CCND1 expression, while this effect could be inverted by miR-1299 inhibitor.. Our data indicated that circ-UBR1 might play a pro-cancer role in BC progression by regulating the miR-1299/CCND1 axis.

Topics: Animals; Apoptosis; Biomarkers, Tumor; Breast Neoplasms; Cell Movement; Cell Proliferation; Cyclin D1; Disease Progression; Female; Gene Expression Regulation, Neoplastic; Humans; Lymphatic Metastasis; Mice; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Prognosis; RNA, Circular; Tumor Cells, Cultured; Ubiquitin-Protein Ligases; Xenograft Model Antitumor Assays

In the present study we aimed to figure out the effect of metformin on the expression of AMPK-alpha, cyclin D1, and Tp53, and apoptosis in primary breast cancer cells (PBCCs).. PBCCs were treated with two doses of metformin (0 mM, 25 mM). Proliferation was determined by BrdU as- say. Real-time PCR was used to assess AMPK-alpha, cyclin D1, and Tp53 gene expressions; apoptotic indexes of PBCCs were analyzed using flow-cytometry.. Twenty-four–hour incubation with 25 mM metformin reduced the proliferation of PBCCs. AMPK-alpha gene expression in PBCCs was not affected by 25 mM metformin treatment compared with the control group. PBCCs treated with 25 mM metformin had lower cyclin D1 expression compared with nontreated cells; however, the difference was not statistically significant. Twenty-five mil- limolar dose of metformin increased p53 expression significantly compared with the nontreated group. The high concentration of met- formin elevated the number of annexin V-positive apoptotic cells, and the increase in the apoptotic index was statistically significant.. Metformin can modulate cyclin D1 and p53 expression through AMPK-alpha-independent mechanism in breast cancer cells, leading to cell proliferation inhibition and apoptosis induction.

Topics: AMP-Activated Protein Kinases; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Humans; Hypoglycemic Agents; Metformin; Middle Aged; Tumor Suppressor Protein p53; Up-Regulation

This study targets to develop curcumin-loaded polyvinyl alcohol/cellulose nanocrystals (PVA/CNCs) membrane as localized delivery system for breast/liver cancer. A novel strategy was developed for enhancing encapsulation capacity and maximizing therapeutic efficiency of curcumin-loaded PVA/CNCs membranes. Membranes were prepared by solution-casting method using citric acid as crosslinker. SEM revealed that PVA/CNCs ratio (80:20) was chosen as the optimum for loading curcumin. FT-IR indicated that, curcumin was incorporated into PVA/CNCs in amorphous-phase via intermolecular hydrogen bond between curcumin and membrane components. Curcumin showed biphasic-release through burst-release of 41% of curcumin during the first hour, followed by sustained-release of 70% and 94% during 24 h and 48 h, respectively. In vitro cytotoxicity of PVA/CNCs/Curcumin membrane exhibited a selective inhibition proliferation of breast and liver cancer cells in a concentration-dependent without any toxic effect on normal cells. At high concentration (8 mg/ml) of PVA/CNCs/Curcumin, reduced viability to 35% and 7% of MCF-7 and Huh-7 cells, respectively; meanwhile high HFB-4 normal cell viability ≥80% was investigated. Antimicrobial activity of PVA/CNCs/Curcumin was investigated by multi-drug-resistant strains, and MIC values. PVA/CNCs/Curcumin membranes with concentration (40 mg/ml) showed broad-spectrum antimicrobial activities, thus inhibited ~96-99% of microbial growth. PVA/CNCs/Curcumin membranes could be as promised anti-infective biomaterials for breast and liver cancer wound healing.

Topics: Antineoplastic Agents, Phytogenic; Biological Dressings; Breast Neoplasms; Carcinoma; Cell Cycle; Cellulose; Curcumin; Cyclin D1; Drug Carriers; Drug Liberation; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Humans; Hydrogels; MCF-7 Cells; Melanocytes; Membranes, Artificial; Models, Molecular; Molecular Docking Simulation; Nanoparticles; Polyvinyl Alcohol; Protein Conformation; Spectroscopy, Fourier Transform Infrared; Wound Healing; X-Ray Diffraction

CCND1 encodes for Cyclin D1 protein and single-nucleotide polymorphisms (SNPs) can modulate its activity. In the present study, the impact of CCND1 SNPs on structure and/or function of Cyclin D1 protein using in silico tools was investigated. Our analysis revealed only one splice site SNP (c.1988+5G

Topics: Amino Acid Sequence; Breast Neoplasms; Computational Biology; Cyclin D1; Female; Frameshift Mutation; Gene Expression Regulation, Neoplastic; Humans; Kaplan-Meier Estimate; Mutation, Missense; Polymorphism, Single Nucleotide; Protein Interaction Maps; Protein Processing, Post-Translational; Protein Structure, Tertiary; RNA Splicing

Breast cancer is the most frequently diagnosed malignancy among women. However, the role of microRNA (miRNA) expression in breast cancer progression is not fully understood. In this study we examined predictive interactions between differentially expressed miRNAs and mRNAs in breast cancer cell lines representative of the common molecular subtypes. Integrative bioinformatics analysis identified miR-193 and miR-210 as potential regulatory biomarkers of mRNA in breast cancer. Several recent studies have investigated these miRNAs in a broad range of tumors, but the mechanism of their involvement in cancer progression has not previously been investigated.. The miRNA-mRNA interactions in breast cancer cell lines were identified by parallel expression analysis and miRNA target prediction programs. The expression profiles of mRNA and miRNAs from luminal (MCF-7, MCF-7/AZ and T47D), HER2 (BT20 and SK-BR3) and triple negative subtypes (Hs578T e MDA-MB-231) could be clearly separated by unsupervised analysis using HB4A cell line as a control. Breast cancer miRNA data from TCGA patients were grouped according to molecular subtypes and then used to validate these findings. Expression of miR-193 and miR-210 was investigated by miRNA transient silencing assays using the MCF7, BT20 and MDA-MB-231 cell lines. Functional studies included, xCELLigence system, ApoTox-Glo triplex assay, flow cytometry and transwell inserts were performed to determine cell proliferation, cytotoxicity, apoptosis, migration and invasion, respectively.. The most evident effects were associated with cell proliferation after miR-210 silencing in triple negative subtype cell line MDA-MB-231. Using in silico prediction algorithms, TNFRSF10 was identified as one of the potential regulated downstream targets for both miRNAs. The TNFRSF10C and TNFRSF10D mRNA expression inversely correlated with the expression levels of miR-193 and miR210 in breast cell lines and breast cancer patients, respectively. Other potential regulated genes whose expression also inversely correlated with both miRNAs were CCND1, a known mediator on invasion and metastasis, and the tumor suppressor gene RUNX3.. In summary, our findings identify miR-193 and miR-210 as potential regulatory miRNA in different molecular subtypes of breast cancer and suggest that miR-210 may have a specific role in MDA-MB-231 proliferation. Our results highlight important new downstream regulated targets that may serve as promising therapeutic pathways for aggressive breast cancers.

Topics: Biomarkers, Tumor; Breast; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Computational Biology; Core Binding Factor Alpha 3 Subunit; Cyclin D1; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; GPI-Linked Proteins; Humans; MicroRNAs; Oligonucleotide Array Sequence Analysis; Receptors, Tumor Necrosis Factor, Member 10c; Tumor Necrosis Factor Decoy Receptors

Emerging evidence indicates that microRNAs (miRNAs) play a critical role in breast cancer development. We recently reported that a higher expression of miR-374b in tumor tissues was associated with a better disease-free survival of triple-negative breast cancer (TNBC). However, the functional significance and molecular mechanisms underlying the role of miR-374b in breast cancer are largely unknown. In this current study, we evaluated the biological functions and potential mechanisms of miR-374b in both TNBC and non-TNBC. We found that miR-374b was significantly downregulated in breast cancer tissues, compared to adjacent tissues. MiR-374b levels were also lower in breast cancer cell lines, as compared to breast epithelial cells. In vitro and in vivo studies demonstrated that miR-374b modulates the malignant behavior of breast cancer cells, such as cell proliferation in 2D and 3D, cell invasion ability, colony-forming ability and tumor growth in mice. By using bioinformatics tools, we predicted that miR-374b plays a role in breast cancer cells through negatively regulating cyclin D1 (CCND1) and transforming growth factor alpha (TGFA). We further confirmed that CCND1 and TGFA contribute to the malignant behavior of breast cancer cells in vitro and in vivo. Our rescue experiments showed that overexpressing CCND1 or TGFA reverses the phenotypes caused by miR-374b overexpression. Taken together, our studies suggest that miR-374b modulates malignant behavior of breast cancer cells by negatively regulating CCND1 and TGFA genes. The newly identified miR-374b-mediated CCND1 and TGFA gene silencing may facilitate a better understanding of the molecular mechanisms of breast cancer progression.

Topics: Animals; Breast Neoplasms; Cell Movement; Cell Proliferation; Cyclin D1; Disease Progression; Female; Gene Expression Regulation, Neoplastic; Heterografts; Humans; MCF-7 Cells; Mice; MicroRNAs; Neoplasm Invasiveness; Transforming Growth Factor alpha

Paeoniflorin (Pae) possesses anti-tumor activity in various malignancies. However, it is unclear whether Pae plays a sensitizer role in breast cancer (BC) and the molecular mechanisms involved in this process. Our oligonucleotide microarray revealed that microRNA (miR)-15b is the most significantly downregulated miRNA in MCF-7/4-hydroxytamoxifen (4-OHT) cells treated with Pae. This paper summarized the relevance of Pae in BC cell endocrine resistance to tamoxifen (Tam) and the molecular mechanisms involved miR-15b expression.. 4-OHT-resistant BC cell lines were developed and treated with different concentrations of Pae. Flow cytometry, lactose dehydrogenase activity, caspase-3 activity, colony formation, and EdU assays were carried out to assess the impact of Pae on BC cells. Differentially expressed miRNAs in BC cells treated with Pae were analyzed by microarray. Targeting mRNAs of screened miR-15b as well as the binding of forkhead box O1 (FOXO1) to the cyclin D1 (CCND1) promoter sequence were predicted through bioinformatics analysis. Finally, the expression of β-catenin signaling-related genes in cells was detected by Western blotting.. Pae (100 μg/mL) inhibited the clonality and viability of BC cells, while enhancing apoptosis in vitro. Pae also repressed miR-15b expression. Overexpression of miR-15b restored the growth and resistance of BC cells to 4-OHT. Moreover, Pae promoted FOXO1 expression by downregulating miR-15b, thereby transcriptionally inhibiting CCND1 and subsequently blocking β-catenin signaling.. Pae inhibits 4-OHT resistance in BC cells by regulating the miR-15b/FOXO1/CCND1/β-catenin pathway.

Topics: Antineoplastic Agents; Apoptosis; beta Catenin; Breast Neoplasms; Cell Proliferation; Cell Survival; Cyclin D1; Down-Regulation; Drug Screening Assays, Antitumor; Female; Forkhead Box Protein O1; Glucosides; Humans; MicroRNAs; Monoterpenes; Tamoxifen; Tumor Cells, Cultured

Poly(A) polymerases add the poly(A) tail at the 3' end of nearly all eukaryotic mRNA, and are associated with proliferation and cancer. To elucidate the role of the most-studied mammalian poly(A) polymerase, poly(A) polymerase α (PAPOLA), in cancer, we assessed its expression in 221 breast cancer samples and found it to correlate strongly with the aggressive triple-negative subtype. Silencing PAPOLA in MCF-7 and MDA-MB-231 breast cancer cells reduced proliferation and anchorage-independent growth by decreasing steady-state cyclin D1 (CCND1) mRNA and protein levels. Whereas the length of the CCND1 mRNA poly(A) tail was not affected, its 3' untranslated region (3'UTR) lengthened. Overexpressing PAPOLA caused CCND1 mRNA 3'UTR shortening with a concomitant increase in the amount of corresponding transcript and protein, resulting in growth arrest in MCF-7 cells and DNA damage in HEK-293 cells. Such overexpression of PAPOLA promoted proliferation in the p53 mutant MDA-MB-231 cells. Our data suggest that PAPOLA is a possible candidate target for the control of tumor growth that is mostly relevant to triple-negative tumors, a group characterized by PAPOLA overexpression and lack of alternative targeted therapies.

Topics: Animals; Breast Neoplasms; Cell Proliferation; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Polyadenylation; RNA, Messenger

of the study was to determine the clinical relevance of cyclin D1 (cD1) and its association with clinicopathological parameters in breast cancer patients treated with hormonal therapy.. The study included 96 primary breast cancer patients with known clinicopathological parameters. In adjuvant setting, 44 patients were tamoxifen-treated and 52 were treated with ovarian irradiation/ablation. The cD1 status (gene amplified/nonamplified) was determined on formalin-fixed paraffin-embedded tumor tissue sections by chromogenic in situ hybridization. Associations between parameters were analyzed by Chi-square and Spearman's rank order correlation tests. Cox proportional hazards regression test was performed. Survival curves for relapse-free survival were constructed according to the Kaplan-Meier method.. There were no significant associations between cyclin D1 and clinicopathological parameters in either patient group. Amplified cyclin D1 associated significantly with the actual relapse incidence in the ovarian ablation patient group (p = 0.01, HR = 3.1), but not in the tamoxifen-treated patient group. Estrogen receptor and cyclin D1 have proven to be independent parameters of poor outcome in the ovarian ablation patient group (p = 0.03, HR = 2.9; and p = 0.009, HR = 2.5; respectively).. Cyclin D1 might be a candidate biomarker of poor outcome in breast cancer patients treated with ovarian ablation, suggesting its possible involvement in acquirement of hormonal resistance. The role of cyclin D1 as potential parameter of response to tamoxifen was not as pronounced.

Topics: Adult; Breast Neoplasms; Cyclin D1; Humans; Middle Aged; Neoplasm Recurrence, Local; Prognosis; Receptors, Estrogen; Tamoxifen

Annotation of structural variations (SVs) and base-level karyotyping in cancer cells remains challenging. Here, we present Integrative Framework for Genome Reconstruction (InfoGenomeR)-a graph-based framework that can reconstruct individual SVs into karyotypes based on whole-genome sequencing data, by integrating SVs, total copy number alterations, allele-specific copy numbers, and haplotype information. Using whole-genome sequencing data sets of patients with breast cancer, glioblastoma multiforme, and ovarian cancer, we demonstrate the analytical potential of InfoGenomeR. We identify recurrent derivative chromosomes derived from chromosomes 11 and 17 in breast cancer samples, with homogeneously staining regions for CCND1 and ERBB2, and double minutes and breakage-fusion-bridge cycles in glioblastoma multiforme and ovarian cancer samples, respectively. Moreover, we show that InfoGenomeR can discriminate private and shared SVs between primary and metastatic cancer sites that could contribute to tumour evolution. These findings indicate that InfoGenomeR can guide targeted therapies by unravelling cancer-specific SVs on a genome-wide scale.

Topics: A549 Cells; Breast Neoplasms; Cell Line, Tumor; Chromosome Aberrations; Cyclin D1; DNA Copy Number Variations; Female; Genome, Human; Genomic Structural Variation; Glioblastoma; HeLa Cells; High-Throughput Nucleotide Sequencing; Humans; Karyotyping; Ovarian Neoplasms; Polyploidy; Receptor, ErbB-2; Sequence Analysis, DNA; Whole Genome Sequencing

We investigated the prognostic influences of BCL1 and BCL2 expression on disease-free survival in breast cancer patients. BCL1 and BCL2 expression statuses were assessed by immunohistochemistry using tissue microarrays from 393 breast cancer patients. The Kaplan-Meier estimator and log-rank test were used for survival analyses. The Cox proportional hazards model was used to calculate hazard ratio (HR) and the 95% confidence interval (CI) of survival analyses. BCL1 expression revealed no impact on survival. The high BCL2 group showed superior disease-free survival compared with the low BCL2 group (p = 0.002), especially regarding local recurrence-free survival (p = 0.045) and systemic recurrence-free survival (p = 0.002). BCL2 expression was a significant prognostic factor by univariable analysis (HR, 0.528; 95% CI, 0.353-0.790; p = 0.002) and by multivariable analysis (HR, 0.547; 95% CI, 0.362-0.826; p = 0.004). High BCL2 expression was associated with higher disease-free survival in the hormone receptor (HRc)-positive and human epidermal growth factor receptor 2 (HER2)-negative (HRc(+)/HER2(-)) subtype only (p = 0.002). The high BCL2 group was associated with positive estrogen receptor (ER), positive progesterone receptor (PR), low histologic grade, and age ≤ 50 years. BCL1 expression had no prognostic impact, but BCL2 expression was a significant independent prognostic factor. High BCL2 expression was associated with higher disease-free survival, especially regarding local recurrence and systemic recurrence. The prognostic effect of BCL2 expression was effective only in the HRc(+)/HER2(-) subtype. Favorable clinicopathologic features and a strong association with the ER/PR status could partly explain the superior prognosis of the high BCL2 group. BCL2 expression could be utilized to assess the prognosis of breast cancer patients in clinical settings.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Breast Neoplasms; Cyclin D1; Disease-Free Survival; Female; Humans; Immunohistochemistry; Middle Aged; Multivariate Analysis; Prognosis; Proto-Oncogene Proteins c-bcl-2; Tissue Array Analysis

Histone deacetylases (HDACs) play a vital role in the epigenetic regulation of gene expression due to their overexpression in several cancer forms. Therefore, these enzymes are considered as a potential anticancer drug target. Different synthetic and natural structures have been studied as HDACs inhibitors; based on available structural design information, the capping group is important for the biological activity due to the different interactions in the active site entrance. The present study aimed to analyze high substituted pyridine as a capping group, which included carrying out the synthesis, antiproliferative activity analysis, and docking studies of these novel compounds.. To achieve the synthesis of these derivatives, four reaction steps were performed, generating desired products 15a-k. Their effects on cell proliferation and gene expression of p21, cyclin D1, and p53 were determined using the sulphorhodamine B (SRB) method and quantitative real-time polymerase chain reaction. The HDAC1, HDAC6, and HDAC8 isoforms were used for performing docking experiments with our 15a-k products.. The products 15a-k were obtained in overall yields of 40-71%. Compounds 15j and 15k showed the highest antiproliferative activity in the breast (BT-474 and MDA-MB-231) and prostate (PC3) cancer cell lines at a concentration of 10 µM. These compounds increased p21 mRNA levels and decreased cyclin D1 and p53 gene expression. The docking study showed an increment in the strength, and in the number of interactions performed by the capping moiety of the tested molecules compared with SAHA; interactions displayed are mainly van der Waals, π-stacking, and hydrogen bond.. The synthesized compounds 2-thiophene (15j) and 2-furan (15k) pyridine displayed cell growth inhibition, regulation of genes related to cell cycle progression in highly metastatic cancer cell lines. The molecular coupling analysis performed with HDAC1, HDAC6 and HDAC8 showed an increment in the number of interactions performed by the capping moiety and consequently in the strength of the capping group interaction.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Epigenesis, Genetic; Female; Furans; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Male; Molecular Docking Simulation; PC-3 Cells; Pregnancy; Prostatic Neoplasms; Pyridines; Thiophenes; Tumor Suppressor Protein p53

In spite of the strong evidence demonstrating the role of overexpression of Ki-67 and Cyclin D1 markers in breast carcinomas, clinical and pathological data remain to be discussed. This can be explained partly by intratumor heterogeneity.. To define the prevalence and clinical significance of Ki-67 and Cyclin D1 overexpression in primary breast tumors ER positive, while highlighting the existence of intratumor heterogeneity in this type of cancer.. 51 ER positive breast cancer tumors were used to evaluate the intratumoral distribution of Ki-67 and Cyclin D1 expression. Image acquisition and visualization of the markers were performed by optical microscopy and stereology sampling method.. The mean Ki-67 labeling index was distributed heterogeneously in the same tumor, from 20.67±6.87 to 45.10±10.65. The coefficient of variation (COV) revealed dispersion values between 13.4% and 42.9%. Associated with positive ER status, all the tumors presented a Cyclin D1 expression with a COV varying between 19% and 28.5% and a mean labeling index fluctuating between 19.40±4.42 and 41.64±10.08 within the same patient showing important intratumor heterogeneous distribution.. In this study, we have adopted a strictly quantitative approach to evaluate and demonstrate intratumor heterogeneity. This establishes one of the main factors for poor response to cancer therapy. To achieve this, intratumor heterogeneity should be usually definable and quantifiable but this domain awaits future progress and methods need to move towards a better understanding of molecular and cellular mechanisms that initiate and maintain this tumor heterogeneity.

Topics: Breast Neoplasms; Cyclin D1; Female; Humans; Ki-67 Antigen; Neoplasm Proteins; Receptors, Estrogen; Tumor Suppressor Proteins

ZFP36L1 is a tandem zinc-finger RNA-binding protein that recognizes conserved adenylate-uridylate-rich elements (ARE) located in 3'untranslated regions (UTR) to mediate mRNA decay. We hypothesized that ZFP36L1 is a negative regulator of a posttranscriptional hub involved in mRNA half-life regulation of cancer-related transcripts. Analysis of

Topics: 3' Untranslated Regions; Animals; Breast Neoplasms; Butyrate Response Factor 1; Carcinogenesis; Cell Cycle; Cell Hypoxia; Cell Line, Tumor; Cyclin D1; E2F1 Transcription Factor; Epigenesis, Genetic; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Mice; Mutation; RNA Processing, Post-Transcriptional; RNA Stability; RNA, Messenger; RNA, Small Interfering; Urinary Bladder Neoplasms; Xenograft Model Antitumor Assays; Zinc Fingers

Breast cancer is one of the main causes of malignancies in females. Many prognostic parameters are verified but they do not give sufficient data about patients' outcome. So, we must search for new prognostic and clinicopathological parameters. This study aimed to evaluate immunohistochemical expression of cyclin D1 and PSA in breast carcinoma and their relation to prognosis. It includes 79 specimens of breast invasive duct carcinoma. Overall survival time was available for all patients. There is a statistically significant association between positive PSA expression and lower tumor stage, nodal stage and tumor grade and between negative PSA expression and presence of metastasis (

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Ductal, Breast; Cyclin D1; Female; Humans; Middle Aged; Prostate-Specific Antigen; Retrospective Studies

Signal transducer and activator of transcription 3 (STAT3) protein frequently overexpressed in many malignancies and plays an essential role in regulating proliferation, apoptosis, migration and invasion in cancer cells. However, No STAT3 inhibitor was used clinically. In this study, we evaluated the toxic potential of a novel STAT3 inhibitor, 6Br-6a in breast cancer cell lines. The anti-cancer effect and underlying mechanism of 6Br-6a on MDA-MB-231 and MCF-7 cells were determined in vitro and in xenograft mouse model. Our data demonstrated that 6Br-6a significantly induced cell cycle arrest and cell apoptosis in breast cancer via blocking the activation of STAT3. Finally, we verified these inhibitory effects of 6Br-6a in the MDA-MB-231 xenograft mouse model. In conclusion, 6Br-6a effectively inhibited activation of STAT3 and induced cell cycle arrest and apoptosis via regulating cyclin D1 and Bcl-2 expression. All of these data indicate that 6Br-6a could be a potential candidate for the treatment of breast cancer.

Topics: Animals; Apoptosis; Breast Neoplasms; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Female; Humans; MCF-7 Cells; Mice, Inbred BALB C; Mice, Nude; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Small Molecule Libraries; STAT3 Transcription Factor; Xenograft Model Antitumor Assays

Topics: Autophagy; Breast Neoplasms; Cyclin D1; Everolimus; G1 Phase Cell Cycle Checkpoints; Humans; Proteolysis

Breast cancer is the most common cause of death in women worldwide. Approximately 5%-10% of instances are attributed to mutations acquired from the parents. Therefore, it is highly recommended to design more potential drugs and drug targets to eradicate such complex diseases. Network-based gene expression profiling is a suggested tool for discovering drug targets by incorporating various factors such as disease states, intensities based on gene expression as well as protein-protein interactions. To find prospective biomarkers in breast cancer, we first identified differentially expressed genes (DEGs) statistical methods p-value and false discovery rate were initially used. Of the total 82 DEGs, 67 were upregulated while the remaining 17 were downregulated. Sub-modules and hub genes include VEGFA with the highest degree, followed by 15 CCND1 and CXCL8 with 12-degree score was found. The survival analysis revealed that all the hub genes have important role in the development and progression of breast cancer. Enrichment analysis revealed that most of these genes are involved in signaling pathways and in the extracellular spaces. We also identified transcription factors and kinases, which regulate proteins in the DEGs PPI. Finally, drugs for each hub genes were identified. These results further expanded the knowledge regarding important biomarkers in breast cancer.

Topics: Biomarkers, Tumor; Breast Neoplasms; Cell Line, Tumor; Computational Biology; Cyclin D1; Drug Discovery; Female; Gene Expression; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Ontology; Gene Regulatory Networks; Humans; Interleukin-8; Models, Biological; Phosphotransferases; Protein Interaction Mapping; Protein Interaction Maps; Signal Transduction; Survival Analysis; Systems Biology; Transcription Factors; Transcriptome; Vascular Endothelial Growth Factor A

The molecular mechanisms governing the secretion of the non-coding genome are poorly understood. We show herein that cyclin D1, the regulatory subunit of the cyclin-dependent kinase that drives cell-cycle progression, governs the secretion and relative proportion of secreted non-coding RNA subtypes (miRNA, rRNA, tRNA, CDBox, scRNA, HAcaBox. scaRNA, piRNA) in human breast cancer. Cyclin D1 induced the secretion of miRNA governing the tumor immune response and oncogenic miRNAs. miR-21 and miR-93, which bind Toll-Like Receptor 8 to trigger a pro-metastatic inflammatory response, represented >85% of the cyclin D1-induced secreted miRNA transcripts. Furthermore, cyclin D1 regulated secretion of the P-element Induced WImpy testis (PIWI)-interacting RNAs (piRNAs) including piR-016658 and piR-016975 that governed stem cell expansion, and increased the abundance of the PIWI member of the Argonaute family, piwil2 in ERα positive breast cancer. The cyclin D1-mediated secretion of pro-tumorigenic immuno-miRs and piRNAs may contribute to tumor initiation and progression.

Topics: Animals; Argonaute Proteins; Breast Neoplasms; Cellular Microenvironment; Cyclin D1; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Mice, Transgenic; MicroRNAs; Neoplastic Stem Cells; RNA, Small Interfering; Signal Transduction

Epithelial‑mesenchymal transition (EMT) serves an important role in tumor migration and invasion. Astragalus polysaccharide (APS), which is the main component of the traditional Chinese medicine Astragalus membranaceus, has been identified to display an antitumor effect. However, the effects and mechanisms of APS during breast cancer migration and invasion are not completely understood. The present study investigated whether APS inhibited breast cancer migration and invasion by modulating the EMT pathway. An MTT assay and a Ki67 immunofluorescence staining assay demonstrated that APS inhibited the proliferation of breast cancer cells. The results of the wound healing and Transwell Matrigel invasion assays suggested that APS decreased the migration and invasion of breast cancer cells. The western blotting and immunofluorescence analyses further demonstrated that APS had a regulatory effect on EMT‑related molecules. APS decreased the expression levels of Snail and vimentin, but increased E‑cadherin expression. APS also downregulated Wnt1, β‑catenin and downstream target expression. Additionally, the present results suggested that APS decreased the proliferation, and EMT‑mediated migration and invasion of cells by inhibiting the Wnt/β‑catenin signaling pathway. The present study suggested that APS may serve as a promising therapeutic agent for breast cancer.

Topics: Astragalus Plant; beta Catenin; Breast Neoplasms; Cadherins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Ki-67 Antigen; Lithium Chloride; Neoplasm Invasiveness; Polysaccharides; Proto-Oncogene Proteins c-myc; Wnt Signaling Pathway; Wnt1 Protein

PPFIA1 is an important regulator of cell migration and invasion, regulating focal adhesion signalling and disassembly. PPFIA1 is frequently amplified in breast cancer, and recent functional studies indicate that PPFIA1 is an important promoter of migration and invasion in breast cancer. This study aims to evaluate the utility of PPFIA1 expression in the luminal breast cancer as a prognostic marker to predict the response to endocrine therapy.. Large, well-characterised cohorts of primary luminal breast cancer patients with long-term follow-up was assessed for the clinical impact of PPFIA1 expression at the transcriptomic and proteomic levels. Prognostic significance of PPFIA1 and its relationship with clinical outcome and benefit of endocrine therapy were analysed. In addition, its association with other related-genes was analysed.. There was significant association between PPFIA1 expression and a member of the liprin family that involves in cell invasion (PPFIBPI), and the cell cycle regulator (CCND1), whereas a negative association was observed with the tumour suppressor gene (CD82). Patients with high PPFIA1 expression were associated with high risk of recurrence, distant metastasis and death from breast cancer (P < 0.05). Importantly, high PPFIA1 expression predicted relapse in a subset of patients who were subject to endocrine treatment alone, and was an independent prognostic marker of unfavourable outcome in these patients (P < 0.05).. These findings support the proposed role for PPFIA1 as a regulator of cell migration in breast cancer and provides definitive evidence for the clinical utility of PPFIA1 expression in patients with luminal breast cancer. Most importantly, our data suggests that PPFIA1 might be a potential predictive marker for poor benefit from endocrine therapy.

Topics: Adaptor Proteins, Signal Transducing; Antineoplastic Agents, Hormonal; Biomarkers, Tumor; Breast Neoplasms; Cyclin D1; Female; Follow-Up Studies; Humans; Neoplasm Recurrence, Local; Prognosis; Proteome; Survival Rate; Transcriptome

Breast cancer is one of the most commonly diagnosed cancer among women globally. Shogaol, the active constituent of many spices belonging to the Zingiberaceae family, has received wide attention among other shogaols in terms of its anticancer activity against different neoplasms. To date, its efficacy at the detailed molecular level against breast cancer has not been established.. In the current study, we investigated the cytotoxic potential and the underlying molecular details of 6-shogaol against breast adenocarcinomacells (MCF-7), and breast ductal carcinoma cells (T47D). Cytotoxicity assay, cell cycle analysis. Real-time PCR (qPCR), apoptosis and autophagy techniques were used for the determination and molecular investigation of its anticancer properties.. 6-Shogaol is a promising candidate to be considered as a treatment of breast cancer.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Autophagy; Breast Neoplasms; Catechols; Cell Proliferation; Cisplatin; Cyclin D1; Dose-Response Relationship, Drug; Female; G2 Phase Cell Cycle Checkpoints; Humans; MCF-7 Cells; Oxaliplatin; Receptors, Notch; Signal Transduction; Transcription Factor HES-1

Topics: Antineoplastic Agents, Phytogenic; Binding Sites; Breast Neoplasms; CDC2 Protein Kinase; Cell Proliferation; Cyclin D1; Female; Humans; Lignans; MCF-7 Cells; Molecular Docking Simulation; Molecular Dynamics Simulation; Protein Binding; Protein Conformation; Proto-Oncogene Proteins c-akt; Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; Signal Transduction; Structure-Activity Relationship

Estrogen receptor (ER) positive accounts for a large proportion of breast cancer. Although there are many targeted therapeutic drugs, the emergence of drug resistance urgently requires the development of new drugs. Arctigenin (Arc), a lignan found in certain plants of the Asteraceae, has the effect on inhibiting breast cancer, but its molecular mechanism has not been clear.. To this end, the current study focuses on understanding the mechanism of Arc on ER-positive breast cancer cells.. Colony formation experiments and sulforhodamine B methods were used to determine the growth-inhibitory effect of Arc. The cell cycle and apoptosis were analyzed by flow cytometry. Alterations of signaling proteins were measured by Western blotting. Protein degradation was determined by comparing protein half-lives and inhibiting proteasome.. The experimental results show that Arc did not induce apoptosis in ER-positive breast cancer cell, rather caused G1 cycle arrest by decreasing cyclin D1 levels without effect on altering CDK4/6 levels. Moreover, we have demonstrated that Arc decreases cyclin D1 levels through prompting Akt/GSK3β-mediated degradation.. These findings warrant the potential of Arc as a candidate treatment for ER-positive breast cancer.

Topics: Breast Neoplasms; Cell Cycle Checkpoints; Cell Proliferation; Cyclin D1; Dose-Response Relationship, Drug; Female; Furans; Glycogen Synthase Kinase 3; Humans; Lignans; MCF-7 Cells; Proteolysis; Receptors, Estrogen

Emerging evidence has demonstrated the limited access to metabolic substrates as an effective approach to block cancer cell growth. The mechanisms remain unclear. Our previous work has revealed that miR-221/222 plays important role in regulating breast cancer development and progression through interaction with target gene p27.. Herein, we determined the miRNA-mRNA interaction in breast cancer cells under induced stress status of starvation. Starvation stimulation attenuated the miR-221/222-p27 interaction in MDA-MB-231 cells, thereby increased p27 expression and suppressed cell proliferation. Through overexpression or knockdown of miR-221/222, we found that starvation-induced stress attenuated the negative regulation of p27 expression by miR-221/222. Similar patterns for miRNA-target mRNA interaction were observed between miR-17-5p and CyclinD1, and between mR-155 and Socs1. Expression of Ago2, one of the key components of RNA-induced silencing complex (RISC), was decreased under starvation-induced stress status, which took responsibility for the impaired miRNA-target interaction since addition of exogenous Ago2 into MDA-MB-231 cells restored the miR-221/222-p27 interaction in starvation condition.. We demonstrated the attenuated interaction between miR-221/222 and p27 by starvation-induced stress in MDA-MB-231 breast cancer cells. The findings add a new page to the general knowledge of negative regulation of gene expression by miRNAs, also demonstrate a novel mechanism through which limited access to nutrients suppresses cancer cell proliferation. These insights provide a basis for development of novel therapeutic options for breast cancer.

Topics: Argonaute Proteins; Breast Neoplasms; Cell Culture Techniques; Cell Line, Tumor; Cell Proliferation; Culture Media; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Fasting; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; MicroRNAs; Stress, Physiological; Suppressor of Cytokine Signaling 1 Protein

Breast cancer is a complex disease and encompassing different types of tumor. Although advances in understanding of the molecular bases of breast cancer biology, the therapeutic proposals available still are not effective. In this scenario, the present study aimed to evaluate the mechanisms associated to antitumor activity of 7-Epiclusianone (7-Epi), a tetraprenylated benzophenone, on luminal A (MCF-7) and claudin-low (Hs 578T) breast cancer cell lines. We found that 7-Epi efficiently inhibited cell proliferation and migration of these cells; however MCF-7 was slightly more responsive than Hs 578T. Cell cycle analysis showed accumulation of cells at G0/G1 phase with drastic reduction of S population in treated cultures. This effect was associated to downregulation of CDKN1A (p21) and cyclin E in both cell lines. In addition, 7-Epi reduced cyclin D1 and p-ERK expression levels in MCF-7 cell line. Cytotoxic effect of 7-Epi on breast cancer cell lines was associated to its ability to increase BAX/BCL-2 ratio. In conclusion, our findings showed that 7-Epi is a promising antitumor agent against breast cancer by modulating critical regulators of the cell cycle and apoptosis.

Topics: Antineoplastic Agents; Apoptosis; Benzophenones; Benzoquinones; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Movement; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Female; Humans; Proto-Oncogene Proteins c-bcl-2

We investigated the function of circular RNA circEIF3M (hsa_circ_0003119) in triple-negative breast cancer. The expression profiles of circRNAs in 3 specimens of triple-negative breast cancer tissues with adjacent nontumor tissues were analyzed by RNA-sequencing. We verified the oncogenic role of circEIF3M in triple-negative breast cancer through a series of biological function experiments. It was found that circEIF3M was markedly upregulated in triple-negative breast cancer as compared to adjacent nontumor tissue, and that circEIF3M promoted triple-negative breast cancer cell proliferation, migration, and invasion. Mechanistic analysis indicated that circEIF3M may act as a competing endogenous RNA for miR-33a that relieves the inhibitory effect of miR-33a on its target cyclin D1. These findings showed that circEIF3M promotes triple-negative breast cancer progression via the circEIF3M/ miR-33a/ cyclin D1 axis.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Disease Progression; Female; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Neoplasm Invasiveness; RNA, Circular; Triple Negative Breast Neoplasms

A growing body of literature has revealed the effective role of miR-34a, as a tumor suppressor and regulator of expression of multiple targets in tumorigenesis and cancer progression. This study aimed at evaluating the potential effects of miR-34a alone or in combination with paclitaxel on breast cancer cells.. After miR-34a transduction by lentiviral vectors in two MCF-7 and MDA-MB-231 cell lines of breast cancer, effects of the elevated expression of miR-34a in the cell viability and the cell proliferation were determined using MTT assay in treated and untreated cells with paclitaxel. The mRNA level of the CCND1 (Cyclin D1)gene was then measured in the two cell lines using the qRT-PCR assay. Finally, the influence of miR-34a and paclitaxel on apoptosis and cell cycle progression were examined by flow cytometry.. The CCND1 mRNA expression levels were significantly down-regulated by overexpressed lentiviral miR-34a in MCF-7 and MDA-MB-231 cells. Combined treatment by miR-34a and paclitaxel reduced the cell viability and proliferation compared to single-drug treatment. In addition, the cell cycle arrest appeared at two phases by the combination of miR-34a and paclitaxel in MDA-MB-231 cells.. Our results suggest that miR34a, in combination with paclitaxel, has a potential for decreasing the cell viability and proliferation. Moreover, it can reduce the expression of CCND1 mRNA independent of the paclitaxel effect.

Topics: Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; DNA; Female; Gene Expression Regulation, Neoplastic; Green Fluorescent Proteins; HEK293 Cells; Humans; Inhibitory Concentration 50; MicroRNAs; Paclitaxel; Plasmids; RNA, Messenger

Breast cancer is the most common malignant tumor and usually occurs in women. Studies have shown that lncRNA nuclear enriched abundant transcript 1 (NEAT1) contributes to breast cancer progression. This study intends to further investigate the molecular mechanism of NEAT1 in breast cancer.. The expression levels of NEAT1, miR-410-3p and Cyclin D1 (CCND1) were detected by quantitative real-time PCR (qRT-PCR) in breast cancer tissues and cells. Kaplan-Meier analysis and the log-rank test were performed to determine the relationship between NEAT1 and overall survival. Cell Counting Kit-8 (CCK-8) assay analyzed cell proliferation. Transwell assay was performed to examine cell migration and invasion. The protein levels of CCND1 and epithelial-mesenchymal transition (EMT)-related proteins (E-cadherin, N-cadherin and Vimentin) were measured by western blot. The target relationship was predicted by bioinformatics analysis, and confirmed by luciferase reporter assay and RNA Immunoprecipitation (RIP) assay. Xenograft analysis was used to evaluate the tumor growth in vivo.. NEAT1 and CCND1 were upregulated, while miR-410-3p was down-regulated in breast cancer tissues and cells. Higher NEAT1 expression level was associated with lower survival rate of breast cancer patients. Knockdown of miR-410-3p restored silenced NEAT1-mediated the inhibition of on proliferation, migration, invasion and EMT of breast cancer cells. In addition, NEAT1 regulated CCND1 expression by sponging miR-410-3p in breast cancer cells. NEAT1 knockdown blocked the tumor growth in vivo.. NEAT1 induced breast cancer progression by regulating the miR-410-3p/CCND1 axis, indicating that NEAT1 may be a potential therapeutic target in breast cancer.

Topics: Animals; Biomarkers, Tumor; Breast; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Computational Biology; Cyclin D1; Disease Progression; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Kaplan-Meier Estimate; Mastectomy; Mice; MicroRNAs; Prognosis; RNA, Long Noncoding; Survival Rate; Up-Regulation; Xenograft Model Antitumor Assays

A significant proportion of patients with oestrogen receptor (ER) positive breast cancers (BC) develop resistance to endocrine treatments (ET) and relapse with metastatic disease. Here we perform whole exome sequencing and gene expression analysis of matched primary breast tumours and bone metastasis-derived patient-derived xenografts (PDX). Transcriptomic analyses reveal enrichment of the G2/M checkpoint and up-regulation of Polo-like kinase 1 (PLK1) in PDX. PLK1 inhibition results in tumour shrinkage in highly proliferating CCND1-driven PDX, including different RB-positive PDX with acquired palbociclib resistance. Mechanistic studies in endocrine resistant cell lines, suggest an ER-independent function of PLK1 in regulating cell proliferation. Finally, in two independent clinical cohorts of ER positive BC, we find a strong association between high expression of PLK1 and a shorter metastases-free survival and poor response to anastrozole. In conclusion, our findings support clinical development of PLK1 inhibitors in patients with advanced CCND1-driven BC, including patients progressing on palbociclib treatment.

Topics: Animals; Breast Neoplasms; Cell Cycle Proteins; Cell Line, Tumor; Cyclin D1; DNA Copy Number Variations; Drug Resistance, Neoplasm; Exome Sequencing; G2 Phase Cell Cycle Checkpoints; Humans; Immunoblotting; Immunohistochemistry; Immunoprecipitation; Mice; Mice, Nude; Piperazines; Polo-Like Kinase 1; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Pteridines; Pyridines

Chemoresistance of tumors often leads to treatment failure in clinical practice, which underscores pivotal needs to uncover novel therapeutic strategies. Accumulating evidences show that microRNAs (miRNAs) are widely involved in carcinogenesis, but their function on chemoresistance remains largely unexplored. In this study, we found that miR-93-5p (miR-93) significantly inhibited cell proliferation, induced G1/S cell cycle arrest and increased chemosensitivity to paclitaxel (PTX) in vitro and in vivo. Moreover, two well-established oncogenes, E2F1 and CCND1, were identified as dual targets of miR-93. Knockdown of E2F1 and CCND1 reduced cell proliferation and PTX-sensitivity, whereas overexpression of them had the opposite effect. More importantly, overexpression of E2F1 and CCND1 antagonized miR-93-mediated cell cycle arrest and apoptosis. Further mechanistic study revealed that miR-93 exhibited its inhibitory role by directly targeting E2F1 and CCND1 to inactivate pRB/E2F1 pathway and AKT phosphorylation. Taken together, our findings suggested that miR-93 greatly improved chemosensitivity and potentially served as a novel therapeutic target for breast cancer treatment.

Topics: Animals; Apoptosis; Breast Neoplasms; Carcinogenesis; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; DNA Methylation; Down-Regulation; Drug Resistance, Neoplasm; E2F1 Transcription Factor; Female; Gene Expression Regulation, Neoplastic; Humans; Mice, Nude; MicroRNAs; Minichromosome Maintenance Complex Component 7; Models, Biological; Paclitaxel; Phosphorylation; Promoter Regions, Genetic; Proto-Oncogene Proteins c-akt; Retinoblastoma Protein

Topics: Animals; Biomarkers, Tumor; BRCA1 Protein; Breast; Breast Neoplasms; Cell Line, Tumor; Chemotherapy, Adjuvant; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Drug Resistance, Neoplasm; Estrogen Receptor alpha; Feedback, Physiological; Female; Gene Expression Regulation, Neoplastic; Humans; Kaplan-Meier Estimate; Mastectomy; Mice; MicroRNAs; Mutagenesis, Site-Directed; Prognosis; Promoter Regions, Genetic; Tamoxifen; Xenograft Model Antitumor Assays

Cyclin D1 encodes the regulatory subunit of a holoenzyme that phosphorylates RB and functions as a collaborative nuclear oncogene. The serine threonine kinase Akt plays a pivotal role in the control of cellular metabolism, survival, and mitogenic signaling. Herein, Akt1-mediated phosphorylation of downstream substrates in the mammary gland is reduced by cyclin D1 genetic deletion and is induced by mammary-gland-targeted cyclin D1 overexpression. Cyclin D1 is associated with Akt1 and augments the rate of onset and maximal cellular Akt1 activity induced by mitogens. Cyclin D1 is identified in a cytoplasmic-membrane-associated pool, and cytoplasmic-membrane-localized cyclin D1-but not nuclear-localized cyclin D1-recapitulates Akt1 transcriptional function. These studies identify a novel extranuclear function of cyclin D1 to enhance proliferative functions via augmenting Akt1 phosphorylation at Ser473.

Topics: 3T3 Cells; Animals; Breast Neoplasms; Cell Membrane; Cyclin D1; Cyclin-Dependent Kinases; Female; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Mammary Glands, Animal; MCF-7 Cells; Mice; Mice, Transgenic; Mitogens; Phosphorylation; Phosphoserine; Protein Binding; Proto-Oncogene Proteins c-akt; Signal Transduction; Transcription, Genetic

Chromothripsis is a form of genome instability by which a presumably single catastrophic event generates extensive genomic rearrangements of one or a few chromosomes. Widely assumed to be an early event in tumor development, this phenomenon plays a prominent role in tumor onset. In this study, an analysis of chromothripsis in 252 human breast cancers from two patient cohorts (149 metastatic breast cancers, 63 untreated primary tumors, 29 local relapses, and 11 longitudinal pairs) using whole-genome and whole-exome sequencing reveals that chromothripsis affects a substantial proportion of human breast cancers, with a prevalence over 60% in a cohort of metastatic cases and 25% in a cohort comprising predominantly luminal breast cancers. In the vast majority of cases, multiple chromosomes per tumor were affected, with most chromothriptic events on chromosomes 11 and 17 including, among other significantly altered drivers,

Topics: Algorithms; Breast Neoplasms; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 17; Chromothripsis; Cyclin D1; Cyclin-Dependent Kinases; DNA Repair; Exome Sequencing; Female; Gene Fusion; Gene Rearrangement; Genes, BRCA1; Genes, BRCA2; Genes, erbB-2; Genes, p53; Humans; INDEL Mutation; Neoplasm Recurrence, Local; Signal Transduction; Whole Genome Sequencing

Breast cancer is the leading cause of cancer mortality in women worldwide. Therefore, it is of great significance to identify the biological mechanism of tumorigenesis and explore the development of breast cancer to achieve a better prognosis for individuals suffering from breast cancer. MicroRNAs (miRNAs) have become a hot topic in cancer research, but the underlying mechanism of its involvement in cancer remains unclear.. The miRNA profile between breast cancer stem cells (BCSCs, CD44. MiR-20b-5p had the highest degree in both the miRNA-gene network and miRNA-GO network to regulate BCSCs. Overexpression of miR-20b-5p significantly promoted the migration and wound healing ability of MCF-7 cells and T47D cells compared with the control (P < 0.05). In addition, miR-20b-5p facilitated the proliferation of MCF-7 cells and T47D-CSCs (P < 0.05) and inhibited the apoptosis of T47D-CSCs (P < 0.05). Moreover, miR-20b-5p promoted xenograft growth compared with the control group (P < 0.05). Accordingly, potential targets of both CCND1 and E2F1 were predicted by bioinformatics analysis. MiR-20b-5p directly targeted both CCND1 and E2F1 in a dual luciferase assay, while antagomir-20b-5p downregulated the protein levels of CCND1 and E2F1.. Oncogenic miR-20b-5p was confirmed to promote the malignant behaviors of breast cancer cells and BCSCs. The underlying mechanism lies in that miR-20b-5p overall enhanced both CCND1 and E2F1 targets via bidirectional regulation probably involving direct downregulation and indirect upregulation.

Topics: Animals; Breast Neoplasms; Cell Proliferation; Cyclin D1; E2F1 Transcription Factor; Female; Humans; Mice; Mice, Nude; Neoplastic Stem Cells; Oncogenes; Transfection

Topics: Agammaglobulinaemia Tyrosine Kinase; Animals; Antineoplastic Agents; Basic Helix-Loop-Helix Transcription Factors; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; CpG Islands; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cytosine; DNA Methylation; ErbB Receptors; Female; Genes, erbB-1; HSP70 Heat-Shock Proteins; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; MAP Kinase Signaling System; Methyltransferases; Mice; Phosphorylation; Retinoblastoma Protein; Signal Transduction; Tumor Hypoxia

Breast cancer is a type of cancer that begins in the breast tissue. Being a woman is the most important factor in the risk of breast cancer. Although men also get the cancer, women are much more likely to get it. This experiment was founded to investigate the effect and mechanism of Cisatracurium on breast cancer cell proliferation, migration and invasion. Breast cancer cells MDA-MB-231 were cultured in vitro. MDA-MB-231 cells were treated with cisatracurium of different concentrations for 48 h. CCK-8method detected cell proliferation, Transwell detected cell migration and invasion, Western Blot method detected the expression levels of CyclinD1, p21, MMP-2andMMP-9protein in cells, RT-qPCR) detected the expression level of miR-3174in cells. After miR-3174 inhibitor was transfected into MDA-MB-231 in order to down-regulate the expression of miR-3174, the same methods as above were used to observe the effect of the down-regulating miR-3174 expression on MDA-MB-231 cell proliferation, migration and invasion as well as the expression levels of CyclinD1, p21, MMP -2 andMMP-9 protein. After different concentrations of Cisatracurium acted on MDA-MB-231 cells, the cell inhibition rate and p21 protein expression were significantly increased (p<0.05), the number of cell migration and invasion and the expression levels of CyclinD1, MMP-2 and MMP-9 were significantly reduced (p<0.05), and the expression of miR-3174 in cells was significantly reduced (p<0.05). After down-regulating the expression of miR-3174, the cell inhibition rate and p21 protein expression were significantly increased (p<0.05), the number of cell migration and invasion and the expression levels of CyclinD1, MMP-2 and MMP-9 were significantly reduced (p<0.05). Up-regulating miR-3174 expression could reverse the effect of Cisatracurium on the proliferation, migration and invasion of MDA-MB-231 cells. Cisatracurium can inhibit the proliferation, migration and invasion of breast cancer MDA-MB-231 cells, and its mechanism is related to the down-regulation of miR-3174 expression in cells.

Topics: Atracurium; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; MicroRNAs; Neoplasm Invasiveness; Up-Regulation

Cyclin D1 is one of the most important oncoproteins that drives cancer cell proliferation and associates with tamoxifen resistance in breast cancer. Here, we identify a lncRNA, DILA1, which interacts with Cyclin D1 and is overexpressed in tamoxifen-resistant breast cancer cells. Mechanistically, DILA1 inhibits the phosphorylation of Cyclin D1 at Thr286 by directly interacting with Thr286 and blocking its subsequent degradation, leading to overexpressed Cyclin D1 protein in breast cancer. Knocking down DILA1 decreases Cyclin D1 protein expression, inhibits cancer cell growth and restores tamoxifen sensitivity both in vitro and in vivo. High expression of DILA1 is associated with overexpressed Cyclin D1 protein and poor prognosis in breast cancer patients who received tamoxifen treatment. This study shows the previously unappreciated importance of post-translational dysregulation of Cyclin D1 contributing to tamoxifen resistance in breast cancer. Moreover, it reveals the novel mechanism of DILA1 in regulating Cyclin D1 protein stability and suggests DILA1 is a specific therapeutic target to downregulate Cyclin D1 protein and reverse tamoxifen resistance in treating breast cancer.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents, Hormonal; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Disease-Free Survival; Drug Resistance, Neoplasm; Female; Follow-Up Studies; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Middle Aged; Prognosis; Protein Processing, Post-Translational; Protein Stability; Proteolysis; RNA, Long Noncoding; Tamoxifen; Xenograft Model Antitumor Assays; Young Adult

The objective of this study is to assess the prognostic and functional role of DSCC1 in breast carcinoma, as well as the potential mechanism. Based upon the TCGA data, the expression pattern and prognostic value of DSCC1 in breast carcinoma was evaluated. The mRNA and protein levels of molecules were determined using qRT-PCR and Western blot. In vitro functional role of DSCC1 in tumor cells was determined using cell counting kit 8, clone formation, and Transwell assays. Gene set enrichment analysis (GSEA) was conducted to determine DSCC1 related gene sets, which are further confirmed by Western blot. The results showed that DSCC1 is overexpressed in breast carcinoma tissues and its high expression was linked to shorter overall survival. Overexpression of DSCC1 facilitated the proliferation, invasion and migration of breast carcinoma cells, while knockdown of DSCC1 showed opposite outcomes. GSEA showed that high DSCC1 expression had a positive correlation with p53, and Wnt signaling-related molecules. Western blot showed that silencing DSCC1 increased the levels of p53 and p-β-catenin, whereas decreased p-GSK-3β and cyclin D1 expression. These observations illustrate that DSCC1 emerges a well value on the diagnosis and prognosis of breast carcinoma, and facilitates the progression of breast carcinoma partly by activating Wnt/b-catenin signaling and inhibiting p53.

Topics: Aged; beta Catenin; Breast Neoplasms; Carrier Proteins; Cyclin D1; Disease Progression; Disease-Free Survival; Female; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3 beta; Humans; MCF-7 Cells; Middle Aged; Nuclear Proteins; RNA, Messenger; Transfection; Tumor Suppressor Protein p53; Wnt Signaling Pathway

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Breast Neoplasms; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Cytochromes c; Dammaranes; Female; Gynostemma; Humans; Hydrolysis; Plant Extracts; Proto-Oncogene Proteins c-bcl-2; Saponins; Triterpenes

The Calcineurin/NFAT (nuclear factor of activated T cells) pathway plays an essential role in the tumorigenic and metastatic properties in breast cancer. The molecular mechanism of the antiproliferative effect of calcineurin inhibition, however, is poorly understood. We found that calcineurin inhibition delayed cell cycle progression at G1/S, and promoted cyclin D1 degradation by inhibiting dephosphorylation at T286. Importantly, overexpression of cyclin D1 partially rescued delayed G1/S progression, thereby revealing cyclin D1 as a key factor downstream of calcineurin inhibition. Cyclin D1 upregulation is observed in human invasive breast cancers, and our findings indicate that dysregulation of T286 phosphorylation could play a role in this phenomenon. We therefore propose that targeting site specific phosphorylation of cyclin D1 could be a potential strategy for clinical intervention of invasive breast cancer.

Topics: Breast Neoplasms; Calcineurin; Cell Line, Tumor; Cyclin D1; Female; G1 Phase; Humans; Neoplasm Invasiveness; Phosphorylation; S Phase

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Collagen; Cyclin D1; Cyclin D3; Drug Combinations; Female; Gene Expression Regulation, Neoplastic; Humans; Laminin; Matrix Metalloproteinase 1; Mice; Phosphatidate Phosphatase; Proteoglycans; RNA, Messenger; Signal Transduction; Transcription Factor AP-1

Recent advances in endocrine therapy have improved the prospects for estrogen receptor-positive breast cancer. Tamoxifen is an effective drug for patients with estrogen receptor-positive breast cancer, but the development of resistance is common. Therefore, discovering ways to enhance the sensitivity of cancer cells to tamoxifen may help improve breast cancer treatment. We studied the biological role of sirtuin 4 (SIRT4) in tamoxifen-treated MCF7 and T47D cells. The levels of the MYC proto-oncogene (MYC) and cyclin D1 (CCND1) were detected by western blotting and quantitative reverse transcription-polymerase chain reaction in breast cancer cells with SIRT4 overexpression or depletion. Immunofluorescence and western blotting were used to assess the phosphorylation status of signal transducer and activator of transcription 3 (STAT3). SIRT4 overexpression decreased the half maximal inhibitory concentration of tamoxifen in MCF7 and T47D cells, while its depletion increased it. Thus, SIRT4 enhances the sensitivity of breast cancer cells to tamoxifen. Moreover, western blotting revealed decreased STAT3 phosphorylation after SIRT4 transfection. The transcription and translation of MYC and CCND1, target genes of the STAT3 pathway, were also blocked. Immunofluorescence revealed that pathway activation and nuclear STAT4 translocation were suppressed when SIRT4 was overexpressed. Furthermore, the effects of SIRT4 overexpression or depletion on proliferation could be offset by STAT3 activation or inhibition. Taken together, these results demonstrate that SIRT4 enhances the tamoxifen sensitivity of breast cancer cells by inhibiting the STAT3 signaling pathway. With this knowledge, therapeutic strategies with reduced drug resistance risk may be developed.

Topics: Antineoplastic Agents, Hormonal; Breast Neoplasms; Cell Line, Tumor; Cyclin D1; Female; Humans; Interleukin-6; Mitochondrial Proteins; Proto-Oncogene Mas; Proto-Oncogene Proteins c-myc; Receptors, Estrogen; Signal Transduction; Sirtuins; STAT3 Transcription Factor; Tamoxifen

Cyclin D1 which is associated with cell cycle regulation is solidly established as an oncogene with an important pathogenetic role in breast carcinomas.. The aim of this study was to relate the Cyclin D1 protein overexpression with the amplification of its gene CCND1 in Estrogen Receptors (ER) positive breast carcinomas, in order to investigate the prognostic effect of their aberrations in relation to ER status, also to correlate the Cyclin D1 overexpression with other prognostic parameters.. Chromogenic in situ hybridization (CISH) was used to identify CCND1 amplification on formalin-fixed paraffin-embedded invasive ductal carcinoma, in which immunohistochemistry (IHC) had previously been performed in order to evaluate the pathological relevance of Cyclin D1 overexpression in human breast cancer (n = 138).. CCND1 amplification was identified in 17/138 (12.3%) tumors and 78/138 (56.5%) tumors have overexpressed Cyclin D1. A significant correlation was identified between CCND1 amplification and Cyclin D1 overexpression (P < 0.001) and both Cyclin D1 and CCND1 were related with ER expression.. Our results show a significant correlation between Cyclin D1 overexpression and CCND1 amplification. Overexpression of Cyclin D1was observed in high proportion of breast cancer which should be considered for routine diagnosis.

Topics: Adult; Aged; Algeria; Breast Neoplasms; Carcinoma, Ductal, Breast; Cyclin D1; Female; Gene Amplification; Humans; Immunohistochemistry; In Situ Hybridization, Fluorescence; Middle Aged; Prognosis

Androgens, through their own receptor, play a protective role on breast tumor development and progression and counterbalance estrogen-dependent growth stimuli which are intimately linked to breast carcinogenesis.. Cell counting by trypan blu exclusion was used to study androgen effect on estrogen-dependent breast tumor growth. Quantitative Real Time RT-PCR, western blotting, transient transfection, protein immunoprecipitation and chromatin immunoprecipitation assays were carried out to investigate how androgen treatment and/or androgen receptor overexpression influences the functional interaction between the steroid receptor coactivator AIB1 and the estrogen- or androgen receptor which, in turn affects the estrogen-induced cyclin D1 gene expression in MCF-7 breast cancer cells. Data were analyzed by ANOVA.. Here we demonstrated, in estrogen receptor α (ERα)-positive breast cancer cells, an androgen-dependent mechanism through which ligand-activated androgen receptor (AR) decreases estradiol-induced cyclin D1 protein, mRNA and gene promoter activity. These effects involve the competition between AR and ERα for the interaction with the steroid receptor coactivator AIB1, a limiting factor in the functional coupling of the ERα with the cyclin D1 promoter. Indeed, AIB1 overexpression is able to reverse the down-regulatory effects exerted by AR on ERα-mediated induction of cyclin D1 promoter activity. Co-immunoprecipitation studies indicated that the preferential interaction of AIB1 with ERα or AR depends on the intracellular expression levels of the two steroid receptors. In addition, ChIP analysis evidenced that androgen administration decreased E. Taken together all these data support the hypothesis that AIB1 sequestration by AR may be an effective mechanism to explain the reduction of estrogen-induced cyclin D1 gene activity. In estrogen-dependent breast cancer cell proliferation, these findings reinforce the possibility that targeting AR signalling may potentiate the effectiveness of anti-estrogen adjuvant therapies.

Topics: Breast Neoplasms; Cyclin D1; Estradiol; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Nuclear Receptor Coactivator 3; Promoter Regions, Genetic; Protein Binding; Receptors, Androgen; RNA, Messenger; Signal Transduction; Transcription Factor AP-1

Objective To investigate the effect of aldosterone reductase family 1 member B10 (AKR1B10) on breast cancer cell proliferation and its mechanism. Methods AKR1B10 was overexpressed in MCF-7 cells and knocked down in BT-20 cells to establish both AKR1B10 overexpression and knockdown cell lines. The effect of AKR1B10 overexpression and knockdown on breast cancer cell proliferation was examined by CCK-8 assay. Real-time quantitative PCR was performed to detect AKR1B10 mRNA levels in breast cancer tissues and paired normal tissues. Western blot analysis was used to determine the protein levels of AKR1B10, β-catenin, cyclin D1, survivin, c-myc in breast cancer tissues and AKR1B10 overexpression/knockdown breast cancer cell lines. Results The expression of AKR1B10 was higher in breast cancer tissues. With AKR1B10 overexpression in MCF-7 cells, cell proliferation was promoted, and the expression levels of β-catenin, cyclin D1, c-myc and survivin were elevated. Meanwhile, knockdown of AKR1B10 in BT-20 breast cancer cells reduced cell proliferation and the expression levels of β-catenin, cyclin D1, c-myc and survivin. Conclusion AKR1B10 is highly expressed in breast cancer and promotes breast cancer cell proliferation by activating Wnt/β-catenin signaling pathway.

Topics: Aldehyde Reductase; Aldo-Keto Reductases; beta Catenin; Breast Neoplasms; Cell Proliferation; Cyclin D1; Humans; MCF-7 Cells; Proto-Oncogene Proteins c-myc; Survivin; Wnt Signaling Pathway

To investigate the therapeutic effects of Jiazhu decoction (JZD) in combination with cyclophosphamide (CTX) on the growth of breast cancer in mice and to explore the possible molecular mechanisms of action.. BALB/c mice were randomly divided into four groups of 10 (untreated model group, JZD group, CTX group, and JZD + CTX group) and subcutaneously injected with 4T1 mouse breast cancer cells. Tumors were allowed to establish for ~7 d before initiation of treatment with CTX (100 mg/kg every week by intraperitoneal injection) and/or JZD (0.015 mL of 1.65 g/mL crude drug, administered daily by gavage). The model group received equivalent volumes of vehicle on the same schedules. Tumor volumes were measured every 3 d. Mice were sacrificed after 3 weeks of treatment, and tumors were excised and subjected to RT-qPCR and western blot analysis to evaluate expression of the Wnt/β-catenin signaling pathway components β-catenin, c-Myc, and cyclin D1 at the mRNA and protein levels.. The mean tumor volume was smaller and the growth rate was slower in the CTX and JZD + CTX groups compared with the model group (P < 0.05), and in the JZD + CTX group compared with the CTX and JZD groups (P < 0.05). Tumor growth was inhibited by 35.4% and 48.1% by CTX and JZD + CTX treatment, respectively (P < 0.001). The expression of β-catenin, c-Myc, and cyclin D1 mRNA and protein in tumors was significantly lower in mice treated with JZD or JZD + CTX compared with the untreated mice (P < 0.05), and was significantly lower in mice treated with JZD + CTX compared with either JZD or CTX alone (P < 0.05).. JZD inhibited the growth of mouse breast cancer cells in vivo, possibly by reducing the expression of β-catenin, c-Myc, and cyclin D1. Combination therapy with JZD plus CTX had a more potent inhibitory effect on breast cancer growth compared with either agent alone.

Topics: Animals; Antineoplastic Agents; beta Catenin; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclophosphamide; Drug Interactions; Drugs, Chinese Herbal; Gene Expression Regulation, Neoplastic; Genes, myc; Mice; RNA, Messenger; Xenograft Model Antitumor Assays

In this study, we aimed to explore the association between miR-99a-5p and CDC25A in breast cancer and the regulatory mechanisms of miR-99a-5p on breast cancer. The expressions of messenger RNA and microRNAs in breast cancer tissues and adjacent tissues were analyzed by the Cancer Genome Atlas microarray analysis. Quantitative real-time polymerase chain reaction was conducted to find out the expression levels of miR-99a-5p and CDC25A. The expression levels of proteins (CDC25A, ki67, cyclin D1, p21, BAX, BCL-2, BCL-XL, MMP2, and MMP9) were determined by Western blot analysis. The relationship between miR-99a-5p and CDC25A was predicted and verified by bioinformatics analysis and dual luciferase assay. After transfection, cell proliferation, invasion, and apoptosis of breast cancer tissues were, respectively, observed by cell counting kit-8 assay, transwell assay, and flow cytometry (FCM). Furthermore, the relationship among miR-99a-5p, CDC25A, and cell-cycle progression was determined by FCM assay. The nude mouse transplantation tumor experiment was performed to verify the influence of miR-99a-5p on breast cancer cell in vivo. The expression of miR-99a-5p in breast cancer tissues and cells was significantly downregulated, whereas CDC25A expression was upregulated. MiR-99a-5p targeted CDC25A and suppressed its expression in breast cancer cells. Overexpression of miR-99a-5p and decreased expression of CDC25A could suppress breast cancer cell proliferation and invasion and facilitate apoptosis. Cell-cycle progression was significantly activated by downregulated miR-99a-5p and upregulated CDC25A. Moreover, miR-99a-5p overexpression repressed the expressions of CDC25A, marker ki67, and Cyclin D1 proteins, whereas it upregulated the expression of p21 protein. MicroRNA-99a-5p suppresses breast cancer progression and cell-cycle pathway through downregulating CDC25A.

Topics: Adult; Animals; Apoptosis; Breast Neoplasms; cdc25 Phosphatases; Cell Cycle; Cell Movement; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Disease Progression; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Humans; Ki-67 Antigen; MCF-7 Cells; Mice, Nude; MicroRNAs; Middle Aged; Neoplasm Invasiveness; Signal Transduction; Tumor Burden

The cell division cycle 25 (CDC25) phosphatases regulate key transitions between cell-cycle phases during normal cell division, and in the case of DNA damage, they are key targets of the checkpoint machinery that ensure genetic stability. Little is known about the mechanisms underlying dysregulation and downstream targets of CDC25. To understand these mechanisms, we silenced the CDC25A gene in breast cancer cell line MDA-MB-231 and studied downstream targets of CDC25A gene. MDA-MB-231 breast cancer cells were transfected and silenced by CDC25A small interfering RNA. Total messenger RNA (mRNA) was extracted and analyzed by quantitative real-time polymerase chain reaction. CDC25A phosphatase level was visualized by Western blot analysis and was analyzed by 2D electrophoresis and LC-ESI-MS/MS. After CDC25A silencing, cell proliferation reduced, and the expression of 12 proteins changed. These proteins are involved in cell-cycle regulation, programmed cell death, cell differentiation, regulation of gene expression, mRNA editing, protein folding, and cell signaling pathways. Five of these proteins, including ribosomal protein lateral stalk subunit P0, growth factor receptor bound protein 2, pyruvate kinase muscle 2, eukaryotic translation elongation factor 2, and calpain small subunit 1 increase the activity of cyclin D1. Our results suggest that CDC25A controls the cell proliferation and tumorigenesis by a change in expression of proteins involved in cyclin D1 regulation and G1/S transition.

Topics: Breast Neoplasms; cdc25 Phosphatases; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; RNA, Small Interfering; Tandem Mass Spectrometry

Breast cancer is a complex disease driven by multiple factors including both genetic and epigenetic alterations. Recent studies revealed that abnormal gene expression induced by epigenetic changes including aberrant promoter methylation plays a critical role in human breast carcinogenesis. Cucurbitacin B has antiproliferative activity against various human breast cancer cells, but the molecular mechanism is not completely understood. In this study, we explore the influence of cucurbitacin B from

Topics: Antineoplastic Agents, Phytogenic; Breast Neoplasms; Cell Line, Tumor; Cyclin D1; DNA (Cytosine-5-)-Methyltransferase 1; DNA Methylation; Female; Gene Expression Regulation, Neoplastic; Humans; Promoter Regions, Genetic; Proto-Oncogene Proteins c-myc; Survivin; Trichosanthes; Triterpenes

Selective estrogen receptor modulators such as tamoxifen (TAM) significantly reduce the risks of developing estrogen receptor-positive (ER+) breast cancer. Low concentrations (nanomolar range) of bisphenol A (BPA) shows estrogenic effects and further promotes the proliferation of hormone-dependent breast cancer cells. However, whether or not BPA can influence TAM-treatment resistance in breast cancer has not drawn much attention. In the current study, low concentrations of BPA reduced TAM-induced cytotoxicity of MCF-7 cells, which was proved by the suppression of cell apoptosis, transition of cell cycle from G1 to S phase, and upregulation of cyclin D1 and ERα. Simultaneously, the mRNA levels of estrogen-related receptor γ (ERRγ) and its coactivators, peroxisome proliferation-activated receptor γ coactivator-1α (PGC-1α), and PGC-1β, were increased. However, the similar effects were not observed in MDA-MB-231 cells. Our results indicated that low concentrations of BPA decreased the sensitivity of TAM in MCF-7 cells rather than in MDA-MB-231 cells. These different actions likely involved the interaction of relative receptors and coactivators. This study provided a possible support that the exposure of BPA in environmental media may potentially induce TAM resistance to breast cancer treatment.

Topics: Apoptosis; Benzhydryl Compounds; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Dose-Response Relationship, Drug; Estrogen Receptor alpha; Estrogens; Female; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Phenols; Selective Estrogen Receptor Modulators; Tamoxifen; Trefoil Factor-1

Topics: Benzhydryl Compounds; Breast Neoplasms; Cell Proliferation; Cyclin D; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 5; Endocrine Disruptors; Estrogen Receptor alpha; Female; G1 Phase Cell Cycle Checkpoints; Humans; MCF-7 Cells; Phenols; Phosphorylation; Retinoblastoma Protein; Signal Transduction

The correlation of genetic alterations with response to neoadjuvant chemotherapy (NAC) has not been fully revealed. In this study, we enrolled 247 breast cancer patients receiving anthracycline-taxane-based NAC treatment. A next generation sequencing (NGS) panel containing 36 hotspot breast cancer-related genes was used in this study. Two different standards for the extent of pathologic complete response (pCR), ypT0/isypN0 and ypT0/is, were used as indicators for NAC treatment. TP53 mutation (n = 149, 60.3%), PIK3CA mutation (n = 109, 44.1%) and MYC amplification (n = 95, 38.5%) were frequently detected in enrolled cases. TP53 mutation (P = 0.019 for ypT0/isypN0 and P = 0.003 for ypT0/is) and ERBB2 amplification (P < 0.001 for both ypT0/isypN0 and ypT0/is) were related to higher pCR rates. PIK3CA mutation (P = 0.040 for ypT0/isypN0) and CCND2 amplification (P = 0.042 for ypT0/is) showed reduced sensitivity to NAC. Patients with MAPK pathway alteration had low pCR rates (P = 0.043 for ypT0/is). Patients with TP53 mutation (-) PIK3CA mutation (-) ERBB2 amplification (+) CCND1 amplification (-), TP53 mutation (+) PIK3CA mutation (-) ERBB2 amplification (+) CCND1 amplification (-) or TP53 mutation (+) PIK3CA mutation (+) ERBB2 amplification (+) CCND1 amplification (-)had significantly higher pCR rates (P < 0.05 for ypT0/isypN0 and ypT0/is) than wild type genotype tumors. Some cancer genetic alterations as well as pathway alterations were associated with chemosensitivity to NAC treatment. Our study may shed light on the molecular characteristics of breast cancer for prediction of NAC expectations when breast cancer is first diagnosed by biopsy.

Topics: Adult; Aged; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Breast Neoplasms; Cell Line, Tumor; Class I Phosphatidylinositol 3-Kinases; Cyclin D1; Drug Resistance, Neoplasm; Female; Gene Amplification; Genetic Variation; High-Throughput Nucleotide Sequencing; Humans; Middle Aged; Mutation; Neoadjuvant Therapy; Receptor, ErbB-2; Signal Transduction; Treatment Outcome; Tumor Burden; Tumor Suppressor Protein p53

Use of cyclin D1 (CCND1) gene amplification as a breast cancer biomarker has been hampered by conflicting assessments of the relationship between cyclin D1 protein levels and patient survival. Here, we aimed to clarify its prognostic and treatment predictive potential through comprehensive long-term survival analyses.. CCND1 amplification was assessed using SNP arrays from two cohorts of 1965 and 340 patients with matching gene expression array and clinical follow-up data of over 15 years. Kaplan-Meier and multivariable Cox regression analyses were used to determine survival differences between CCND1 amplified vs. non-amplified tumours in clinically relevant patient sets, within PAM50 subtypes and within treatment-specific subgroups. Boxplots and differential gene expression analyses were performed to assess differences between amplified vs. non-amplified tumours within PAM50 subtypes.. When combining both cohorts, worse survival was found for patients with CCND1-amplified tumours in luminal A (HR = 1.68; 95% CI, 1.15-2.46), luminal B (1.37; 1.01-1.86) and ER+/LN-/HER2- (1.66; 1.14-2.41) subgroups. In gene expression analysis, CCND1-amplified luminal A tumours showed increased proliferation (P < 0.001) and decreased progesterone (P = 0.002) levels along with a large overlap in differentially expressed genes when comparing luminal A and B-amplified vs. non-amplified tumours.. Our results indicate that CCND1 amplification is associated with worse 15-year survival in ER+/LN-/HER2-, luminal A and luminal B patients. Moreover, luminal A CCND1-amplified tumours display gene expression changes consistent with a more aggressive phenotype. These novel findings highlight the potential of CCND1 to identify patients that could benefit from long-term treatment strategies.

Topics: Adult; Biomarkers, Tumor; Breast Neoplasms; Cyclin D1; Female; Follow-Up Studies; Gene Amplification; Genetic Testing; Humans; Lymph Nodes; Middle Aged; Predictive Value of Tests; Prognosis; Receptor, ErbB-2; Receptors, Estrogen; Retrospective Studies; Survival Analysis

Long noncoding RNAs (lncRNAs) are critical regulators in tumorigenesis. However, their roles in breast cancer remain unclear. Here, we found that lncRNA LINC00473 is significantly upregulated in breast cancer cells. Loss- or gain-of-function experiments show that LINC00473 promotes cell proliferation. Mechanistically, LINC00473 is required for the activation of cyclin D1 (CCND1) expression through recruitment of phosphorylated CREB and histone acetylation to the CCND1 promoter. Interestingly, we found that LINC00473 is also required for maintaining the expression levels of the noncoding RNA

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Promoter Regions, Genetic; RNA-Binding Protein FUS; RNA, Long Noncoding; Transcriptional Activation

Cancer-associated fibroblasts (CAFs) remain active even in the absence of cancer cells. However, the molecular mechanism underlying the sustained active status of CAFs is largely unrevealed. We found that in CAFs, DNMT3B was not only a target of miR-200b, miR-200c and miR-221, but was able to induce DNA methylation of miR-200s promoters. DNMT3B eventually reached a stably high level by the counteracting effect of decreasing miR-200b/c and increasing miR-221 in normal fibroblasts (NFs) with long-term exogenous TGF-β1 treatment, and DNMT3B further led to a low level of miR-200s which established CAF activation. Meanwhile, miR-200s/miR-221/DNMT3B signaling sustained autocrine TGF-β1 maintaining active CAF status. Destruction of the autocrine TGF-β1/miR-200s/miR-221/DNMT3B signaling led to demethylation of miR-200s promoters and further restored the NF phenotypes. Moreover, we confirmed that TCF12, the target of miR-141, stimulated c-Myc/Cyclin D1 axis in breast cancer cells to promote cancer growth by enhancing CXCL12 of CAFs. The current study reveals that the TGF-β1/miR-200s/miR-221/DNMT3B regulatory loop is responsible for the maintenance of CAFs status and is also necessary for CAF function in promoting malignance of breast cancer, which provides a potential target for CAF-driven therapeutic strategy.

Topics: Basic Helix-Loop-Helix Transcription Factors; Breast Neoplasms; Cancer-Associated Fibroblasts; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; DNA Methyltransferase 3B; Female; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Humans; MCF-7 Cells; MicroRNAs; Proto-Oncogene Proteins c-myc; Signal Transduction; Transforming Growth Factor beta1

Using an ORF kinome screen in MCF-7 cells treated with the CDK4/6 inhibitor ribociclib plus fulvestrant, we identified FGFR1 as a mechanism of drug resistance. FGFR1-amplified/ER+ breast cancer cells and MCF-7 cells transduced with FGFR1 were resistant to fulvestrant ± ribociclib or palbociclib. This resistance was abrogated by treatment with the FGFR tyrosine kinase inhibitor (TKI) lucitanib. Addition of the FGFR TKI erdafitinib to palbociclib/fulvestrant induced complete responses of FGFR1-amplified/ER+ patient-derived-xenografts. Next generation sequencing of circulating tumor DNA (ctDNA) in 34 patients after progression on CDK4/6 inhibitors identified FGFR1/2 amplification or activating mutations in 14/34 (41%) post-progression specimens. Finally, ctDNA from patients enrolled in MONALEESA-2, the registration trial of ribociclib, showed that patients with FGFR1 amplification exhibited a shorter progression-free survival compared to patients with wild type FGFR1. Thus, we propose breast cancers with FGFR pathway alterations should be considered for trials using combinations of ER, CDK4/6 and FGFR antagonists.

Topics: Aminopyridines; Animals; Antineoplastic Agents, Hormonal; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Circulating Tumor DNA; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Drug Resistance, Neoplasm; Female; Fulvestrant; High-Throughput Nucleotide Sequencing; Humans; MCF-7 Cells; Mice; Mutation; Naphthalenes; Piperazines; Progression-Free Survival; Proportional Hazards Models; Protein Kinase Inhibitors; Purines; Pyrazoles; Pyridines; Quinolines; Quinoxalines; Receptor, Fibroblast Growth Factor, Type 1; Receptor, Fibroblast Growth Factor, Type 2; Receptors, Estrogen; Signal Transduction; Xenograft Model Antitumor Assays

Traditional Chinese medicine (TCM) are both historically important therapeutic agents and important source of new drugs. Halofuginone (HF), a small molecule alkaloid derived from febrifugine, has been shown to exert strong antiproliferative effects that differ markedly among various cell lines. However, whether HF inhibits MCF-7 cell growth in vitro and underlying mechanisms of this process are not yet clear. Here, we offer the strong evidence of the connection between HF treatment, exosome production and proliferation of MCF-7 cells. Our results showed that HF inhibits MCF-7 cell growth in both time- and dose-dependent manner. Further microRNA (miRNA) profiles analysis in HF treated and nontreated MCF-7 cell and exosomes observed that six miRNAs are particularly abundant and sorted in exosomes. miRNAs knockdown experiment in exosomes and the MCF-7 growth inhibition assay showed that exosomal microRNA-31 (miR-31) modulates MCF-7 cells growth by specially targeting the histone deacetylase 2 (HDAC2), which increases the levels of cyclin-dependent kinases 2 (CDK2) and cyclin D1 and suppresses the expression of p21. In conclusion, these data indicate that inhibition of exosome production reduces exosomal miR-31, which targets the HDAC2 and further regulates the level of cell cycle regulatory proteins, contributing to the anticancer functions of HF. Our data suggest a new role for HF and the exosome production in tumorigenesis and may provide novel insights into prevention and treatment of breast cancer.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p21; Exosomes; Female; Histone Deacetylase 2; Humans; MCF-7 Cells; Medicine, Chinese Traditional; MicroRNAs; Piperidines; Quinazolinones

Cancer stem cells (CSC) generate and sustain tumors due to tumor-initiating potential, resulting in recurrence or metastasis. We showed that knockout of the cell-cycle inhibitor, p21CIP1, in the PyMT mammary tumor model inhibits metastasis; however the mechanism remained unknown. Here, we show a pivotal role for p21 in potentiating a cancer stem-like phenotype. p21 knockout in PyMT mammary tumor cells caused dramatic suppression of CSC properties involving tumorsphere formation, ALDH1 activity, and tumor-initiating potential, which were in turn rescued by p21 overexpression into PyMT/p21 knockout cells. Interestingly, p21 knockout dramatically suppresses Wnt/β-catenin signaling activity, leading to striking inhibition of LEF1 and TCF1 expression. TCF1 knockdown in PyMT cells suppressed tumorsphere formation due to Cyclin D1 attenuation. These data demonstrate that p21 promotes a CSC-like phenotype via activation of Wnt/TCF1/Cyclin D1 signaling. IMPLICATIONS: p21 is a strong promoter of mammary CSCs.

Topics: Aldehyde Dehydrogenase 1 Family; Animals; Breast; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Female; Gene Knockout Techniques; Hepatocyte Nuclear Factor 1-alpha; Humans; Lymphoid Enhancer-Binding Factor 1; Mammary Neoplasms, Animal; Neoplasm Recurrence, Local; Neoplastic Stem Cells; Retinal Dehydrogenase; Wnt Signaling Pathway

C/EBPα is an essential transcription factor involved in regulating the expression or function of certain cell-cycle regulators, including in breast cancer cells. Although protein arginine methyltransferases have been shown to play oncogenic roles in a variety of cancers, little is known about the role of arginine methylation in regulating the antiproliferation activity of C/EBPα. Here, we report that the protein arginine methyltransferase 1 (PRMT1) is overexpressed in human breast cancer and that elevated PRMT1 correlates with cancer malignancy. RNA-sequencing analysis revealed that knockdown of PRMT1 in breast cancer cells is accompanied by a decrease in the expression of pro-proliferative genes, including cyclin D1. Furthermore, tandem affinity purification followed by mass spectrometry identified PRMT1 as a component of the C/EBPα complex. C/EBPα associated with and was methylated by PRMT1 at three arginine residues (R35, R156, and R165). PRMT1-dependent methylation of C/EBPα promoted the expression of cyclin D1 by blocking the interaction between C/EBPα and its corepressor HDAC3, which resulted in rapid growth of tumor cells during the pathogenesis of breast cancer. Inhibition of PRMT1 significantly impeded the growth of cancer cells from patients with triple-negative breast cancer. This evidence that PRMT1 mediates C/EBPα methylation sheds light on a novel pathway and potential therapeutic target in breast cancer. SIGNIFICANCE: This study provides novel mechanistic insight of the role of the arginine methyltransferase PRMT1 in breast cancer pathogenesis.

Topics: Animals; Arginine; Breast Neoplasms; CCAAT-Enhancer-Binding Proteins; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Histone Deacetylases; Humans; Methylation; Mice, Inbred BALB C; Protein-Arginine N-Methyltransferases; Repressor Proteins; Xenograft Model Antitumor Assays

Effector CD8. The interactions between T cell activation status and either BRCA1 or CCND1 expression were evaluated using Kaplan-Meier survival curves and multivariate Cox regression models in a public dataset with 1088 breast cancer patients.. Among the patients with low BRCA1 or CCND1 expression, the Activation group showed better overall survival than the Exhaustion group. Adjusted hazards ratios were 0.43 (95% CI: 0.20-0.93) in patients with a low BRCA1 level, and 0.39 (95% CI: 0.19-0.81) in patients with a low CCND1 level, respectively. There was a significant trend in both subgroups (p-trend = 0.011 in the low BRCA1 group, and p-trend = 0.009 in the low CCND1 group). In contrast, there is no significant association in patients with either high BRCA1 or high CCND1 levels. There is a significant interaction between T cell activation status and BRCA1 level (p = 0.009), but not between T cell activation status and CCND1 level (p = 0.135).. BRCA1 expression modified the effect of T cell activation status on patient survival in breast cancer, suggesting that the existence of neoantigens and the enhancement of neoantigen presentation in combination with immune checkpoint blockade may have synergistic effects on patient outcome.

Topics: Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; BRCA1 Protein; Breast Neoplasms; Carcinogenesis; Cyclin D1; Disease-Free Survival; Female; Gene Expression Regulation, Neoplastic; Genomic Instability; Humans; Lymphocyte Activation; Middle Aged; Prognosis; RNA, Messenger; T-Lymphocytes

β-Thujaplicin, a natural monoterpenoid, has been demonstrated to exert health beneficial activities in chronic diseases. However, it has not been studied in regulating estrogen receptor (ER) negative breast cancer. Here, we investigated the effect of β-thujaplicin on inhibiting ER-negative basal-like breast cancer and the underlying mechanism of action using an in vitro and in vivo xenograft animal model. β-Thujaplicin induced G0/G1 phase cell cycle arrest and regulated cell cycle mediators, cyclin D1, cyclin E, and cyclin-dependent kinase 4 (CDK 4), leading to the inhibition of the proliferation of ER-negative basal-like MCF10DCIS.com human breast cancer cells. It also modulated the phosphorylation of protein kinase B (AKT) and glycogen synthase kinase (GSK-3β) and the protein level of β-catenin. In an MCF10DCIS.com xenograft animal model, β-thujaplicin significantly inhibited tumor growth, reduced tumor weight, and regulated the expression of cell cycle proteins, phosphorylation of AKT and GSK-3β, and protein level of β-catenin in the tumor tissues. These results demonstrate that β-thujaplicin can suppress basal-like mammary tumor growth by regulating GSK-3β/β-catenin signaling, suggesting that β-thujaplicin may be a potent chemopreventive agent against the basal-like subtype of breast cancer.

Topics: Animals; beta Catenin; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Chamaecyparis; Cyclin D1; Cyclin-Dependent Kinase 4; Drugs, Chinese Herbal; Female; G1 Phase Cell Cycle Checkpoints; Glycogen Synthase Kinase 3 beta; Humans; Mice, Inbred BALB C; Monoterpenes; Phosphorylation; Signal Transduction; Thuja; Tropolone

In this study, we investigated the relationships of pathological response after neoadjuvant chemo-endocrine therapy with alterations in the Ki67 labeling index (LI), expression of cyclin D1 (CCND1) and progesterone receptor (PgR), and estrogen receptor (ER) activity in breast cancer. A total of 43 Japanese post-menopausal ER-positive and human epidermal growth factor receptor 2-negative invasive breast cancer patients with tumors >2 cm or positive lymph nodes were enrolled. Exemestane alone was administered for 2 months. Neoadjuvant chemo-endocrine therapy included four cycles of doxorubicin plus paclitaxel followed by weekly paclitaxel. The immunohistochemical expression of Ki67 LI, CCND1, and PgR, and ER activity were evaluated using core needle biopsy samples at the pretreatment and post-exemestane alone stages. ER activity was evaluated through transfection of an adenovirus vector carrying an estrogen-response element-green fluorescent protein gene. In current study, marked pathological responses (including 4.7% with pathological complete response) were obtained in 34.9% of patients. Ki67 LI and PgR expression were significantly decreased after treatment. High Ki67 LI at pretreatment was a significant predictive factor of marked pathological response. At the stage of post-exemestane alone, Ki67 LI was not significantly associated with pathological response; however, high CCND1 expression was significantly correlated with high Ki67 LI. Moreover, low-level ER activity at the post-exemestane alone stage was significantly associated with marked pathological response. In conclusions, pretreatment Ki67 LI was a predictor of response to neoadjuvant chemo-endocrine therapy. CCND1 expression and ER activity at the post-endocrine therapy alone stage may be useful in determining further treatments.

Topics: Aged; Breast Neoplasms; Cyclin D1; Female; Humans; Ki-67 Antigen; Middle Aged; Neoadjuvant Therapy; Postmenopause; Prognosis; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone

Everolimus inhibits mammalian target of rapamycin complex 1 (mTORC1) and is known to cause induction of autophagy and G

Topics: Antineoplastic Agents; Autophagy; Autophagy-Related Protein 7; Breast Neoplasms; Cell Proliferation; Cyclin D1; Everolimus; Female; G1 Phase Cell Cycle Checkpoints; Humans; MCF-7 Cells; Mechanistic Target of Rapamycin Complex 1; Protein Kinase Inhibitors; Proteolysis; Signal Transduction; Tissue Culture Techniques

The family of long noncoding mitochondrial RNAs (ncmtRNAs), comprising sense (SncmtRNA), and antisense (ASncmtRNA-1 and ASncmtRNA-2) members, are differentially expressed according to cell proliferative status; SncmtRNA is expressed in all proliferating cells, while ASncmtRNAs are expressed in normal proliferating cells, but is downregulated in tumor cells. ASncmtRNA knockdown with an antisense oligonucleotide induces massive apoptosis in tumor cell lines, without affecting healthy cells. Apoptotic death is preceded by proliferation blockage, suggesting that these transcripts are involved in cell cycle regulation. Here, we show that ASncmtRNA knockdown induces cell death preceded by proliferative blockage in three different human breast cancer cell lines. This effect is mediated by downregulation of the key cell cycle progression factors cyclin B1, cyclin D1, CDK1, CDK4, and survivin, the latter also constituting an essential inhibitor of apoptosis, underlying additionally the onset of apoptosis. The treatment also induces an increase in the microRNA hsa-miR-4485-3p, whose sequence maps to ASncmtRNA-2 and transfection of MDA-MB-231 cells with a mimic of this miRNA induces cyclin B1 and D1 downregulation. Other miRNAs that are upregulated include nuclear-encoded hsa-miR-5096 and hsa-miR-3609, whose mimics downregulate CDK1. Our results suggest that ASncmtRNA targeting blocks tumor cell proliferation through reduction of essential cell cycle proteins, mediated by mitochondrial and nuclear miRNAs. This work adds to the elucidation of the molecular mechanisms behind cell cycle arrest preceding tumor cell apoptosis induced by ASncmtRNA knockdown. As proof-of-concept, we show that in vivo knockdown of ASncmtRNAs results in drastic inhibition of tumor growth in a xenograft model of MDA-MB-231 subcutaneous tumors, further supporting this approach for the development of new therapeutic strategies against breast cancer.

Topics: Animals; Antagomirs; Apoptosis; Breast Neoplasms; CDC2 Protein Kinase; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin B1; Cyclin D1; Down-Regulation; Female; Humans; Mice; Mice, Inbred BALB C; MicroRNAs; Mitochondria; RNA Interference; RNA, Long Noncoding; RNA, Small Interfering

Natural products are considered recently as one of the source for production of efficient therapeutical agents for breast cancer treatment. In this study, a sesquiterpene lactone, 13-O-acetylsolstitialin A (13ASA), isolated from Centaurea cyanus, showed cytotoxic activities against MCF-7 and MDA-MB-231 breast cancer cell lines using standard 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. To find the mechanism of action of cytotoxicity, annexin V/propidium iodide (PI) staining was performed for evaluation of apoptosis. This process was further confirmed by immunoblotting of anti- and proapoptotic, Bcl-2 and Bax, proteins. Cell cycle arrest was evaluated by measurement of fluorescence intensity of PI dye and further confirmed by immunoblotting of Cdk-4 and cyclin D1. Mitochondrial transmembrane potential (ΔΨm) and generation of reactive oxygen species (ROS) were measured using the JC-1 and DCFDA fluorescence probes, respectively. These experiments showed that 13ASA is a potent cytotoxic agent, which activates apoptosis-mediated cell death. In response to this compound, Bax/Bcl-2 ratio was noticeably increased in MCF-7 and MDA-MB-231 cells. Moreover, 13ASA induced cell cycle arrest at subG1 and G1 phases by decreasing protein levels of cyclin D1 and Cdk-4. It was done possibly through the decrease of ΔΨm and increase of ROS levels which induce apoptosis. In conclusion, this study mentioned that 13ASA inhibit the growth of MCF-7 and MDA-MB-231 breast cancer cell lines through the induction of cell cycle arrest, which triggers apoptotic pathways. 13ASA can be considered as a susceptible compound for further investigation in breast cancer study.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Centaurea; Cyclin D1; Cyclin-Dependent Kinase 4; G1 Phase Cell Cycle Checkpoints; Humans; Lactones; MCF-7 Cells; Membrane Potential, Mitochondrial; Molecular Structure; Plant Extracts; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Sesquiterpenes

BACKGROUND The role of the ubiquitin-specific peptidase 9 X-linked (USP9X) gene in breast cancer remains poorly understood. This study aimed to investigate the role of USP9X in breast cancer tissue and cell lines. MATERIAL AND METHODS Immunohistochemistry was used to examine the expression levels of USP9X in 102 breast cancer tissue samples and 41 normal breast tissue samples. Overexpression of USP9X in MCF-7 and MDA-MB-231 breast cancer cell lines were studied by USP9X lentivirus vector transfection. Clustered regularly interspaced short palindromic repeats (CRISPR)/caspase-9 USP9X gene knockout was performed. Cell proliferation, growth, and survival were examined using the cell counting kit-8 (CCK-8) assay, the colony formation assay, flow cytometry assays, and a tumor xenograft study. RESULTS Immunohistochemistry showed that USP9X was significantly overexpressed in 93 of 102 (91.1%) breast cancer tissue samples compared with 41 normal breast tissue samples and was associated with tumor size ≥5.0 cm (P<0.05). USP9X overexpression in MCF-7 and MDA-MB-231 breast cancer increased cell proliferation and survival, significantly reduced the number of cells in the G1-phase cells and increased the number of cells in the S-phase cells, which were reversed by CRISPR/caspase-9 USP9X gene knockout. Overexpression of USP9X upregulated the CCND1 gene encoding cyclin D1 and downregulated cyclin-dependent inhibitor kinase 1A (CDKN1A) gene in breast cancer cells, which were reversed by USP9X knockout. CONCLUSIONS Overexpression of USP9X was associated with upregulation of the CCND1 gene and downregulation of the CDKN1A gene in breast cancer tissue and cell lines.

Topics: Adult; Animals; Breast Neoplasms; Carcinogenesis; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Female; Gene Expression Regulation, Neoplastic; Heterografts; Humans; Immunohistochemistry; MCF-7 Cells; Mice; Mice, Inbred BALB C; Mice, Nude; Ubiquitin Thiolesterase

Long noncoding RNA (lncRNAs) frequently exhibited abnormal levels in numerous tumors and other diseases in current biological researches. LINC01287, a newly discovered lncRNA, has been found to act as an oncogene in hepatocellular carcinoma. The aim of this research was to explore the expressions and functions of LINC01287 in breast cancer (BC).. The relative expressions of LINC01287 in BC tissues and cells were determined using RT-PCR. The associations between the LINC01287 expression, the clinicopathological factors, and the overall survival of BC patients were statistically examined. The apoptosis and proliferation abilities of MCF-7 and MDA-MB-468 cells were analyzed by MTT and flow cytometry assay after LINC01287 knockdown. The effects of LINC01287 in migration and invasion were determined using wound-healing and transwell assays. The protein expressions of the Wnt/β-catenin pathway were determined using Western blot.. We showed that the levels of LINC01287 were significantly upregulated in BC tissues and BC cell lines, and the abnormal expressions of LINC01287 were correlated with TNM stage and lymph node metastasis. A distinct difference was observed and indicated that BC patients with higher LINC01287 expressions had significantly shorter overall survival than patients with lower LINC01287 expressions. The multivariate analysis demonstrated that LINC01287 expression was independently correlated with the overall survival. Si-LINC01287 transfection significantly inhibited the proliferation and metastasis of BC cells, and further promoted apoptosis. Besides, the knockdown of LINC01287 suppressed Wnt/β-catenin activation and affected the expressions of β-catenin, cyclin D1, and c-myc.. Our findings indicated that the new lncRNA LINC01287 was correlated with poor clinical outcome and may function as a novel prognostic biomarker and therapeutic target in the development of antineoplastic therapies for BC.

Topics: Apoptosis; beta Catenin; Breast Neoplasms; Case-Control Studies; Cell Line, Tumor; Cell Movement; Cell Proliferation; China; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Lymphatic Metastasis; Middle Aged; Neoplasm Staging; Prognosis; Proto-Oncogene Proteins c-myc; RNA, Long Noncoding; Survival Analysis; Wnt Signaling Pathway

Circular RNAs (circRNAs) that form through non-canonical backsplicing events of pre-mRNA transcripts are evolutionarily conserved and abundantly expressed across species. However, the functional relevance of circRNAs remains a topic of debate.. We identified one of the highly expressed circRNA (circANKRD12) in cancer cell lines and characterized it validated it by Sanger sequencing, Real-Time PCR. siRNA mediated silencing of the circular junction of circANKRD12 was followed by RNA Seq analysis of circANKRD12 silenced cells and control cells to identify the differentially regulated genes. A series of cell biology and molecular biology techniques (MTS assay, Migration analysis, 3D organotypic models, Real-Time PCR, Cell cycle analysis, Western blot analysis, and Seahorse Oxygen Consumption Rate analysis) were performed to elucidate the function, and underlying mechanisms involved in circANKRD12 silenced breast and ovarian cancer cells.. In this study, we identified and characterized a circular RNA derived from Exon 2 and Exon 8 of the ANKRD12 gene, termed here as circANKRD12. We show that this circRNA is abundantly expressed in breast and ovarian cancers. The circANKRD12 is RNase R resistant and predominantly localized in the cytoplasm in contrast to its source mRNA. We confirmed the expression of this circRNA across a variety of cancer cell lines and provided evidence for its functional relevance through downstream regulation of several tumor invasion genes. Silencing of circANKRD12 induces a strong phenotypic change by significantly regulating cell cycle, increasing invasion and migration and altering the metabolism in cancer cells. These results reveal the functional significance of circANKRD12 and provide evidence of a regulatory role for this circRNA in cancer progression.. Our study demonstrates the functional relevance of circANKRD12 in various cancer cell types and, based on its expression pattern, has the potential to become a new clinical biomarker.

Topics: Biomarkers, Tumor; Breast; Breast Neoplasms; Cell Movement; Cyclin D1; Exons; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Lung; Lung Neoplasms; MCF-7 Cells; Neoplasm Invasiveness; Nuclear Proteins; Phenotype; RNA, Circular; RNA, Small Interfering; Transfection

Previously, several protein-coding tumor suppressors localized at 1p36 have been reported. In the present work, we focus on functional long non-coding RNAs (lncRNAs) embedded in this locus. Small interfering RNA was used to identify lncRNA candidates with growth-suppressive activities in breast cancer. The mechanism involved was also explored. LINC01355 were downregulated in breast cancer cells relative to non-malignant breast epithelial cells. Overexpression of LINC01355 significantly inhibited proliferation, colony formation, and tumorigenesis of breast cancer cells. LINC01355 arrested breast cancer cells at the G0/G1 phase by repressing CCND1. Moreover, LINC01355 interacted with and stabilized FOXO3 protein, leading to transcriptional repression of CCND1. Importantly, LINC01355-mediated suppression of breast cancer growth was reversed by knockdown of FOXO3 or overexpression of CCND1. Clinically, LINC01355 was downregulated in breast cancer specimens and correlated with more aggressive features. There was a negative correlation between LINC01355 and CCND1 expression in breast cancer samples. LINC01355 acts as a tumor suppressor in breast cancer, which is ascribed to enhancement of FOXO3-mediated transcriptional repression of CCND1. Re-expression of LINC01355 may provide a potential therapeutic strategy to block breast cancer growth and progression.

Topics: Animals; Blotting, Western; Breast Neoplasms; Cell Proliferation; Chromatin Immunoprecipitation; Cyclin D1; Female; Flow Cytometry; Forkhead Box Protein O3; G1 Phase; Gene Expression Regulation, Neoplastic; Humans; In Vitro Techniques; Mice; Mice, Inbred BALB C; Mice, Nude; Plasmids; Real-Time Polymerase Chain Reaction; Resting Phase, Cell Cycle; RNA, Long Noncoding; Xenograft Model Antitumor Assays

To identify the role and to explore the mechanism of extracellular 5'-nucleotidase (CD73) in human breast cancer growth, CD73 expression was measured firstly in breast cancer tissues and cell lines, and then interfered with or over-expressed by recombinant lentivirus in cell lines. Impacts of CD73 on breast cancer cell proliferation and cell cycle were investigated with colony formation assay, CCK-8 and flow cytometry. The relationship between CD73 and AKT/GSK-3β/β-catenin pathway was assessed with adenosine, adenosine 2A receptor antagonist (SCH-58261), adenosine 2A receptor agonist (NECA), CD73 enzyme inhibitor (APCP) and Akt inhibitor (MK-2206). Moreover, the effect of CD73 on breast cancer growth in vivo was examined with human breast cancer transplanting model of nude mice. The results showed that the expression of CD73 was high in breast cancer tissues and increased with advanced tumor grades and lympho-node status. CD73 expression was higher in more malignant cells, and CD73 overexpression promoted breast cancer cell proliferation in both in vivo and in vitro. It activated AKT/GSK-3β/β-catenin/cyclinD1 signaling pathway through CD73 enzyme activity and other mechanism.

Topics: 5'-Nucleotidase; Animals; Apoptosis; beta Catenin; Breast Neoplasms; Cell Cycle; Cell Movement; Cell Proliferation; Cyclin D1; Female; Glycogen Synthase Kinase 3 beta; GPI-Linked Proteins; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Prognosis; Proto-Oncogene Proteins c-akt; Signal Transduction; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Hormone receptor (HR)-positive breast cancer (BC) shows a poor response to neoadjuvant chemotherapy (NACT). New treatment targets like the Cyclin D1-CDK4/CDK6 complex are promising adjuvant/post-neoadjuvant therapeutic strategies. Evaluating Cyclin D1 overexpression in residual tumor could recognize those patients that benefit most from such post-neoadjuvant treatment. In this study, we determined Cyclin D1 expression in residual BC after NACT. Secondary aims were to correlate Cyclin D1 expression levels with clinicopathological parameters and to assess its prognostic value after NACT.. We retrospectively assessed the nuclear expression of Cyclin D1 on tissue microarrays with residual tumor from 284 patients treated in the neoadjuvant GeparTrio (n = 186) and GeparQuattro (n = 98) trials. Evaluation was performed with a standardized immunoreactive score (IRS) after selecting a cut-off value.. A high expression level (IRS ≥ 6) of Cyclin D1 was found in 37.3% of the assessed specimens. An increased Cyclin D1 expression was observed in HR-positive tumors, compared to HR-negative tumors (p = 0.02). Low Cyclin D1 levels correlated with clinical tumor stage 1-3 (p = 0.03). Among patients with HR-positive/Her2-negative tumors and high Cyclin D1 expression, a better disease-free survival (DFS) was graphically suggested, but not significant (p = 0.21).. Our study demonstrates a measurable nuclear expression of Cyclin D1 in post-neoadjuvant residual tumor tissue of HR-positive BC. Cyclin D1 expression was not prognostic for DFS after NACT. Our results and defined cut-off suggest that the marker can be used to stratify tumors according to protein expression levels. Based on this, a prospective evaluation is currently performed in the ongoing Penelope-B trial.

Topics: Adult; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Breast; Breast Neoplasms; Cell Nucleus; Cyclin D1; Disease-Free Survival; Female; Gene Expression Profiling; Humans; Mastectomy; Middle Aged; Neoadjuvant Therapy; Neoplasm, Residual; Prognosis; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone; Retrospective Studies; Tissue Array Analysis

To investigate anticancer activity of the DNA binding domain of SMAR1 (His 5) in vitro and in vivo.. His 5 was conjugated to hydrothermally synthesized carbon nanospheres (CNs). Anticancer activity of CNs-His 5 was evaluated in vitro and in vivo.. CNs- His 5 significantly reduced cyclin D1 levels in MDA-MB-231 cells. Tumor bearing Balb/c mice injected with CNs-His 5 showed approximately 62% tumor regression and significantly reduced. DNA binding domain of the SMAR1 protein (His 5) has potent anticancer activity and its CNs mediated delivery could control breast tumor in mice model.

Topics: Animals; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Carbon; Cell Cycle Proteins; Cell Line, Tumor; Cell Survival; Cyclin D1; DNA-Binding Proteins; Drug Carriers; Drug Liberation; Female; Humans; Mice, Inbred BALB C; Nanospheres; Nuclear Proteins; Protein Domains; Recombinant Proteins; Tissue Distribution

Mucuna pruriens (L.) DC (MP) is an ancient Indian medicinal plant traditionally used to treat Parkinson's disease. L-Dopa (LD), precursor of dopamine is abundantly found in the seeds of MP. L-dopa is a natural inhibitor of prolactin (PRL) hormone which is required to maintain lactation in women but it's over production (hyperprolactinemia) plays critical role in advancement of breast cancer.. We aim to examine the pharmacological effect of LD and MP on this hyperprolactinemia associated breast cancer and related signaling for effective management of the disease. We also investigated chemo-sensitizing effect of MP on hyperprolactinemia-mediated cisplatin resistance.. Methanolic seed extract of MP were prepared and analysed using HPLC. Effect of LD and MP on the cellular viability of breast cancer cells (T47D, MCF-7, MDA-MB-468 and MDA-MB-231) were evaluated using MTT assay. Further, effect of LD and MP on colony forming potential, DNA damage, cell cycle distribution and apoptosis was determined using agar/agarose method, comet assay and annexin and PI method followed by FACS analysis. To reveal the molecular mechanism involved in the anti-cancer activity of MP, transcriptional and translational level analysis of the key proteins involved in the PRL-mediated signaling, was performed using RT-PCR and western blot analysis. The effect of MP extract on PRL-mediated signaling was validated using dopaminergic agonist bromocriptine. MP extract and cisplatin was given in different combination with appropriate controls to check their effect on chemo-resistivity of breast cancer cells.. Our results demonstrated that MP seed extract has the potential to inhibit cellular proliferation of PRL expressing T47D and MCF-7 breast cancer cells via induction of DNA damage, G1 phase of cell cycle arrest and apoptosis more effectively as compare to LD. Further, MP-mediated anti-cancerous effect was associated with the downregulation of PRL expression, further suppressing the JAK2/STAT5A/Cyclin D1 signaling pathway which has been validated using dopaminergic agonist bromocriptine. Cancer-related hyperprolactinemia confers cisplatin resistance, we observed that MP via PRL inhibition, enhances cisplatin efficacy after their combinatorial treatment in breast cancer cells.. Collectively, our study suggests that MP could be recommended as dietary supplement along with the chemotherapeutic agents against breast cancer.

Topics: Antineoplastic Agents, Phytogenic; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Breast Neoplasms; Cell Cycle Checkpoints; Cell Proliferation; Cisplatin; Cyclin D1; Dose-Response Relationship, Drug; Down-Regulation; Drug Repositioning; Drug Resistance, Neoplasm; Female; Humans; Hyperprolactinemia; Janus Kinase 2; Levodopa; MCF-7 Cells; Mucuna; Phytotherapy; Plant Extracts; Plants, Medicinal; Prolactin; Signal Transduction; STAT5 Transcription Factor; Tumor Suppressor Proteins

Drug resistance to approved systemic therapies in estrogen receptor-positive (ER+) breast cancer remains common. We hypothesized that factors present in the human tumor microenvironment (TME) drive drug resistance. Screening of a library of recombinant secreted microenvironmental proteins revealed fibroblast growth factor 2 (FGF2) as a potent mediator of resistance to anti-estrogens, mTORC1 inhibition, and phosphatidylinositol 3-kinase inhibition in ER+ breast cancer. Phosphoproteomic analyses identified ERK1/2 as a major output of FGF2 signaling via FGF receptors (FGFRs), with consequent up-regulation of Cyclin D1 and down-regulation of Bim as mediators of drug resistance. FGF2-driven drug resistance in anti-estrogen-sensitive and -resistant models, including patient-derived xenografts, was reverted by neutralizing FGF2 or FGFRs. A transcriptomic signature of FGF2 signaling in primary tumors predicted shorter recurrence-free survival independently of age, grade, stage, and FGFR amplification status. These findings delineate FGF2 signaling as a ligand-based drug resistance mechanism and highlights an underdeveloped aspect of precision oncology: characterizing and treating patients according to their TME constitution.

Topics: Animals; Apoptosis; Bcl-2-Like Protein 11; Breast Neoplasms; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cytokines; Down-Regulation; Drug Resistance, Neoplasm; Female; Fibroblast Growth Factor 2; Humans; Ligands; Mechanistic Target of Rapamycin Complex 1; Mice; Models, Biological; Molecular Targeted Therapy; Neoplasm Recurrence, Local; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Receptors, Estrogen; Receptors, Fibroblast Growth Factor; Signal Transduction; Transcriptome; Treatment Outcome; Tumor Microenvironment; Up-Regulation

Objective: To explore the possibility of a novel chemopreventive strategy for improving breast cancer treatment,\ the anticancer effects of a combination two natural compounds, Chrysin and Metformin, against T47D breast cancer\ cells were investigated. Materials and Methods: After treatment of T47D cells with Metformin, Chrysin and the two\ drugs in combination, toxicity to cancer cells was evaluated by MTT assay. Real time PCR was then used to determine\ the expression levels of hTERT and cyclin D1 genes. Results: The MTT test findings showed that the combination of\ metformin and chrysin had high synergistic effects in killing cancer cells. In addition PCR demonstrated a significant\ decrease in cyclin D1 and hTERT gene expression in the T47D breast cancer cell line. Conclusion: The conmbination\ of metformin and chrysin suppressing hTERT and cyclin D1 gene expression might offer an appropriate approach for\ breast cancer therapy.

Topics: Apoptosis; Breast Neoplasms; Cell Proliferation; Cyclin D1; Drug Combinations; Drug Synergism; Female; Flavonoids; Gene Expression Regulation, Neoplastic; Humans; Hypoglycemic Agents; Metformin; Telomerase; Tumor Cells, Cultured

Steroid sulfatase is detectable in most hormone-dependent breast cancers. STX64, an STS inhibitor, induced tumor reduction in animal assay. Despite success in phase І clinical trial, the results of phase II trial were not that significant. Breast Cancer epithelial cells (MCF-7 and T47D) were treated with two STS inhibitors (STX64 and EM1913). Cell proliferation, cell cycle, and the concentrations of estradiol and 5α-dihydrotestosterone were measured to determine the endocrinological mechanism of sulfatase inhibition. Comparisons were made with inhibitions of reductive 17β-hydroxysteroid dehydrogenases (17β-HSDs). Proliferation studies showed that DNA synthesis in cancer cells was modestly decreased (approximately 20%), accompanied by an up to 6.5% in cells in the G0/G1 phase and cyclin D1 expression reduction. The concentrations of estradiol and 5α-dihydrotestosterone were decreased by 26% and 3% respectively. However, supplementation of 5α-dihydrotestosterone produced a significant increase (approximately 35.6%) in the anti-proliferative effect of sulfatase inhibition. This study has clarified sex-hormone control by sulfatase in BC, suggesting that the different roles of estradiol and 5α-dihydrotestosterone can lead to a reduction in the effect of sulfatase inhibition when compared with 17β-HSD7 inhibition. This suggests that combined treatment of sulfatase inhibitors with 17β-HSD inhibitors such as the type7 inhibitor could hold promise for hormone-dependent breast cancer.

Topics: Aromatase Inhibitors; Breast Neoplasms; Cell Cycle; Cell Proliferation; Cyclin D1; Dihydrotestosterone; Drug Therapy, Combination; Estradiol; Estradiol Dehydrogenases; Female; Gene Expression Regulation, Neoplastic; Humans; Neoplasms, Hormone-Dependent; Steryl-Sulfatase; Sulfonic Acids; Tumor Cells, Cultured

Breast cancer is the most common cancer among women worldwide. Butyl benzyl phthalate (BBP) is ubiquitous in human's environment, and is strongly linked to breast cancer development. microRNA (miRNA) is an important regulator of target genes. So far, no studies have been reported yet to reveal the action of miRNAs in BBP-mediated breast cancer cell proliferation. In this study, we showed that BBP induced proliferation of both ER(+) MCF-7 and ER(-) MDA-MB-231 breast cancer cells, proved by increased cell viability, transition of cell cycle from G1 to S phase, upregulation of proliferating cell nuclear antigen (PCNA) and Cyclin D1, and downregulation of p21. Meanwhile, the expression of oncogenic miR-19a/b and PTEN/AKT/p21 axis was also modulated by BBP. Furthermore, for the first time we revealed that miR-19 played crucial role in the promoting effect of BBP on breast cancer cells through targeting PTEN 3'UTR. Findings from this study could provide an important new perspective on the molecular mechanisms through which BBP exerts its promoting effect on breast cancer as well as its target intervention.

Topics: 3' Untranslated Regions; Binding Sites; Breast Neoplasms; Cell Proliferation; Cell Survival; Cell Transformation, Neoplastic; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Dose-Response Relationship, Drug; Female; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; MicroRNAs; Phthalic Acids; Plasticizers; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Receptors, Estrogen; Signal Transduction; Time Factors

Topics: Blotting, Western; Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Proliferation; Cyclin D1; Endopeptidases; Female; Humans; Immunohistochemistry; Neoplasm Proteins; RNA, Messenger; Transfection; Tumor Cells, Cultured; Ubiquitin Thiolesterase

There is a growing body of data that chemotherapeutic combination strategies would be more effective in reducing drug toxicity, inhibiting tumor progression in comparison to either drug alone.. To explore a chemopreventive strategy for improving breast cancer treatment efficacy, the anticancer effects of a combination of Metformin (MET) and Silibinin (SIL) were investigated in T47D breast cancer cells.. Cytotoxicity of the drugs individually and in combination was evaluated using MTT assay. The precise nature of the interaction between MET and SIL was further analyzed through the median-effect method. In addition, qRT-PCR was applied to determine the expression levels of hTERT and cyclin D1 genes after 48 h drug exposure.. MTT assays showed that MET and SIL individually inhibited the cell viability in a dose and time-dependent manner, and the obtained combination indices (CIs) were<1 for all the combination treatments, indicating that the anticancer agents synergistically induced growth inhibition in the breast cancer cells. qPCR findings revealed that the drug combination also synergistically down-regulated the expression levels of hTERT and cyclin D1 at all used concentrations compared with the drugs used alone after 48 h treatment (P≤0.05).. The results provide evidence that synergistic antiproliferative effects of MET and SIL, linking to the down-regulation of Cyclin D1 and hTERT genes, and propose that MET+SIL may have therapeutic value in breast cancer therapy.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Drug Screening Assays, Antitumor; Drug Synergism; Female; Gene Expression Regulation, Neoplastic; Humans; Metformin; Silybin; Telomerase

Characterising the activated oncogenic signalling that leads to advanced breast cancer is of clinical importance. Here, we showed that SET domain, bifurcated 1 (SETDB1), a histone H3 lysine 9 methyltransferase, is aberrantly expressed and behaves as an oncogenic driver in breast cancer. SETDB1 enhances c-MYC and cyclin D1 expression by promoting the internal ribosome entry site (IRES)-mediated translation of MYC/CCND1 mRNA, resulting in prominent signalling of c-MYC to promote cell cycle progression, and provides a growth/self-renewal advantage to breast cancer cells. The activated c-MYC-BMI1 axis is essential for SETDB1-mediated breast tumourigenesis, because silencing of either c-MYC or BMI1 profoundly impairs the enhanced growth/colony formation conferred by SETDB1. Furthermore, c-MYC directly binds to the SETDB1 promoter region and enhances its transcription, suggesting a positive regulatory interplay between SETDB1 and c-MYC. In this study, we identified SETDB1 as a prominent oncogene and characterised the underlying mechanism whereby SETDB1 drives breast cancer, providing a therapeutic rationale for targeting SETDB1-BMI1 signalling in breast cancer. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Topics: Animals; Breast Neoplasms; Carcinogenesis; Cell Cycle; Cell Proliferation; Cyclin D1; Estrogen Receptor alpha; Gene Expression Regulation, Neoplastic; HEK293 Cells; Histone-Lysine N-Methyltransferase; Humans; MCF-7 Cells; Mice; Oncogenes; Polycomb Repressive Complex 1; Protein Methyltransferases; Proto-Oncogene Proteins c-myc; Signal Transduction; Transcriptional Activation

The peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor that plays a key role in regulating cellular metabolism, and is a therapeutic target for cancer therapy. To search for potential PPARγ activators, a compound library comprising 11 marine compounds was examined. Among them, a sterol, 3β,11-dihydroxy-9,11-secogorgost-5-en-9-one (compound

Topics: Animals; Anthozoa; Apoptosis; Autophagy; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 6; Extracellular Signal-Regulated MAP Kinases; Female; Humans; MCF-7 Cells; Phosphorylation; PPAR gamma; Reactive Oxygen Species; Sterols

Controversy exists for the use of Ki67 protein expression as a predictive marker to select patients who do or do not derive benefit from adjuvant endocrine therapy. Whether other proliferation markers, like Cyclin D1, and mitotic count can also be used to identify those estrogen receptor α (ERα) positive breast cancer patients that derive benefit from tamoxifen is not well established. We tested the predictive value of these markers for tamoxifen benefit in ERα positive postmenopausal breast cancer patients.. We collected primary tumor blocks from 563 ERα positive patients who had been randomized between tamoxifen (1 to 3 years) vs. no adjuvant therapy (IKA trial) with a median follow-up of 7.8 years. Mitotic count, Ki67 and Cyclin D1 protein expression were centrally assessed by immunohistochemistry on tissue microarrays. In addition, we tested the predictive value of CCND1 gene copy number variation using MLPA technology. Multivariate Cox proportional hazard models including interaction between marker and treatment were used to test the predictive value of these markers.. Patients with high Ki67 (≥5%) as well as low (< 5%) expressing tumors equally benefitted from adjuvant tamoxifen (adjusted hazard ratio (HR) 0.5 for both groups)(p for interaction 0.97). We did not observe a significant interaction between either Cyclin D1 or Ki67 and tamoxifen, indicating that the relative benefit from tamoxifen was not dependent on the level of these markers. Patients with tumors with low mitotic count derived substantial benefit from tamoxifen (adjusted HR 0.24, p <  0.0001), while patients with tumors with high mitotic count derived no significant benefit (adjusted HR 0.64, p = 0.14) (p for interaction 0.03).. Postmenopausal breast cancer patients with high Ki67 counts do significantly benefit from adjuvant tamoxifen, while those with high mitotic count do not. Mitotic count is a better selection marker for reduced tamoxifen benefit than Ki67.

Topics: Antineoplastic Agents, Hormonal; Breast Neoplasms; Cell Proliferation; Cluster Analysis; Cyclin D1; Female; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Ki-67 Antigen; Middle Aged; Mitosis; Postmenopause; Retrospective Studies; Tamoxifen

Epithelial-to-mesenchymal transition (EMT) plays a critical role in cancer metastasis, and is relevant to the inflammatory microenvironment. Lipopolysaccharide (LPS), a cell wall constituent of gram-negative bacteria, has been reported to induce EMT of cancer cells through TLR4 signal. We previously reported that LPS promoted metastasis of mesenchymallike breast cancer cells with high expression of cyclin D1b. However, the role of cyclin D1b in LPS-induced EMT has not been fully elucidated. In the present study, we described that cyclin D1b augmented EMT induced by LPS in MCF-7 breast cancer cells. Cyclin D1b markedly amplified integrin αvβ3 expression, which was further up-regulated under LPS stimulation. Our results showed ectopic expression of cyclin D1b promoted invasiveness of epithelial-like MCF-7 cells under LPS stimulation. Additionally, LPS-induced metastasis and EMT in MCF-7-D1b cells might depend on αvβ3 expression. Further exploration indicated that cyclin D1b cooperated with HoxD3, a transcription factor promoting αvβ3 expression, to promote LPSinduced EMT. Knockout of HoxD3 repressed LPS-induced EMT and αvβ3 over-expression in MCF-7 cells with high expression of cyclin D1b. Specifically, all these effects were in a cyclin Dla independent manner. Taken all together, LPS up-regulated integrin αvβ3 expression in MCF-7 cells with high expression of cyclin D1b and induced EMT in breast cancer cells, which highlights that cyclin D1b may act as an endogenous pathway participating in exogenous signal inducing EMT in breast cancer cells.

Topics: Alternative Splicing; Breast Neoplasms; Cell Adhesion; Cell Movement; Cyclin D1; Epithelial-Mesenchymal Transition; Female; Fibrinogen; Homeodomain Proteins; Humans; Integrin alphaVbeta3; Lipopolysaccharides; MCF-7 Cells; Neoplasm Invasiveness; Neoplasm Metastasis; Transcription Factors; Transfection; Up-Regulation

The aims of this study were to detect \ CCND1\ , \ C-MYC\ , and \ FGFR1\ amplification using qPCR, confirmation with \ FISH, and to further assess their clinicopathological relevance.. Thirty-five breast tumor samples were analyzed for amplification of the selected genes using modified SYBR \ Green qPCR. The accuracy of the qPCR was assessed by FISH as a gold-standard method.. CCND1\ , \ C-MYC\ , and \ FGFR1 \ amplifications were observed in 34.28%, 28.57%, and 17.14% of the 35 samples, respectively.\ qPCR results were significantly confirmed by FISH and qPCR and FISH showed excellent correlation (P = 0.000). \ CCND1\ amplification \ with tumor stage (P = 0.044), positive metastatic status (P = 0.042), positive family history (P = 0.042), and \ C-MYC\ status (P = 0.005); \ C-MYC\ amplification with tumor size (P = 0.021), tumor grade (P = 0.018), tumor stage (P = 0.032), and \ FGFR1\ status (P < 0.000); and \ FGFR1\ amplification with tumor size (P = 0.041) and positive \ ER\ status (P = 0.042) were statistically associated.. Our findings revealed that the applied qPCR approach could precisely quantify the relative gene copy number. More \ studies with a larger sample size are suggested to confirm the clinicopathological value of \ CCND1\ , \ C-MYC\ , and \ FGFR1\ amplification.

Topics: Adolescent; Adult; Benzothiazoles; Breast Neoplasms; Carcinoma, Ductal, Breast; Cyclin D1; Diamines; DNA-Binding Proteins; Female; Gene Amplification; Gene Dosage; Humans; In Situ Hybridization, Fluorescence; Middle Aged; Neoplasm Grading; Neoplasm Staging; Organic Chemicals; Quinolines; Real-Time Polymerase Chain Reaction; Receptor, Fibroblast Growth Factor, Type 1; Reproducibility of Results; Transcription Factors; Young Adult

Zinc is a transition metal and catalytic cofactor involved in many biological processes including proliferation, development, differentiation, and metabolism. Zinc transporters (ZnTs) play a fundamental role in cellular zinc homeostasis. ZnTs are responsible of zinc efflux and are encoded by 10 genes belonging to solute carrier family 30A (SLC30A1-10), while zinc-regulated transporter (ZRT)/iron-regulated transporter (IRT)-like protein (ZIP) transporters are responsible for the influx of zinc into the cytoplasm and are encoded by 14 genes belonging to solute carrier family 39A (SLC39A1-14). In this study, we analyzed, by transcriptome analysis, the microRNA levels of ZnT-encoding and ZIP-encoding genes in colorectal cancer (CRC) samples matched to normal colon tissues and in CRC cell lines. Results revealed an upregulation of specific ZnT and ZIP transcripts in CRC. Upregulation of SLC30A5, SLC30A6, SLC30A7 transcripts, encoding zinc efflux transporters ZnT5, ZnT6, ZnT7, localized on endoplasmic reticulum membranes, might be part of a coordinated transcriptional program associated to the increased activity of the early secretory pathway, while transcriptional upregulation of several specific ZIP transporters (SLC39A6, SLC39A7, SLC39A9, SLC39A10, and SLC39A11) could contribute in meeting the increased demand of zinc in cancer cells. Moreover, exon-level analysis of SLC30A9, a nuclear receptor coactivator involved in the transcriptional regulation of Wnt-responsive genes, revealed the differential expression of alternative transcripts in CRC and normal colonic mucosa.

Topics: Aged; Breast Neoplasms; Cation Transport Proteins; Cell Cycle Proteins; Colorectal Neoplasms; Cyclin D1; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Male; Middle Aged; Nuclear Proteins; Transcription Factors; Wnt Signaling Pathway; Zinc

Complex clusters of rearrangements are a challenge in interpretation of cancer genomes. Some clusters of rearrangements demarcate clear amplifications of driver oncogenes but others are less well understood. A detailed analysis of rearrangements within these complex clusters could reveal new insights into selection and underlying mutational mechanisms.. Here, we systematically investigate rearrangements that are densely clustered in individual tumours in a cohort of 560 breast cancers. Applying an agnostic approach, we identify 21 hotspots where clustered rearrangements recur across cancers.. Some hotspots coincide with known oncogene loci including CCND1, ERBB2, ZNF217, chr8:ZNF703/FGFR1, IGF1R, and MYC. Others contain cancer genes not typically associated with breast cancer: MCL1, PTP4A1, and MYB. Intriguingly, we identify clustered rearrangements that physically connect distant hotspots. In particular, we observe simultaneous amplification of chr8:ZNF703/FGFR1 and chr11:CCND1 where deep analysis reveals that a chr8-chr11 translocation is likely to be an early, critical, initiating event.. We present an overview of complex rearrangements in breast cancer, highlighting a potential new way for detecting drivers and revealing novel mechanistic insights into the formation of two common amplicons.

Topics: Adult; Age Distribution; Aged; Aged, 80 and over; Algorithms; Breast; Breast Neoplasms; Carrier Proteins; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 8; Cyclin D1; Datasets as Topic; Female; Gene Amplification; Genetic Loci; Genomics; Humans; Middle Aged; Oncogenes; Receptor, Fibroblast Growth Factor, Type 1; Translocation, Genetic; Whole Genome Sequencing

Genetic research provides the basis for sporadic and hereditary breast cancer diagnoses. Several mutated genes in sporadic breast cancer (eg, ERBB2 and myc) and hereditary breast cancer (eg, BRCA1 and BRCA2) influence how health care professionals assess breast cancer screenings and risks. The knowledge of these genetic mutations in the context of risk management, genetic counseling, genetic testing, and ethics might improve breast cancer treatment, prevention, and awareness.

Topics: Breast; Breast Neoplasms; Breast Self-Examination; Cell Division; Chromosomes, Human; Cortactin; Cyclin D1; Female; Fibroblast Growth Factor 3; Genes, BRCA1; Genes, BRCA2; Genetic Counseling; Genetic Predisposition to Disease; Genetic Testing; Humans; Male; Mammography; Mutation; Proto-Oncogene Proteins c-myc; Receptor, ErbB-2; Tumor Suppressor Protein p53; Tumor Virus Infections

Trefoil factor 3 (TFF3) expression is positively associated with advanced clinicopathological features of mammary carcinoma (MC). Herein, we provide evidence for a functional role of TFF3 in oncogenic transformation of immortalized, but otherwise normal human mammary epithelial cells (HMECs), namely, HMEC-hTERT, MCF10A, and MCF12A. Forced expression of TFF3 in immortalized-HMECs enhanced cell proliferation, cell survival, anchorage-independent growth, produced highly disorganised three-dimensional (3D) acinar structures and generated tumours in immunocompromised mice. Forced expression of TFF3 in immortalized-HMECs stimulated STAT3 activity that was required for TFF3-stimulated cell proliferation, survival, and anchorage-independent growth. TFF3 specifically utilised STAT3 activity to govern a transcriptional program, which was required for TFF3-stimulated oncogenic transformation of immortalized-HMECs, including transcriptional upregulation of CCND1 and BCL2. siRNA-mediated depletion or functional inhibition of STAT3 significantly inhibited the TFF3-stimulated transcription of CCND1 and BCL2 and oncogenicity in immortalized-HMECs. Furthermore, DOX-inducible expression of TFF3 in HMEC-hTERT cells also permitted anchorage-independent growth and produced disorganized acinar structures in 3D Matrigel culture. Removal of DOX-induced expression of TFF3 in HMEC-hTERT cells, previously grown with DOX, resulted in efficient normalisation of the disorganized acinar architecture and attenuated cell viability in Matrigel culture. Cumulatively, these findings suggest that TFF3 is a potent oncogene and its increased expression along with hTERT in HMECs is sufficient to produce oncogenic transformation.

Topics: Animals; Breast; Breast Neoplasms; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin D1; Doxycycline; Epithelial Cells; Female; Gene Expression Regulation, Neoplastic; Humans; Mammary Glands, Human; Mice; Proto-Oncogene Proteins c-bcl-2; STAT3 Transcription Factor; Telomerase; Trefoil Factor-3

The aim of this study was to determine whether gold nanoparticles conjugated cytotoxic protein NKCT1 (GNP-NKCT1) acted through the estrogen receptor mediated pathway in MCF-7 cells and to establish the MAPK and PI3k/Akt signal transduction pathway.. Apoptosis was done by flow cytometry. BrdU incorporation and nuclear proliferating antigen was measured by flow cytometry. Wound healing assay along with matrigel chamber invasion and migration was done. Expression of MMP9 was checked by flow cytometry and also by gelatin zymography. To analyze the regulation of signaling protein, western blot was done. MTT assay was done to evaluate the ligand receptor pathway using the estrogen receptor negative cell line (MDA-MB-231) for inhibitor effects.. The data suggested that GNP-NKCT1 induced MCF7 cell inhibition may occur through estrogen receptor pathway via inactivation of CDK4 and inactivation of PI3K/Akt, ERK1/2 and p38 MAPK signaling pathway with inhibitory effects on NF-κB, reducing the activity of MMP9. This result provides a new mechanism to explain the role of gold nanoparticles conjugated NKCT1 as a potent anti-metastatic agent in MCF7 cells.

Topics: Apoptosis; Breast Neoplasms; Cell Movement; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p21; Down-Regulation; Elapid Venoms; Female; G1 Phase Cell Cycle Checkpoints; Gold; Humans; MAP Kinase Signaling System; Matrix Metalloproteinase 9; MCF-7 Cells; Metal Nanoparticles; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nanoconjugates; Neoplasm Invasiveness; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proto-Oncogene Proteins c-akt; Receptors, Estrogen; Tamoxifen

Long non-coding RNAs (lncRNAs) function in the development and progression of cancer, but only a small portion of lncRNAs have been characterized to date. A novel lncRNA transcript, 2.53 kb in length, was identified by transcriptome sequencing analysis, and was named p53-inducible cancer-associated RNA transcript 1 (PICART1). PICART1 was found to be upregulated by p53 through a p53-binding site at -1808 to -1783 bp. In breast and colorectal cancer cells and tissues, PICART1 expression was found to be decreased. Ectopic expression of PICART1 suppressed the growth, proliferation, migration, and invasion of MCF7, MDA-MB-231 and HCT116 cells whereas silencing of PICART1 stimulated cell growth and migration. In these cells, the expression of PICART1 suppressed levels of p-AKT (Thr308 and Ser473) and p-GSK3β (Ser9), and accordingly, β-catenin, cyclin D1 and c-Myc expression were decreased, while p21Waf/cip1 expression was increased. Together these data suggest that PICART1 is a novel p53-inducible tumor-suppressor lncRNA, functioning through the AKT/GSK3β/β-catenin signaling cascade.

Topics: beta Catenin; Breast Neoplasms; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3 beta; HCT116 Cells; Humans; MCF-7 Cells; Oncogene Protein v-akt; RNA, Long Noncoding; Tumor Suppressor Protein p53

Breast cancer stem cells (CSCs) are a small population of tumour cells with the ability of self-renewal and resistance to chemotherapy. Targeting CSCs is a promising strategy for treatment of cancer. A recent study demonstrated that adenosine receptor agonists inhibit glioblastoma CSCs proliferation. At present, the effect of adenosine on breast CSCs has not been reported. Therefore, this study was designed to evaluate the effect of adenosine and its signalling pathways in breast CSCs.. Anti-proliferative effect of adenosine on breast CSCs was evaluated by mammosphere formation and MTS assay. The effect of adenosine on cell cycle progression was examined using flow cytometry. Detection of apoptosis was conducted by Annexin V-FITC. The expression levels of cell cycle and apoptosis regulatory proteins as well as ERK1/2, and GLI-1 were measured by Western blot.. Adenosine reduced CSCs population and mammosphere formation in breast CSCs. Adenosine induced G1 cell cycle arrest in breast CSCs in conjunction with a marked down-regulation of cyclin D1 and CDK4. Adenosine also induced apoptosis by regulation of Bax/Bcl-2 ratio, mitochondrial membrane potential depletion and activation of caspase-6. Moreover, adenosine inhibited ERK1/2 phosphorylation and GLI-1 protein expression.. These findings indicated that adenosine induces cell cycle arrest and apoptosis through inhibition of GLI-1 and ERK1/2 pathways in breast CSCs.

Topics: Adenosine; Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Caspase 6; CD24 Antigen; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase 4; Down-Regulation; Female; G1 Phase Cell Cycle Checkpoints; Humans; Hyaluronan Receptors; MAP Kinase Signaling System; MCF-7 Cells; Membrane Potential, Mitochondrial; Neoplastic Stem Cells; Proto-Oncogene Proteins c-bcl-2; Zinc Finger Protein GLI1

Epidemiological studies have convincingly suggested that obesity is an important risk factor for postmenopausal breast cancer, but the mechanisms responsible for this relationship are still not fully understood. We hypothesize that obesity creates a low-grade inflammatory microenvironment, which stimulates Wnt-signaling and thereby promotes the development of breast cancer. To test this hypothesis, we evaluated the correlations between expression of multiple inflammatory cytokines and Wnt pathway downstream genes in mammary tissues from women (age ≥ 50) undergoing reduction mammoplasty. Moreover, we specifically examined the role of tumor necrosis factor-α (TNF-α), an important proinflammatory cytokine associated with obesity and a possible modulator of the Wnt pathway. The regulatory effects of TNF-α on Wnt pathway targets were measured in an ex vivo culture of breast tissue treated with anti-TNF-α antibody or TNF-α recombinant protein. We found that BMI was positively associated with the secretion of inflammatory cytokines IL-1β, IL-6 and TNF-α, all of which were negatively correlated with the expression of SFRP1. The transcriptional expression of Wnt-signaling targets, AXIN2 and CYCLIN D1, were higher in mammary tissue from women with BMI ≥ 30 compared to those with BMI < 30. Our ex vivo work confirmed that TNF-α is causally linked to the up-regulation of active β-CATENIN, a key component in the Wnt pathway, and several Wnt-signaling target genes (i.e. CYCLIN D1, AXIN2, P53 and COX-2). Collectively, these findings indicate that obesity-driven inflammation elevates Wnt-signaling in mammary tissue and thereby creates a microenvironment conducive to the development of breast cancer.

Topics: Antibodies, Blocking; Axin Protein; beta Catenin; Breast Neoplasms; Cell Proliferation; Cells, Cultured; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Inflammation; Intercellular Signaling Peptides and Proteins; Interleukin-1beta; Interleukin-6; Mammary Glands, Human; Membrane Proteins; Middle Aged; Obesity; Tumor Microenvironment; Tumor Necrosis Factor-alpha; Wnt Signaling Pathway

Tamoxifen is still the most commonly used endocrine therapy drug for estrogen receptor (ER)-positive breast cancer patients and has an excellent outcome, but tamoxifen resistance remains a great impediment to successful treatment. Recent studies have prompted an anti-tumor effect of aspirin. Here, we demonstrated that aspirin not only inhibits the growth of ER-positive breast cancer cell line MCF-7, especially when combined with tamoxifen, but also has a potential function to overcome tamoxifen resistance in MCF-7/TAM. Aspirin combined with tamoxifen can down regulate cyclinD1 and block cell cycle in G0/G1 phase. Besides, tamoxifen alone represses c-myc, progesterone receptor (PR) and cyclinD1 in MCF-7 cell line but not in MCF-7/TAM, while aspirin combined with tamoxifen can inhibit the expression of these proteins in the resistant cell line. When knocking down c-myc in MCF-7/TAM, cells become more sensitive to tamoxifen, cell cycle is blocked as well, indicating that aspirin can regulate c-myc and cyclinD1 proteins to overcome tamoxifen resistance. Our study discovered a novel role of aspirin based on its anti-tumor effect, and put forward some kinds of possible mechanisms of tamoxifen resistance in ER-positive breast cancer cells, providing a new strategy for the treatment of ER-positive breast carcinoma.

Topics: Antineoplastic Agents, Hormonal; Aspirin; Biomarkers; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Drug Resistance, Neoplasm; Drug Synergism; Female; Humans; Hydroxytestosterones; Proto-Oncogene Proteins c-myc; Receptors, Estrogen; Tamoxifen

Since the incidence of breast cancer increases dramatically all over the world, the search for effective treatment is an urgent need. Metformin has demonstrated anti-tumorigenic effect both in vivo and in vitro in different cancer types. This work was designed to examine on molecular level the mode of action of metformin in mice bearing solid Ehrlich carcinoma and to evaluate the use of metformin in conjunction with doxorubicin as a combined therapy against solid Ehrlich carcinoma. Ehrlich ascites carcinoma cells were inoculated in 60 female mice as a model of breast cancer. The mice were divided into four equal groups: Control tumor, metformin, doxorubicin, and co-treatment. Metformin (15 mg/kg) and doxorubicin (4 mg/kg) were given intraperitoneally (i.p.) for four cycles every 5 days starting on day 12 of inoculation. The anti-tumorigenic effect of metformin was mediated by enhancement of adenosine monophosphate protein kinase activity and elevation of P53 protein as well as the suppression of nuclear factor-kappa B, DNA contents, and cyclin D1 gene expression. Metformin and doxorubicin mono-treatments exhibited opposing action regarding cyclin D1 gene expression, phosphorylated adenosine monophosphate protein kinase, and nuclear factor-kappa B levels. Co-treatment markedly decreased tumor volume, increased survival rate, and improved other parameters compared to doxorubicin group. In parallel, the histopathological findings demonstrated enhanced apoptosis and absence of necrosis in tumor tissue of co-treatment group. Metformin proved chemotherapeutic effect which could be mediated by the activation of adenosine monophosphate protein kinase and related pathways. Combining metformin and doxorubicin, which exhibited different mechanisms of action, produced greater efficacy as anticancer therapeutic regimen.

Topics: AMP-Activated Protein Kinases; Animals; Apoptosis; Breast Neoplasms; Carcinoma, Ehrlich Tumor; Cell Proliferation; Cyclin D1; Doxorubicin; Female; Gene Expression Regulation, Neoplastic; Humans; Mammary Neoplasms, Animal; Metformin; Mice; NF-kappa B; Signal Transduction; Tumor Suppressor Protein p53

Overexpression of Annexin A2 (Anxa2) is positively correlated with breast cancer progression, drug resistance, and poor prognosis of patients with breast cancer. Tyr23 Phosphorylation by Src-family tyrosine kinase is an important post-translational modification of Anxa2. This modification regulates the subcellular localization and functions of Anxa2 and has significant effects on cell proliferation, migration, and invasion. This study aims at revealing the association of Anxa2-Tyr23 phosphorylation in Anxa2-mediated acceleration of breast cancer progression and their elaborate molecular mechanisms.. Cell biological function experiments were performed to determine the effects of Anxa2-Tyr23 Phosphorylation on breast cancer cell proliferation and invasion in vitro and metastasis in vivo. The interaction of Tyr23 phosphorylated Anxa2 and STAT3 was verified by co-immunoprecipitation assay. Related mRNA and protein expression levels of cyclin D1 and MMP2/9 and phosphorylation level of STAT3 were detected.. Anxa2-Tyr23 phosphorylation is necessary for proliferation, invasion, and metastasis of breast cancer cells in vitro and in vivo. Tyr23 phosphorylated Anxa2 binds and enhances the sensitivity of STAT3 activation in response to IL-6, thereby increasing the protein and mRNA expression levels of cyclin D1 and MMP2/9 which are STAT3 key target genes and serve pivotal regulatory functions in cell proliferation and invasion, respectively.. Our findings further confirmed the regulatory role of Anxa2 and revealed the direct relationship between Anxa2-Tyr23 phosphorylation and activation of STAT3. Moreover, this study provides novel insights into the function of Anxa2-Tyr23 phosphorylation in signal transduction for further understanding of the mechanism through which Anxa2 promotes the progression of breast cancer.

Topics: Animals; Annexin A2; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Humans; Lung Neoplasms; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Neoplasm Invasiveness; Neoplasm Transplantation; Phosphorylation; STAT3 Transcription Factor

Topics: Animals; Apoptosis; beta Carotene; Breast Neoplasms; Cell Cycle; Cell Death; Cell Proliferation; Cyclin D1; Drug Synergism; Female; Humans; Lutein; Oxidative Stress; Penaeidae; Plant Extracts; Proto-Oncogene Proteins c-bcl-2; Spinacia oleracea

Together with estrogen receptors ERα and ERβ, the G protein-coupled estrogen receptor (GPER) mediates important pathophysiological signaling pathways induced by estrogens and is currently regarded as a promising target for ER-negative (ER-) and triple-negative (TN) breast cancer. Only a few selective GPER modulators have been reported to date, and their use in cancer cell lines has often led to contradictory results. Herein we report the application of virtual screening and cell-based studies for the identification of new chemical scaffolds with a specific antiproliferative effect against GPER-expressing breast cancer cell lines. Out of the four different scaffolds identified, 8-chloro-4-(4-chlorophenyl)pyrrolo[1,2-a]quinoxaline 14 c was found to be the most promising compound able to induce: 1) antiproliferative activity in GPER-expressing cell lines (MCF7 and SKBR3), similarly to G15; 2) no effect on cells that do not express GPER (HEK293); 3) a decrease in cyclin D1 expression; and 4) a sustained induction of cell-cycle negative regulators p53 and p21.

Topics: Antineoplastic Agents; Binding Sites; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Estrogen Receptor alpha; Estrogen Receptor beta; Female; HEK293 Cells; Humans; MCF-7 Cells; Molecular Docking Simulation; Protein Structure, Tertiary; Quinoxalines; Receptors, G-Protein-Coupled; Tumor Suppressor Protein p53

Autophagy activated after DNA damage or other stresses mitigates cellular damage by removing damaged proteins, lipids, and organelles. Activation of the master metabolic kinase AMPK enhances autophagy. Here we report that cyclin D1 restrains autophagy by modulating the activation of AMPK. In cell models of human breast cancer or in a cyclin D1-deficient model, we observed a cyclin D1-mediated reduction in AMPK activation. Mechanistic investigations showed that cyclin D1 inhibited mitochondrial function, promoted glycolysis, and reduced activation of AMPK (pT172), possibly through a mechanism that involves cyclin D1-Cdk4/Cdk6 phosphorylation of LKB1. Our findings suggest how AMPK activation by cyclin D1 may couple cell proliferation to energy homeostasis.

Topics: 3T3 Cells; AMP-Activated Protein Kinase Kinases; AMP-Activated Protein Kinases; Animals; Autophagy; Breast Neoplasms; Cell Proliferation; Cyclin D1; Female; Humans; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Protein Serine-Threonine Kinases; Signal Transduction; Transfection

Cyclin D1 has a central role in cell cycle control and is an important component of estrogen regulation of cell cycle progression. We have previously shown that high cyclin D expression is related to aggressive features of ER-positive but not ER-negative breast cancer. The aims of the present study were to validate this differential ER-related effect and furthermore explore the relationship between cyclin D overexpression and CCND1 gene amplification status in a node-negative breast cancer case-control study.. Immunohistochemical nuclear expression of cyclin D1 (n = 364) and amplification of the gene CCND1 by fluorescent in situ hybridization (n = 255) was performed on tissue microarray sections from patients with T1-2N0M0 breast cancer. Patients given adjuvant chemotherapy were excluded. The primary event was defined as breast cancer death. Breast cancer-specific survival was analyzed in univariate and multivariable models using conditional logistic regression.. Expression of cyclin D1 above the median (61.7%) in ER breast cancer was associated with an increased risk for breast cancer death (OR 3.2 95% CI 1.5-6.8) also when adjusted for tumor size and grade (OR 3.1). No significant prognostic impact of cyclin D1 expression was found among ER-negative cases. Cyclin D1 overexpression was significantly associated to high expression of the proliferation markers cyclins A (ρ 0.19, p = 0.006) and B (ρ 0.18, p = 0.003) in ER-positive tumors, but not in ER-negative cases. There was a significant association between CCND1 amplification and cyclin D1 expression (p = 0.003), but CCND1 amplification was not statistically significantly prognostic (HR 1.4, 95% CI 0.4-4.4).. We confirmed our previous observation that high cyclin D1 expression is associated to high proliferation and a threefold higher risk of death from breast cancer in ER-positive breast cancer.

Topics: Adult; Aged; Aged, 80 and over; Breast Neoplasms; Cell Proliferation; Cyclin A; Cyclin B; Cyclin D1; Female; Gene Amplification; Gene Expression Regulation, Neoplastic; Humans; Middle Aged; Neoplasm Staging; Receptors, Estrogen; Tissue Array Analysis; Tumor Burden; Up-Regulation

Total choline (tCho) was documented as a biomarker for breast cancer diagnosis by in vivo MRS. To understand the molecular mechanisms behind elevated tCho in breast cancer, an association of tCho with β-catenin and cyclin D1 was evaluated. Hundred fractions from 20 malignant, 10 benign and 20 non-involved breast tissues were isolated. Cytosolic and nuclear expressions of β-catenin and cyclin D1 were estimated using ELISA. Higher tCho was seen in malignant compared to benign tissues. Malignant tissues showed higher cytosolic and nuclear β-catenin expressions than benign and non-involved tissues. Within malignant tissues, β-catenin and cyclin D1 expressions were higher in the nucleus than cytosol. Cyclin D1 expression was higher in the cytosolic fractions of benign and non-involved than malignant tissues. Furthermore, in malignant tissues, tCho showed a positive correlation with the cytosolic and nuclear expression of β-catenin and cyclin D1 and also a correlation between nuclear expressions of both these proteins was seen. Higher cytosolic β-catenin expression was seen in progesterone receptor negative than positive patients. Results provide an evidence of correlation between non-invasive biomarker, tCho and the Wnt/β-catenin pathway. The findings explain the molecular mechanism of tCho elevation which may facilitate exploration of additional therapeutic targets for breast cancer.

Topics: Adult; Aged; beta Catenin; Breast Neoplasms; Choline; Cyclin D1; Enzyme-Linked Immunosorbent Assay; Female; Gene Expression; Humans; Magnetic Resonance Spectroscopy; Middle Aged; Models, Biological; Neoplasm Staging; Pilot Projects; Receptors, Cell Surface; Signal Transduction

Topics: Antineoplastic Agents, Phytogenic; Biosensing Techniques; Breast Neoplasms; Cerium; Cyclin D1; Drugs, Chinese Herbal; Electrodes; Electroplating; Female; Ferrosoferric Oxide; Humans; Limit of Detection; Luminescent Measurements; MCF-7 Cells; Nanocomposites; Quantum Theory; Silver; Up-Regulation

IQ-domain GTPase-activating protein 1 is a scaffolding protein with multidomain which plays a role in modulating dishevelled (Dvl) nuclear translocation in canonical Wnt pathway. However, the biological function and mechanism of IQ-domain GTPase-activating protein 1 in invasive ductal carcinoma (IDC) remain unknown. In this study, we found that IQ-domain GTPase-activating protein 1 expression was elevated in invasive ductal carcinoma, which was positively correlated with tumor grade, lymphatic metastasis, and poor prognosis. Coexpression of IQ-domain GTPase-activating protein 1 and Dvl in the nucleus and cytoplasm of invasive ductal carcinoma was significantly correlated but not in the membrane. Postoperative survival in the patients with their coexpression in the nucleus and cytoplasm was obviously lower than that without coexpression. The positive expression rates of c-myc and cyclin D1 were significantly higher in the patients with nuclear coexpression of Dvl and IQ-domain GTPase-activating protein 1 than that with cytoplasmic coexpression, correlating with poor prognosis. IQ-domain GTPase-activating protein 1 significantly enhanced cell proliferation and invasion in invasive ductal carcinoma cell lines by interacting with Dvl in cytoplasm to promote Dvl nuclear translocation so as to upregulate the expression of c-myc and cyclin D1. Collectively, our data suggest that IQ-domain GTPase-activating protein 1 may promote the malignant phenotype of invasive ductal carcinoma via canonical Wnt signaling, and it could be used as a potential prognostic biomarker for breast cancer patients.

Topics: Adult; Aged; beta Catenin; Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Ductal; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Lymphatic Metastasis; Middle Aged; Neoplasm Invasiveness; Prognosis; Proto-Oncogene Proteins c-myc; ras GTPase-Activating Proteins; Survival Analysis; Wnt Signaling Pathway

Noxin (also called chromosome 11 open reading frame 82 or DNA damage-induced apoptosis suppressor) is associated with anti-apoptosis and cell proliferation in response to stress signals. However, to our knowledge, the role of Noxin in regulating cell proliferation is still controversial and there are no reports of the function and clinicopathological association in breast cancer. In this study, immunohistochemistry results showed that Noxin expression was significantly correlated with advanced tumor-node-metastasis stage ( p = 0.027), positive regional lymph node metastasis ( p = 0.002), and poor overall survival ( p = 0.002). Proliferation assay results showed that Noxin obviously promoted the ability of proliferation of normal breast cells. Subsequent western blot results revealed that Cyclin D1 and Cyclin E1 were upregulated by overexpressing Noxin, whereas Cyclin D1 and Cyclin E1 were downregulated after depleting Noxin. The levels of phosphorylated P38 and activating transcription factor 2 were obviously increased after overexpressing Noxin, and their expression was downregulated accordingly by transfecting Noxin-small interfering RNA. Moreover, P38 inhibitor counteracted the elevating expression of phosphorylated activating transcription factor 2, Cyclin D1, and Cyclin E1 induced by Noxin overexpression and thereby reversed the effect of Noxin overexpression on facilitating cell growth. Taken together, our studies indicated that Noxin was overexpressed in breast cancer and its positive expression was significantly correlated with advance tumor-node-metastasis stage, positive lymph node metastasis, and poor prognosis. Noxin facilitated the expression of Cyclin D1 and Cyclin E1 through activating P38-activating transcription factor 2 signaling pathway, thus enhanced cell growth of breast cancer.

Topics: Activating Transcription Factor 2; Adult; Apoptosis; Apoptosis Regulatory Proteins; Breast Neoplasms; Cell Cycle; Cell Cycle Proteins; Cell Proliferation; Cyclin D1; Cyclin E; Female; Gene Expression Regulation, Neoplastic; Humans; Lymphatic Metastasis; MCF-7 Cells; Middle Aged; Neoplasm Staging; Oncogene Proteins; p38 Mitogen-Activated Protein Kinases; RNA, Small Interfering; Signal Transduction

Our previous study reported several alternative splicing variants of arginine N-methyltransferase 2 (PRMT2), which lose different exons in the C-terminals of the wild-type PRMT2 gene. Particularly, due to frame-shifting, PRMT2β encodes a novel amino acid sequence at the C-terminus of the protein, the function of which is not understood. In the present study, we determined the role of PRMT2β in breast cancer cell proliferation, apoptosis and its effect on the Akt signaling pathway. Stable breast cancer MCF7 cell line with lentivirus-mediated PRMT2β overexpression was obtained after selection by puromycin for 2 weeks. The effect of lentivirus-mediated PRMT2β overexpression on breast cancer cellular oncogenic properties was evaluated by MTT, colony formation, cell cycle analysis and apoptosis assays in MCF7 cells. Luciferase activity assay and western blot analysis were performed to characterize the effects of PRMT2β on cyclin D1 promoter activities and the Akt signaling pathway. Tissue microarray was performed to investigate the association of PRMT2β with breast cancer progression. Lentivirus-mediated PRMT2β overexpression suppressed the cell proliferation and colony formation of breast cancer MCF7 cells. PRMT2β overexpression induced cell cycle arrest and apoptosis of MCF7 cells. Furthermore, PRMT2β was revealed to suppress the transcription activity of the cyclin D1 promoter, and PRMT2β was also found to inhibit cyclin D1 expression via the suppression of Akt/GSK-3β signaling in breast cancer cells. Clinically, it was revealed that PRMT2β expression was negatively correlated with human epidermal growth factor receptor 2 (HER2) (p=0.033) in breast tumors. Our results revealed that PRMT2β, a novel splice variant of PRMT2, plays potential antitumor effect by suppressing cyclin D1 expression and inhibiting Akt signaling activity. This also opens a new avenue for treating breast cancer.

Topics: Alternative Splicing; Apoptosis; Biomarkers, Tumor; Breast Neoplasms; Case-Control Studies; Cell Proliferation; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Intracellular Signaling Peptides and Proteins; Prognosis; Protein Isoforms; Protein-Arginine N-Methyltransferases; Receptor, ErbB-2; Tumor Cells, Cultured

Acquisition of resistance to paclitaxel is one of the most important problems in treatment of breast cancer patients, but the molecular mechanisms underlying sensitivity to paclitaxel remains elusive. Emerging evidence has demonstrated that microRNAs (miRNAs) play important roles in regulation of cell growth, migration and invasion through inhibiting the expression of its target genes. In our previous studies, we have shown that microRNA-335 (miR‑335) decreased obviously between paclitaxel-resistant (PR) and parental breast cancer cells through miRNA microarray. However, the roles of miR‑335 in breast cancer progression and metastasis are still largely unknown. NT21MP was designed and synthesized as an antagonist with CXCR4 to inhibit cellular proliferation and induce apoptosis. Therefore, the aim of this study was to explore the underlying mechanism of miR‑335 and NT21MP in reverse PR in breast cancer cells. In this study, we found that miR‑335 expression is significantly lower in PR MCF‑7 and SKBR-3 cells (MCF‑7/PR and SKBR-3/PR) compared with their parental MCF‑7 and SKBR-3 cells. Functional experiments showed that overexpression of miR‑335 and NT21MP increased the number of apoptosis cells, arrested cells in G0/G1 phase transition, and suppressed cell migration and invasion in vitro. Dual luciferase assays revealed that SETD8 is a direct target gene of miR‑335. Furthermore, miR‑335 markedly inhibited expression of SETD8 via Wnt/β‑catenin signaling and subsequently inhibited the expression of its downstream genes cyclin D1, and c‑Myc. Additionally, ectopic expression of miR‑335 or depletion of its target gene SETD8 could enhance the sensitivity of PR cells to paclitaxel. Taken together, these date elucidated that NT21MP and miR‑335 mediated PR of breast cancer cells partly through regulation of Wnt/β‑catenin signaling pathway. Activation of miR‑335 or inactivation of SETD8 could be a novel approach for the treatment of breast cancer.

Topics: Apoptosis; Breast Neoplasms; Cell Movement; Cell Proliferation; Chemokines; Cyclin D1; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Histone-Lysine N-Methyltransferase; Humans; MCF-7 Cells; MicroRNAs; Paclitaxel; Peptides; Receptors, CXCR4

Breast cancer risk is strongly associated with an intergenic region on 11q13. We have previously shown that the strongest risk-associated SNPs fall within a distal enhancer that regulates CCND1. Here, we report that, in addition to regulating CCND1, this enhancer regulates two estrogen-regulated long noncoding RNAs, CUPID1 and CUPID2. We provide evidence that the risk-associated SNPs are associated with reduced chromatin looping between the enhancer and the CUPID1 and CUPID2 bidirectional promoter. We further show that CUPID1 and CUPID2 are predominantly expressed in hormone-receptor-positive breast tumors and play a role in modulating pathway choice for the repair of double-strand breaks. These data reveal a mechanism for the involvement of this region in breast cancer.

Topics: Breast Neoplasms; Cell Line, Tumor; Chromatin; Chromosomes, Human, Pair 11; Cyclin D1; DNA Breaks, Double-Stranded; DNA Damage; DNA Repair; Enhancer Elements, Genetic; Estrogens; Female; Gene Expression Regulation, Neoplastic; Genetic Predisposition to Disease; Humans; MCF-7 Cells; Polymorphism, Single Nucleotide; Promoter Regions, Genetic; RNA Interference; RNA, Guide, Kinetoplastida; RNA, Long Noncoding; RNA, Small Interfering

Recent studies have identified many long non-coding RNAs (lncRNAs) with critical roles in various biological processes including tumorigenesis. Taurine-upregulated gene 1 (TUG1), is an lncRNA recently reported to be involved in the progression of several human cancers. This study aimed to investigate the clinical significance and biological functions of TUG1 in breast cancer. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to measure TUG1 expression in cells from breast cancer cell lines and in 58 matched pairs of breast cancer and normal tissue samples from patients with clinicopathological comparisons. Gain-and loss-of-function experiments were performed in vitro to investigate the biological role of TUG1. TUG1 expression was significantly downregulated in both breast cancer tissues and cell lines compared to controls, and low TUG1 expression was significantly correlated with mutant p53 expression (p=0.037) and lymph node metastasis (p=0.044). In vitro experiments revealed that TUG1 overexpression significantly suppressed cell proliferation by causing cell cycle arrest and inducing apoptosis in breast cancer cells, while TUG1 knockdown caused increased cell growth via promoting cell cycle progression and regulating the expression of cyclinD1 and CDK4. Further functional assays indicated that TUG1 overexpression significantly promoted cell migration and invasion while TUG1 knockdown had the opposite effects. Our findings indicate that the lncRNA TUG1 is a tumor suppressor in breast cancer, and may serve as a novel prognostic biomarker and potential therapeutic target for patients with breast cancer.

Topics: Apoptosis; Breast Neoplasms; Case-Control Studies; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Disease Progression; Down-Regulation; Female; Gene Knockdown Techniques; Humans; Middle Aged; Neoplasm Invasiveness; RNA, Long Noncoding

Abnormal proliferation is significantly associated with the promotion of malignant tumor. Growing evidence suggest that the signal pathways of p21. We evaluated PAK5 and p65 staining in breast cancer tissues (BCTs) and paired non-cancerous tissues (NTs) using tissue microarray (TMA) technology. The functions of PAK5 were studied in vitro and in vivo. Cell Counting Kit-8 (CCK-8) and flow cytometry were performed to determine proliferation of breast cancer cells. Phosphorylation assay and co-immunoprecipitation (co-IP) were employed to identify the regulation mechanism of p65 by PAK5. The activation of Cyclin D1 promoter was measured with luciferase reporter assay. Xenograft models in nude mice were established to explore the roles of PAK5 in breast cancer growth.. In this study, we show that PAK5 is highly expressed in breast cancer tissues and the increased PAK5 is significantly associated with breast cancer progression. Overexpression of PAK5 promotes the proliferation and cell-cycle progression by increasing the expression of Cyclin D1 in vitro and in vivo. Mechanistic studies demonstrated that PAK5 can promote the phosphorylation and the nuclear translocation of p65 subunit of nuclear factor-kappaB (NF-κB). Furthermore, p65 can directly bind to the promoter of Cyclin D1 and mediate an increase in its protein expression.. Taken together, our findings suggest that PAK5 may serve as a potential prognosis marker and therapeutic target for human breast cancer.

Topics: Animals; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Disease Models, Animal; Female; Follow-Up Studies; Gene Expression; Genes, Reporter; Heterografts; Humans; Immunohistochemistry; Mice; Neoplasm Staging; p21-Activated Kinases; Phosphorylation; Prognosis; Promoter Regions, Genetic; Protein Transport; Signal Transduction; Transcription Factor RelA; Transcriptional Activation; Tumor Burden

Estrogen receptor (ER) plays important roles in cell growth, development and tumorigenesis. Although ER-regulated genes have been extensively investigated, little is known about roles of ER-regulated lncRNAs in breast cancer. Here, we conducted genome-wide study of ER-regulated lncRNAs by using RNA-seq, ChIP-seq and TCGA data. A total of identified 114 ER-regulated lncRNAs were identified, many of them were overexpressed in ER+ breast cancer and co-expressed with some key regulators. Silencing one of most prominent lncRNA, AP000439.3, resulted in inhibition of cell cycle progression and proliferation. Further study revealed AP000439.3 can regulate expression of CCND1 through enhancing estrogen receptor induction of CCND1. This finding revealed lncRNAs may serve as important effectors of ER in regulation of gene expression and cell phenotype in breast cancer.

Topics: Breast Neoplasms; Carcinogenesis; Cell Cycle; Cell Proliferation; Cyclin D1; Estrogen Receptor alpha; Estrogens; Female; Gene Expression Regulation, Neoplastic; Genome, Human; High-Throughput Nucleotide Sequencing; Humans; MCF-7 Cells; RNA, Long Noncoding

In this study, resveratrol-loaded solid lipid nanoparticles (Res-SLNs) were successfully designed to treat MDA-MB-231 cells. The Res-SLNs were prepared using emulsification and low-temperature solidification method. The Res-SLNs were spherical, with small size, negative charge, and narrow size distribution. Compared with free resveratrol, the Res-SLNs displayed a superior ability in inhibiting the proliferation of MDA-MB-231 cells. In addition, Res-SLNs exhibited much stronger inhibitory effects on the invasion and migration of MDA-MB-231 cells. Western blot analysis revealed that Res-SLNs could promote the ratio of Bax/Bcl-2 but decreased the expression of cyclinD1 and c-Myc. These results indicate that the Res-SLN may have great potential for breast cancer treatment.

Topics: Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Lipids; Nanoparticles; Particle Size; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; Resveratrol; Stilbenes

The oncogenic capacity of cyclin D1 has long been established in breast cancer. CCND1 amplification has been identified in a subset of patients with poor prognosis, but there are conflicting data regarding the predictive value of cyclin D1 protein overexpression. This study was designed to analyze the expression of cyclin D1 and its correlation with CCND1 amplification and their prognostic implications in invasive breast cancer. By using the tissue microarray technique, we performed an immunohistochemical study of ER, PR, HER2, p53, cyclin D1, Ki67 and p16 in 179 invasive breast carcinoma cases. The FISH method was performed to detect HER2/Neu and CCND1 amplification. High cyclin D1 expression was identified in 94/179 (52%) of invasive breast cancers. Cyclin D1 overexpression and CCND1 amplification were significantly associated (p = 0.010). Overexpression of cyclin D1 correlated with ER expression, PR expression and Luminal subtypes (p<0.001), with a favorable impact on overall survival in the whole series. However, in the Luminal A group, high expression of cyclin D1 correlated with shorter disease-free survival, suggesting that the prognostic role of cyclin D1 depends on the molecular subtype. CCND1 gene amplification was detected in 17 cases (9%) and correlated significantly with high tumor grade (p = 0.038), high Ki-67 protein expression (p = 0.002), and the Luminal B subtype (p = 0.002). Patients with tumors with high amplification of CCND1 had an increased risk of recurrence (HR = 2.5; 95% CI, 1.2-4.9, p = 0.01). These findings suggest that CCND1 amplification could be useful for predicting recurrence in invasive breast cancer.

Topics: Breast Neoplasms; Cyclin D1; Gene Amplification; Humans; In Situ Hybridization, Fluorescence; Ki-67 Antigen; Neoplasm Invasiveness; Prognosis; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone; Tumor Suppressor Protein p53

ESE-1/Elf3 controls transformation properties in mammary epithelial cells, and is most clinically relevant in HER2. We used cell proliferation, clonogenicity, viability, and soft agar assays to measure the effects of ESE-1 knockdown in cell lines.. ESE-1 knockdown in the resistant cell lines inhibited HER2 and other downstream effectors in a cell-type specific manner, but caused down-regulation of pAkt and cyclin D1 in both sublines. In parental BT474 and SKBR3 ESE-1 silencing revealed a potent anti-proliferative effect that mimics the trastuzumab-mediated growth inhibition but did not enhance trastuzumab sensitivity in the resistant sublines.. This study provides rationale to study ESE-1 as a novel mean to treat HER2

Topics: Antineoplastic Agents, Immunological; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; DNA-Binding Proteins; Drug Resistance, Neoplasm; Female; Humans; Immunoblotting; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-ets; Receptor, ErbB-2; RNA Interference; Transcription Factors; Trastuzumab

A growing amount of evidence has indicated that PSAT1 is an oncogene that plays an important role in cancer progression and metastasis. In this study, we explored the expression and function of PSAT1 in estrogen receptor (ER)-negative breast cancer.. The expression level of PSAT1 in breast cancer tissues and cells was analyzed using real-time-PCR (RT-PCR), TCGA datasets or immunohistochemistry (IHC). The overall survival of patients with ER-negative breast cancer stratified by the PSAT1 expression levels was evaluated using Kaplan-Meier analysis. The function of PSAT1 was analyzed using a series of in vitro assays. Moreover, a nude mouse model was used to evaluate the function of PSAT1 in vivo. qRT-PCR and western blot assays were used to evaluate gene and protein expression, respectively, in the indicated cells. In addition, we demonstrated that PSAT1 was activated by ATF4 by chromatin immunoprecipitation (ChIP) assays.. mRNA expression of PSAT1 was up-regulated in ER-negative breast cancer. A tissue microarray that included 297 specimens of ER-negative breast cancer was subjected to an immunohistochemistry assay, which demonstrated that PSAT1 was overexpressed and predicted a poor clinical outcome of patients with this disease. Our data showed that PSAT1 promoted cell proliferation and tumorigenesis in vitro and in vivo. We further found that PSAT1 induced up-regulation of cyclin D1 via the GSK3β/β-catenin pathway, which eventually led to the acceleration of cell cycle progression. Furthermore, ATF4 was also overexpressed in ER-negative breast cancers, and a positive correlation between the ATF4 and PSAT1 mRNA levels was observed in ER-negative breast cancers. We further demonstrated that knockdown of ATF4 by siRNA reduced PSAT1 expression. Finally, chromatin immunoprecipitation (ChIP) assays showed that PSAT1 was a target of ATF4.. PSAT1, which is overexpressed in ER-negative breast cancers, is activated by ATF4 and promotes cell cycle progression via regulation of the GSK3β/β-catenin/cyclin D1 pathway.

Topics: Activating Transcription Factor 4; Animals; beta Catenin; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Glycogen Synthase Kinase 3 beta; Heterografts; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Receptors, Estrogen; RNA, Messenger; Signal Transduction; Transaminases

Breast carcinoma proliferative activity, histological grade and commercial molecular tests are all important in prognostication and treatment. There is a particular need for improved, standardised techniques for subclassification of grade 2 breast cancers into low-risk and high-risk prognostic groups. In this study we investigated whether gene expression profiling of five proliferation genes was feasible using breast cancer tissue in a clinical setting and whether these profiles could enhance pathological assessment.. Expression of five proliferation gene mRNAs; Ki-67, STK 15, CCNB1, CCND1 and MYBL2, was quantified in 27 breast carcinomas and compared with Ki-67 proliferation index (PI) and Nottingham mitotic score.. Expression of Ki-67, STK15 and MYBL2 mRNA showed moderate Spearman's correlation with Ki-67 PI (p<0.01), but CCND1 and CCNB1 showed weak, non-significant correlation. Individual gene expression did not associate with mitotic score but combined mRNA expression correlated with both Ki-67 PI (p=0.018) and mitotic score (p=0.03; 0.007).. This study confirms mRNA analysis in breast carcinoma formalin-fixed, paraffin-embedded samples is feasible and suggests gene expression profiling, using a small set of five proliferation genes, has potential in aiding histological grading or assessment of proliferative activity of breast cancers. To fully evaluate the clinical applicability of this approach, a larger cohort study with long-term follow-up data is required.

Topics: Aurora Kinase A; Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Cycle Proteins; Cyclin B1; Cyclin D1; Feasibility Studies; Female; Formaldehyde; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Ki-67 Antigen; Mitotic Index; Paraffin Embedding; Prognosis; Tissue Fixation; Trans-Activators

Fludioxonil is an antifungal agent used in agricultural applications that is present at measurable amounts in fruits and vegetables. In this study, the effects of fludioxonil on cancer cell viability, epithelial-mesenchymal transition (EMT), and metastasis were examined in MCF-7 clonal variant breast cancer cell (MCF-7 CV cells) with estrogen receptors (ERs). MCF-7 CV cells were cultured with 0.1% DMSO (control), 17β-estradiol (E2; 1 ×10

Topics: Animals; Antigens, CD; Antineoplastic Agents; Breast Neoplasms; Cadherins; Cathepsin D; Cell Movement; Cell Proliferation; Cell Shape; Cyclin D1; Dioxoles; Epithelial-Mesenchymal Transition; Female; Humans; MCF-7 Cells; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Metastasis; Pyrroles; Receptors, Estrogen; Signal Transduction; Tumor Burden; Xenograft Model Antitumor Assays

Molecular networks governing responses to targeted therapies in cancer cells are complex dynamic systems that demonstrate nonintuitive behaviors. We applied a novel computational strategy to infer probabilistic causal relationships between network components based on gene expression. We constructed a model comprised of an ensemble of networks using multidimensional data from cell line models of cell-cycle arrest caused by inhibition of MEK1/2. Through simulation of a reverse-engineered Bayesian network model, we generated predictions of G

Topics: Bayes Theorem; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Survival; Cyclin D1; Female; Humans; Intracellular Signaling Peptides and Proteins; Mitogen-Activated Protein Kinase Kinases; NF-kappa B; Phosphatidylinositol 3-Kinases; Promoter Regions, Genetic; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction

Intrinsic or acquired drug resistance is a major impediment to the successful treatment of women with breast cancer using chemotherapy. We have observed that MCF-7 breast tumor cells selected for resistance to doxorubicin or epirubicin (MCF-7DOX2 and MCF-7EPI cells, respectively) exhibited increased expression of several members of the aldo-keto reductase (AKR) gene family (in particular AKR1C3 and AKR1B10) relative to control MCF-7CC cells selected by propagation in the absence of drug. Normal cellular roles for the AKRs include the promotion of estrogen (E2) synthesis from estrone (E1) and the hydroxylation and detoxification of exogenous xenobiotics such as anthracycline chemotherapy drugs. While hydroxylation of anthracyclines strongly attenuates their cytotoxicity, it is unclear whether the enhanced AKR expression in the above anthracycline-resistant cells promotes E2 synthesis and/or alterations in E2 signalling pathways and whether such changes contribute to enhanced survival and anthracycline resistance. To determine the role of AKRs and E2 pathways in doxorubicin resistance, we examined changes in the expression of E2-related genes and proteins upon acquisition of doxorubicin resistance. We also assessed the effects of AKR overexpression or downregulation or the effects of activators or inhibitors of E2-dependent pathways on previously acquired resistance to doxorubicin. In this study we observed that the enhanced AKR expression upon acquisition of anthracycline resistance was, in fact, associated with enhanced E2 production. However, the expression of estrogen receptor α (ERα) was reduced by 2- to 5-fold at the gene transcript level and 2- to 20-fold at the protein level upon acquisition of anthracycline resistance. This was accompanied by an even stronger reduction in ERα phosphorylation and activity, including highly suppressed expression of two proteins under E2-dependent control (Bcl-2 and cyclin D1). The diminished Bcl-2 and cyclin D1 expression would be expected to reduce the growth rate of the cells, a hypothesis which was confirmed in subsequent cell proliferation experiments. AKR1C3 or AKR1B10 overexpression alone had no effect on doxorubicin sensitivity in MCF-7CC cells, while siRNA-mediated knockdown of AKR1C3 and/or AKR1B10 expression had no significant effect on sensitivity to doxorubicin in MCF-7DOX2 or MCF-7EPI cells. This suggested that enhanced or reduced AKR expression/activity is insufficient to confer anthracycline resistance

Topics: 3-Hydroxysteroid Dehydrogenases; Aldehyde Reductase; Aldo-Keto Reductase Family 1 Member C3; Aldo-Keto Reductases; Breast Neoplasms; Cyclin D1; Doxorubicin; Drug Resistance, Neoplasm; Estrogens; Female; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Hydroxyprostaglandin Dehydrogenases; Proto-Oncogene Proteins c-bcl-2; Signal Transduction

The purpose of this study was to elucidate the mechanisms associated with the specific effects of AKT1 and AKT2 isoforms in breast cancer progression. We modulated the abundance of specific AKT isoforms in IBH-6 and T47D human breast cancer cell lines and showed that AKT1 promoted cell proliferation, through S6 and cyclin D1 upregulation, but it inhibited cell migration and invasion through β1-integrin and focal adhesion kinase (FAK) downregulation. In contrast, AKT2 promoted cell migration and invasion through F-actin and vimentin induction. Thus, while overexpression of AKT1 promoted local tumor growth, downregulation of AKT1 or overexpression of AKT2 promoted peritumoral invasion and lung metastasis. Furthermore, we evaluated The Cancer Genome Atlas (TCGA) dataset for invasive breast carcinomas and found that increased AKT2 but not AKT1 mRNA levels correlated with a worse clinical outcome. We conclude that AKT isoforms play specific roles in different steps of breast cancer progression, with AKT1 involved in the local tumor growth and AKT2 involved in the distant tumor dissemination, having AKT2 a poorer prognostic value and consequently being a worthwhile target for therapy.

Topics: Actins; Animals; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Disease Progression; Female; Gene Expression Regulation, Neoplastic; Humans; Mice, Inbred NOD; Mice, Knockout; Mice, SCID; Neoplasm Invasiveness; Proto-Oncogene Proteins c-akt; Survival Analysis; Transplantation, Heterologous

Cyclin D1 (CCND1) gene amplification is a molecular key alteration in breast cancer and was suggested to predict resistance to antihormonal therapy. As tissue heterogeneity may affect diagnostic accuracy of predictive biomarkers, CCND1 genetic heterogeneity was assessed in this study. A novel tissue microarray (TMA) platform was manufactured for this purpose.. Primary breast carcinomas from 147 patients were sampled in a "heterogeneity-TMA" by taking eight different tissue cores from 4 to 8 tumor-containing blocks per case. Additional tissue samples were taken from 1 to 4 corresponding nodal metastases in 35 of these patients. CCND1 amplification was assessed by fluorescence in situ hybridization (FISH).. CCND1 amplification was seen in 28 of 133 (21.05 %) informative patients. Amplification was significantly associated with high tumor grade (p = 0.042), but unrelated to tumor type (p = 0.307), stage (p = 0.540) and ER (p = 0.061) or PR (p = 0.871) expression. A discordant Cyclin D1 amplification status was detected in 6 out of 28 (21.43 %) amplified tumors by heterogeneity-TMA analysis. Re-testing on large sections revealed three patients with true heterogeneity of high-level CCND1 amplification and another three patients with variable interpretation of borderline FISH ratios ranging between 1.7 and 2.3. No discrepancies were detected between 22 primary tumors and their matched lymph node metastases.. The high degree of homogeneity seen for CCND1 amplification suggests that this alteration is an early event in the development of a small subset of breast cancers.

Topics: Breast Neoplasms; Carcinoma, Lobular; Cyclin D1; Female; Gene Amplification; Humans; In Situ Hybridization, Fluorescence; Neoplasm Grading; Neoplasm Staging; Tissue Array Analysis

A subset of patients with ductal carcinoma in situ (DCIS) experience recurrence or progression to invasive cancer. Current clinical practice is not reliably guided by DCIS recurrence prediction, although recurrence risk for invasive breast cancer can now be assessed. We analyzed a panel of biomarkers (estrogen receptor, Her2, Ki67, p53, cyclin D1, COX-2, caveolin-1, survivin, and PPAR-γ) and DCIS histologic and clinical features to determine associations with DCIS recurrence.. Seventy DCIS cases diagnosed between 1995 and 2010 were divided into 2 groups: 52 had DCIS without known recurrence after excision and 18 had DCIS with subsequent recurrence after excision as DCIS or invasive carcinoma in the ipsilateral or contralateral breast. Tissue microarrays were prepared, immunohistochemistry performed, and expression of the biomarkers scored semiquantitatively. Variables analyzed included age, tumor size, margin status, DCIS grade, necrosis, histologic type, and immunohistochemistry scores. Differences between groups were evaluated using t tests for continuous variables and Fisher exact tests for categorical variables.. Intraductal necrosis was associated with increased recurrence risk: 46% of nonrecurrent cases showed necrosis compared with 83% of those who recurred (P=0.007). Her2 (human epidermal growth factor receptor 2) and Ki67 expression distributions were significantly different between nonrecurrent and recurrent cases. Her2 was overexpressed in 14% of nonrecurrent cases compared with 50% in the recurrent cases (P=0.03). A total of 87% of nonrecurrent cases had low Ki67 staining (0% to 10%) compared with 50% among the recurrent cases (P=0.002).. Our results suggest that Her2 and Ki67 immunohistochemistry and the presence of intraductal necrosis aid in DCIS risk stratification.

Topics: Adult; Aged; Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Caveolin 1; Cyclin D1; Cyclooxygenase 2; Estrogen Receptor alpha; Female; Gene Expression; Humans; Inhibitor of Apoptosis Proteins; Ki-67 Antigen; Middle Aged; Necrosis; PPAR gamma; Prognosis; Receptor, ErbB-2; Recurrence; Retrospective Studies; Risk Factors; Survivin; Treatment Outcome; Tumor Suppressor Protein p53

The Cyclin D1 protein has been extensively studied over the last decades, for its various roles in physiological processes, both in normal and cancer cells. Gene amplifications and overexpression of CCND1 are frequently reported in several types of cancers, including breast carcinomas, showing the increasing relevance of Cyclin D1 in tumorigenesis. Little is known about the role of this protein in the metastatic process, and the main objective of this study was to evaluate the importance of the CCND1 as a potential marker of tumor progression in breast carcinomas, in a sample collected in Southern Brazil. We studied 41 samples of formalin-fixed paraffin-embedded tissue sections from invasive ductal breast carcinomas subdivided into metastatic (n = 19) and non-metastatic (n = 22) tumors. Gene expression analysis was performed through Quantitative Real-Time PCR and immunohistochemistry. In spite of the higher expression levels of CCND1 mRNA and protein in tumors when compared with the control samples, no differences were observed between the metastatic and non-metastatic groups, suggesting that, in these samples, the expression of CCND1 has no significant influence on the metastatic process. Further studies must be performed in an attempt to clarify the diagnostic and prognostic value of Cyclin D1 in breast cancers, as well as the mechanisms that trigger its overexpression in tumors.

Topics: Adult; Aged; Brazil; Breast Neoplasms; Carcinoma, Ductal, Breast; Cyclin D1; Disease Progression; Female; Gene Expression; Gene Expression Regulation, Neoplastic; Humans; Lymphatic Metastasis; Middle Aged; Prognosis

To explore the expression levels and clinical significance of cyclin D1 and estrogen receptor (ER) in breast cancer.. Immunohistochemical staining was performed to detect the expression levels of cyclin D1 and ER in 102 breast cancers and 60 normal breast tissue specimens. Referring to clinicopathological parameters and 5-year follow-up data, we analyzed the correlations between cyclin D1, ER and their prognosis.. The expression levels of both cyclin D1 and ER were significantly different between breast cancer and normal breast tissues. Cyclin D1 expression was related to tumor histological grade, TNM stage and ER levels. Spearman correlation analysis showed that cyclin D1 and ER were positively related. Moreover, Kaplan-Meier analysis showed that patients who expressed high levels of cyclin D1 had a longer overall survival compared with those with low expressions. Cyclin D1, ER, and TNM stage were independent prognosis factors according to Cox analysis.. Cyclin D1 has a higher expression in breast cancer, positively correlated with ER and better prognosis.

Topics: Adult; Aged; Aged, 80 and over; Breast Neoplasms; Cyclin D1; Estrogen Receptor alpha; Estrogen Receptor beta; Female; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Middle Aged; Neoplasm Staging; Prognosis; Proportional Hazards Models

Deregulated signal transducer and activator of transcription 3 (STAT3) signaling has been well documented in certain cancers. Alterations in specific negative regulators, such as protein inhibitor of activated STAT3 (PIAS3), may contribute to cancer development.. The expression of total PIAS3 was determined in 100 paired cancerous and non-cancerous breast tissues by immunoblotting and was statistically analyzed along with the clinicopathological characteristics and overall survival of the patients. XTT, immunoblotting, and chromatin immunoprecipitation (Chip) were used to examine the biological effect of PIAS3 in breast cancer cells.. Hormone therapy failed to improve the overall survival in patients presenting with increased PIAS3 expression. Ectopic PIAS3 overexpression increased the proliferation and expression of cyclin D1 in estrogen receptor (ER)-positive MCF-7 and T47D cells, but decreased those in ER-negative MDA-MB-231 and SKBR3 cells. Furthermore, PIAS3 overexpression attenuated cytotoxicity of tamoxifen and increased proliferation and cyclin D1 expression in MCF-7 cells. PIAS3 also decreased the binding of itself on the cyclin D1 promoter and this decreased binding was not affected by tamoxifen.. PIAS3 may serve as a biomarker for predicting hormone therapy stratification, although it is limited to those breast cancer patients receiving hormone therapy.

Topics: Breast Neoplasms; Cell Proliferation; Cyclin D1; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Humans; Lymphatic Metastasis; MCF-7 Cells; Molecular Chaperones; Protein Inhibitors of Activated STAT; STAT3 Transcription Factor; Tamoxifen

The mechanisms of action of 2α-hydroxyursolic acid in inhibiting cell proliferation and inducing apoptosis in MDA-MB-231 human breast cancer cells were investigated. The antiproliferative activity and cytotoxicity were determined by the methylene blue assay. The expression of proteins was determined using Western blot. 2α-Hydroxyursolic acid significantly inhibited MDA-MB-231 cell proliferation, and no cytotoxicity was observed at concentrations below 30 μM. 2α-Hydroxyursolic acid significantly down-regulated expressions of TRAF2, PCNA, cyclin D1, and CDK4 and up-regulated the expressions of p-ASK1, p-p38, p-p53, and p-21. 2α-Hydroxyursolic acid induced apoptosis in MDA-MB-231 cells by significantly increasing the Bax/Bcl-2 ratio and inducing the cleaved caspase-3. Additionally, treatment of SB203580, a p38 MAPK specific inhibitor, reversed the inhibition of PCNA, cyclin D1, and Bcl-2 expression induced by 2α-hydroxyursolic acid in MDA-MB-231 cells. These results suggested that 2α-hydroxyursolic acid exhibited anticancer activity through the inhibition of cell proliferation and the induction of apoptosis by regulating the p38/MAPK signal transduction pathway.

Topics: Apoptosis; Breast Neoplasms; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; Female; Humans; p38 Mitogen-Activated Protein Kinases; Signal Transduction; Triterpenes

Ductal carcinoma in situ (DCIS) is a non-invasive form of breast cancer. It is often associated with invasive ductal carcinoma (IDC), and is considered to be a non-obligate precursor of IDC. It is not clear to what extent these two forms of cancer share low-risk susceptibility loci, or whether there are differences in the strength of association for shared loci.. To identify genetic polymorphisms that predispose to DCIS, we pooled data from 38 studies comprising 5,067 cases of DCIS, 24,584 cases of IDC and 37,467 controls, all genotyped using the iCOGS chip.. Most (67 %) of the 76 known breast cancer predisposition loci showed an association with DCIS in the same direction as previously reported for invasive breast cancer. Case-only analysis showed no evidence for differences between associations for IDC and DCIS after considering multiple testing. Analysis by estrogen receptor (ER) status confirmed that loci associated with ER positive IDC were also associated with ER positive DCIS. Analysis of DCIS by grade suggested that two independent SNPs at 11q13.3 near CCND1 were specific to low/intermediate grade DCIS (rs75915166, rs554219). These associations with grade remained after adjusting for ER status and were also found in IDC. We found no novel DCIS-specific loci at a genome wide significance level of P < 5.0x10(-8).. In conclusion, this study provides the strongest evidence to date of a shared genetic susceptibility for IDC and DCIS. Studies with larger numbers of DCIS are needed to determine if IDC or DCIS specific loci exist.

Topics: Adult; Aged; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Intraductal, Noninfiltrating; Cyclin D1; Female; Genetic Association Studies; Genotype; Humans; Ki-67 Antigen; Middle Aged; Neoplasm Proteins; Polymorphism, Single Nucleotide; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone

Recent high-throughput studies revealed recurrent RUNX1 mutations in breast cancer, specifically in oestrogen receptor-positive (ER(+)) tumours. However, mechanisms underlying the implied RUNX1-mediated tumour suppression remain elusive. Here, by depleting mammary epithelial cells of RUNX1 in vivo and in vitro, we demonstrate combinatorial regulation of AXIN1 by RUNX1 and oestrogen. RUNX1 and ER occupy adjacent elements in AXIN1's second intron, and RUNX1 antagonizes oestrogen-mediated AXIN1 suppression. Accordingly, RNA-seq and immunohistochemical analyses demonstrate an ER-dependent correlation between RUNX1 and AXIN1 in tumour biopsies. RUNX1 loss in ER(+) mammary epithelial cells increases β-catenin, deregulates mitosis and stimulates cell proliferation and expression of stem cell markers. However, it does not stimulate LEF/TCF, c-Myc or CCND1, and it does not accelerate G1/S cell cycle phase transition. Finally, RUNX1 loss-mediated deregulation of β-catenin and mitosis is ameliorated by AXIN1 stabilization in vitro, highlighting AXIN1 as a potential target for the management of ER(+) breast cancer.

Topics: Animals; Axin Protein; beta Catenin; Blotting, Western; Breast Neoplasms; Core Binding Factor Alpha 2 Subunit; Cyclin D1; Estrogens; Female; G1 Phase Cell Cycle Checkpoints; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; MCF-7 Cells; Mice; Proto-Oncogene Proteins c-myc; Receptors, Estrogen; Reverse Transcriptase Polymerase Chain Reaction; TCF Transcription Factors

Multifaceted activities of class I phosphatidylinositol 3-kinase (PI3K) inhibitor ZSTK474 were investigated on human breast cancer cell MCF-7. ZSTK474 inhibited proliferation of MCF-7 cells potently. Flow cytometric analysis indicated that ZSTK474 induced cell cycle arrest at G1 phase, but no obvious apoptosis occurred. Western blot analysis suggested that blockade of PI3K/Akt/GSK-3β/cyclin D1/p-Rb pathway might contribute to the G1 arrest induced. Moreover, we demonstrated that ZSTK474 induced autophagy in MCF-7 cells by use of various assays including monodansylcadaverine (MDC) staining, transmission electron microscopy (TEM), tandem mRFP-GFP-LC3 fluorescence microscopy, and western blot detection of the autophagy protein markers of LC3B II, p62 and Atg 5. Inhibition of class I PI3K and the downstream mTOR might be involved in the autophagy-inducing effect. Combinational use of ZSTK474 and autophagy inhibitors enhanced cell viability, suggesting ZSTK474-induced autophagy might contribute to the antitumor activity. Our report supports the application of ZSTK474, which is being evaluated in clinical trials, for breast cancer therapy.

Topics: Autophagy; Blotting, Western; Breast Neoplasms; Cell Proliferation; Cell Survival; Class I Phosphatidylinositol 3-Kinases; Cyclin D1; G1 Phase Cell Cycle Checkpoints; Glycogen Synthase Kinase 3 beta; Humans; MCF-7 Cells; Microscopy, Electron, Transmission; Microscopy, Fluorescence; Proto-Oncogene Proteins c-akt; Signal Transduction; TOR Serine-Threonine Kinases; Triazines

Syzygium cumini (jamun) is perhaps the only berry that has the diversity of anthocyanidins of blueberry and bilberry and the abundance of ellagitannins/ellagic acid of black raspberry. Here, we report the potential of jamun against 17β-estrogen-mediated breast cancer and the role of miRNAs and other targets in disease inhibition.. Female August-Copenhagen Irish rats were given AIN-93M diet or diet supplemented with jamun. Two weeks later, animals received 17β-estradiol and were palpated weekly for the mammary tumors. At the end of 26 weeks, the jamun-diet significantly delayed the first tumor appearance by 21 days, and reduced the tumor incidence (65% versus 96%), tumor burden (313 ± 95 versus 661 ± 123 mm(3) ) and tumor multiplicity (1.8 ± 0.3 versus 4.2 ± 0.4 tumors/rat) compared to control. The experimental diet significantly reduced the estrogen-associated growth of pituitary prolactinomas, circulating prolactin and estradiol levels and offset estrogen-associated increases in mammary cell-proliferation, estrogen receptor-alpha (ER-α), and cyclinD1. miRNAs that were either overexpressed (miR-182 and miR-375) or underexpressed (miR-127 and miR-206) following estrogen-treatment were significantly protected by jamun diet.. Together, our data show that jamun significantly offset estrogen-mediated alterations in mammary cell-proliferation, ER-α, cyclinD1, and candidate miRNAs, and that the modulation of these biomarkers correlated with a reduction in mammary carcinogenicity.

Topics: Animals; Anthocyanins; Breast Neoplasms; Cell Proliferation; Cyclin D1; Diet; Ellagic Acid; Estradiol; Estrogen Receptor alpha; Female; Fruit; Gene Expression Regulation, Neoplastic; Hydrolyzable Tannins; Mammary Neoplasms, Experimental; MicroRNAs; Plant Preparations; Rats; Rats, Inbred Strains; Syzygium

Invasive lobular carcinoma (ILC) comprises approximately ~10-20% of breast cancers. In general, multifocal/multicentric (MF/MC) breast cancer has been associated with an increased rate of regional lymph node metastases. Tumor heterogeneity between foci represents a largely unstudied source of genomic variation in those rare patients with MF/MC ILC.. We characterized gene expression and copy number in 2 or more foci from 11 patients with MF/MC ILC (all ER+, HER2-) and adjacent normal tissue. RNA and DNA were extracted from 3x1.5 mm cores from all foci. Gene expression (730 genes) and copy number (80 genes) were measured using Nanostring PanCancer and Cancer CNV panels. Linear mixed models were employed to compare expression in tumor versus normal samples from the same patient, and to assess heterogeneity (variability) in expression among multiple ILC within an individual.. 35 and 34 genes were upregulated (FC>2) and down-regulated (FC<0.5) respectively in ILC tumor relative to adjacent normal tissue, q<0.05. 9/34 down-regulated genes (FIGF, RELN, PROM1, SFRP1, MMP7, NTRK2, LAMB3, SPRY2, KIT) had changes larger than CDH1, a hallmark of ILC. Copy number changes in these patients were relatively few but consistent across foci within each patient. Amplification of three genes (CCND1, FADD, ORAOV1) at 11q13.3 was present in 2/11 patients in both foci. We observed significant evidence of within-patient between-foci variability (heterogeneity) in gene expression for 466 genes (p<0.05 with FDR 8%), including CDH1, FIGF, RELN, SFRP1, MMP7, NTRK2, LAMB3, SPRY2 and KIT.. There was substantial variation in gene expression between ILC foci within patients, including known markers of ILC, suggesting an additional level of complexity that should be addressed.

Topics: Adult; Breast; Breast Neoplasms; Carcinoma, Lobular; Chromosomes, Human, Pair 11; Cyclin D1; DNA, Neoplasm; Female; Gene Dosage; Genetic Loci; Humans; Lymphatic Metastasis; Reelin Protein; RNA, Neoplasm; Transcriptome

Cyclin D1 (Ccnd1) is a proto-oncogen amplified in many different cancers and nuclear accumulation of Ccnd1 is a characteristic of tumor cells. Ccnd1 activates the transcription of a large set of genes involved in cell cycle progress and proliferation. However, Ccnd1 also targets cytoplasmic proteins involved in the regulation of cell migration and invasion. In this work, we have analyzed by immunohistochemistry the localization of Ccnd1 in endometrial, breast, prostate and colon carcinomas with different types of invasion. The number of cells displaying membranous or cytoplasmic Ccnd1 was significantly higher in peripheral cells than in inner cells in both collective and pushing invasion patterns of endometrial carcinoma, and in collective invasion pattern of colon carcinoma. Also, the cytoplasmic localization of Ccnd1 was higher when tumors infiltrated as single cells, budding or small clusters of cells. To evaluate cytoplasmic function of cyclin D1, we have built a variant (Ccnd1-CAAX) that remains attached to the cell membrane therefore sequestering this cyclin in the cytoplasm. Tumor cells harboring Ccnd1-CAAX showed high levels of invasiveness and metastatic potential compared to those containing the wild type allele of Ccnd1. However, Ccnd1-CAAX expression did not alter proliferative rates of tumor cells. We hypothesize that the role of Ccnd1 in the cytoplasm is mainly associated with the invasive capability of tumor cells. Moreover, we propose that subcellular localization of Ccnd1 is an interesting guideline to measure cancer outcome.

Topics: Amino Acid Motifs; Animals; Biomarkers, Tumor; Breast Neoplasms; Cell Line, Tumor; Cell Membrane; Cells, Cultured; Colonic Neoplasms; Cyclin D1; Cytoplasm; Endometrial Neoplasms; Female; Humans; Immunohistochemistry; Male; Mice, Nude; Mice, SCID; Microscopy, Confocal; Neoplasm Invasiveness; Neoplasms; Prostatic Neoplasms

The Aurora protein kinase (AURKA) and the Polo-like kinase-1 (PLK1) activate the cell cycle, and they are considered promising druggable targets in cancer therapy. However, resistance to chemotherapy and to specific small‑molecule inhibitors is common in cancer patients; thus alternative therapeutic approaches are needed to overcome clinical resistance. Here, we showed that the dietary compound resveratrol suppressed the cell cycle by targeting AURKA and PLK1 kinases. First, we identified genes modulated by resveratrol using a genome-wide analysis of gene expression in MDA-MB-231 breast cancer cells. Transcriptional profiling indicated that 375 genes were modulated at 24 h after resveratrol intervention, whereas 579 genes were regulated at 48 h. Of these, 290 genes were deregulated in common at 24 and 48 h. Interestingly, a significant decrease in the expression of genes involved in the cell cycle, DNA repair, cytoskeleton organization, and angiogenesis was detected. In particular, AURKA and PLK1 kinases were downregulated by resveratrol at 24 h. In addition the BRCA1 gene, an AURKA/PLK1 inhibitor, was upregulated at 24 h of treatment. Moreover, two well-known resveratrol effectors, cyclin D1 (CCND1) and cyclin B1 (CCNB1), were also repressed at both times. Congruently, we found that resveratrol impaired G1/S phase transition in both MDA-MB-231 and MCF-7 cells. By western blot assays, we confirmed that resveratrol suppressed AURKA, CCND1 and CCNB1 at 24 and 48 h. In summary, we showed for the first time that resveratrol regulates cell cycle progression by targeting AURKA and PLK1. Our findings highlight the potential use of resveratrol as an adjuvant therapy for breast cancer.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Aurora Kinase A; BRCA1 Protein; Breast Neoplasms; Cell Cycle Proteins; Cell Line, Tumor; Cell Movement; Cyclin B1; Cyclin D1; Female; G1 Phase Cell Cycle Checkpoints; Humans; MCF-7 Cells; Oligonucleotide Array Sequence Analysis; Polo-Like Kinase 1; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Resveratrol; Stilbenes; Transcriptome

As a main active compound in the bark of waxberry (Myrica rubra), myricetin is a macrocyclic diarylheptanoid, and can trigger the apoptosis of HeLa and PC3 cells. The aim of the present study was to elucidate the anticancer effect of myricetin on human breast cancer MCF-7 cells and to explore the possible mechanisms of action. MCF-7 cells were treated with different concentrations of myricetin (0-80 µM) for 12, 24 and 48 h. In the present study, we found that myricetin suppressed the cell viability of the MCF-7 cells at least partly through the induction of apoptosis as determined by MTT assay and flow cytometry. Western blot analysis revealed that myricetin effectively suppressed the protein expression of p21-activated kinase 1 (PAK1), MEK and phosphorylated extracellular mitogen-activated protein kinase (ERK1/2). In addition, treatment of myricetin activated glycogen synthase kinase-3β (GSK3β) and Bax protein expression, and inhibited β-catenin/cyclin D1/proliferating cell nuclear antigen (PCNA)/survivin and promoted caspase-3 activity in the MCF-7 cells. These results demonstrated that myricetin suppressed the cell viability of human breast cancer MCF-7 cells through PAK1/MEK/ERK/GSK3β/β-catenin/cyclin D1/PCNA/survivin/Bax-caspase-3 signaling.

Topics: Apoptosis; bcl-2-Associated X Protein; beta Catenin; Breast Neoplasms; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Flavonoids; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3; Humans; Inhibitor of Apoptosis Proteins; MAP Kinase Signaling System; MCF-7 Cells; Mitogen-Activated Protein Kinases; p21-Activated Kinases; Proliferating Cell Nuclear Antigen; Signal Transduction; Survivin

The Ras-Raf-MEK-ERK1/2 signaling pathway regulates fundamental processes in malignant cells. However, the exact contributions of MEK1 and MEK2 to the development of cancer remain to be established. We studied the effects of MEK small-molecule inhibitors (PD98059 and U0126) and MEK1 and MEK2 knock-down on cell proliferation, apoptosis and MAPK activation. We showed a diminution of cell viability that was associated with a downregulation of cyclin D1 expression and an increase of apoptosis marker in MEK2 silenced cells; by contrast, a slight increase of cell survival was observed in the absence of MEK1 that correlated with an augment of cyclin D1 expression. These data indicate that MEK2 but not MEK1 is essential for MDA-MB-231 cell survival. Importantly, the role of MEK2 in cell survival appeared independent on ERK1/2 phosphorylation since its absence did not alter the level of activated ERK1/2. Indeed, we have reported an unrevealed link between MEK2 and MKK3/MKK6-p38 MAPK axis where MEK2 was essential for the phosphorylation of MKK3/MKK6 and p38 MAPK that directly impacted on cyclin D1 expression. Importantly, the MEK1 inhibitor PD98059, like MEK1 silencing, induced an augment of cyclin D1 expression that correlated with an increase of MDA-MB-231 cell proliferation suggesting that MEK1 may play a regulatory role in these cells. In sum, the crucial role of MEK2 in MDA-MB-231 cell viability and the unknown relationship between MEK2 and MKK3/MKK6-p38 axis here revealed may open new therapeutic strategies for aggressive breast cancer.

Topics: Breast Neoplasms; Butadienes; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Down-Regulation; Enzyme Activation; Female; Flavonoids; Gene Knockdown Techniques; Gene Silencing; Humans; MAP Kinase Kinase 2; MAP Kinase Kinase 3; MAP Kinase Kinase 6; Nitriles; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Signal Transduction

The interrelationship among genetic variations between the developing process of carcinoma and the order of occurrence has not been completely understood. Interpreting the mechanisms of copy number variation (CNV) is absolutely necessary for understanding the etiology of genetic disorders. Oncogenetic tree is a special phylogenetic tree inferential pictorial representation of oncogenesis. In our present study, we constructed oncogenetic tree to imitate the occurrence of genetic and cytogenetic alterations in human breast cancer. The oncogenetic tree model was built on CNV of ErbB2, AKT2, KRAS, PIK3CA, PTEN, and CCND1 genes in 963 cases of tumors with sequencing and CNA data of human breast cancer from TCGA. Results from the oncogenetic tree model indicate that ErbB2 copy number variation is the frequent early event of human breast cancer. The oncogenetic tree model based on the phylogenetic tree is a type of mathematical model that may eventually provide a better way to understand the process of oncogenesis.

Topics: Breast Neoplasms; Class I Phosphatidylinositol 3-Kinases; Cyclin D1; Disease Progression; Female; Gene Dosage; Humans; Models, Theoretical; Phosphatidylinositol 3-Kinases; Phylogeny; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins p21(ras); PTEN Phosphohydrolase; Receptor, ErbB-2

EF hand (EFh) domain-containing proteins have been implicated in malignant progression, but their precise functional contributions are uncertain. Here, we report evidence that the EFh protein IBA2 promotes the proliferation of breast cancer cells by facilitating their transit through the G1-S cell-cycle transition. Mechanistic investigations revealed that IBA2 acted at the transcriptional level to promote the expression of the critical cell-cycle regulator cyclin D1. Clinically, we found that levels of IBA2 were significantly upregulated in breast cancer specimens, where its expression correlated positively with histologic grade. Our results suggest a key role for IBA2 in mammary tumorigenesis. Cancer Res; 76(15); 4535-45. ©2016 AACR.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; EF Hand Motifs; Female; Humans; Mice; Mice, Inbred BALB C; Transfection; Xenograft Model Antitumor Assays

MicroRNAs (miRNAs) are short noncoding RNAs derived from the 3' and 5' ends of the same precursor. However, the biological function and mechanism of miRNA arm expression preference remain unclear in breast cancer. We found significant decreases in the expression levels of miR-193a-5p but no significant differences in those of miR-193a-3p in breast cancer. MiR-193a-3p suppressed breast cancer cell growth and migration and invasion abilities, whereas miR-193a-5p suppressed cell growth but did not influence cell motility. Furthermore, NLN and CCND1, PLAU, and SEPN1 were directly targeted by miR-193a-5p and miR-193a-3p, respectively, in breast cancer cells. The endogenous levels of miR-193a-5p and miR-193a-3p were significantly increased by transfecting breast cancer cells with the 3'UTR of their direct targets. Comprehensive analysis of The Cancer Genome Atlas database revealed significant differences in the arm expression preferences of several miRNAs between breast cancer and adjacent normal tissues. Our results collectively indicate that the arm expression preference phenomenon may be attributable to the target gene amount during breast cancer progression. The miRNA arm expression preference may be a means of modulating miRNA function, further complicating the mRNA regulatory network. Our findings provide a new insight into miRNA regulation and an application for breast cancer therapy.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Female; Humans; MCF-7 Cells; Membrane Proteins; MicroRNAs; Muscle Proteins; Neoplasm Invasiveness; Real-Time Polymerase Chain Reaction; RNA Interference; RNA, Small Interfering; Selenoproteins

Previous studies regarding the association between serum 25-hydroxyvitamin D (25OHD3) and breast cancer risk have not been conclusive. The aim of this study was to investigate the potential association between pre-diagnostic serum 25OHD3 levels and the risk of different subtypes of breast cancer.. The study was based on The Malmö Diet and Cancer Study recruiting 17,035 women from 1991 to 1996. A total of 764 incident breast cancers with matched controls were analysed for 25OHD3 in samples collected at baseline, before diagnosis. A logistic regression analysis was used to calculate odds ratios with 95% confidence intervals for tertiles of 25OHD3 in relation to different subtypes of breast cancer, i.e. defined according to tumour type, tumour size, lymph node involvement, histological grade, oestrogen receptor (ER) status, progesterone receptor (PgR) status, Ki67, cyclin D1 and p27.. As compared to the 1st tertile of 25OHD3, the second tertile had a statistically significantly lower risk of ER negative tumours, PgR negative tumours and tumours with a high expression of Ki67, A similar pattern was seen in relation to large tumours (≥21 mm), grade III tumours, and tumours with low p27 expression, but these associations did not reach statistical significance. The third tertile had a similar risk as the first tertile.. We found that women with low levels of 25OHD3, as compare to women in the middle tertile, had a high risk of breast tumours with an unfavourable prognosis.

Topics: Breast Neoplasms; Calcifediol; Case-Control Studies; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Female; Humans; Ki-67 Antigen; Lymphatic Metastasis; Middle Aged; Neoplasm Grading; Receptors, Estrogen; Receptors, Progesterone; Risk Factors; Tumor Burden

Persistent activation of signal transducer and activator of transcription 3 (STAT3) is associated with the progression of a range of tumors. In this report, we present the anticancer activity of 2-(1-(4-(2-cyanophenyl)1-benzyl‑1H-indol-3-yl)-5-(4-methoxy-phenyl)-1-oxa-3-azaspiro(5,5)undecane (CIMO) against breast cancer cells. We observed that CIMO suppresses the proliferation of both estrogen receptor-negative (ER-) (BT-549, MDA-MB‑231) and estrogen receptor-positive (ER+) (MCF-7, and BT-474) breast cancer (BC) cells with IC50 of 3.05, 3.41, 4.12 and 4.19 µM, respectively, and without significantly affecting the viability of normal cells. CIMO was observed to mediate its anti-proliferative effect in ER- BC cells by inhibiting the phosphorylation of JAK2 and STAT3 proteins. Quantitative PCR analysis demonstrated that CIMO decreases the relative mRNA expression of genes that are involved in cell cycle progression (CCND1) and cell survival (BCL2, BCL-xL, BAD, CASP 3/7/9, and TP53). In addition, CIMO was observed to arrest BC cells at G0/G1 phase and of the cell cycle. Furthermore, CIMO suppressed BC cell migration and invasion with concordant regulation of genes involved in epithelial to mesechymal transition (CDH1, CDH2, OCLN and VIM). Thus, we report the utility of a synthetic azaspirane which targets the JAK-STAT pathway in ER- BC.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Cyclin D1; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Janus Kinase 2; MCF-7 Cells; Phosphorylation; Signal Transduction; Spiro Compounds; STAT3 Transcription Factor

Anticancer agents consisting of hybrid molecules are used to improve effectiveness and diminish drug resistance. The current study aimed to introduce newly synthesized hetero-steroids of promising anticancer effects. Besides, the pro-apoptotic effects of new compounds were investigated extensively. Several pyrimidino-, triazolopyrimidino-, pyridazino-, and curcumin-steroid derivatives were synthesized, elucidated and confirmed using the spectral and analytical data. The synthesized hetero-steroids, compounds 9, 10, 11, 12, 13, 14, 15, 18, 20, 21, 22 and 24, were tested for their cytotoxic effects versus human breast cancer cells (MCF-7) using neutral red supravital dye uptake assay. Compound 24 (IC50=18μM) showed more inhibitory influence on MCF-7 growth. Using QRT-PCR (Quantitative real time-polymerase chain reaction), CCND1, Survivin, BCL-2, CDC2, P21 and P53, genes expression levels were investigated. The study results disclose that compounds 4, 7, 18, 24 knocked down the expression levels of CCND1, Survivin, BCL-2 and CDC2. However, P21 and P53 were up-regulated by compounds 21, 22. This study introduced promising pro-apoptotic anticancer agents acting through the modulation of key regulators of apoptosis and cell cycle genes.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; CDC2 Protein Kinase; Curcumin; Cyclin D1; Cyclin-Dependent Kinases; Female; Humans; MCF-7 Cells; Proto-Oncogene Proteins c-bcl-2; Steroids, Heterocyclic; Tumor Suppressor Protein p53

Phosphatidylinositol analogs (PIAs) were originally designed to bind competitively to the Akt PH domain and prevent membrane translocation and activation. d-3-Deoxy-dioctanoylphosphatidylinositol (d-3-deoxy-diC

Topics: Breast Neoplasms; Cell Death; Cyclin D1; Down-Regulation; Female; Humans; Magnetic Resonance Spectroscopy; MCF-7 Cells; p38 Mitogen-Activated Protein Kinases; Phosphatidic Acids; Phosphatidylinositols; Phosphorylation; Pleckstrin Homology Domains; Proto-Oncogene Proteins c-akt; Retinoblastoma Protein; RNA, Small Interfering; Signal Transduction

Thrombin activates its G-coupled seven transmembrane protease-activated receptor (PAR-1) by cleaving the receptor's N-terminal end. In several human cancers, PAR1 expression and activation correlates with tumor progression and metastatization. This provides compelling evidence for the effectiveness of an appropriate antithrombin agent for the adjuvant treatment of patients with cancer. Dabigatran is a selective direct thrombin inhibitor that reversibly binds to thrombin. In this study, we aimed to explore if dabigatran may affect mechanisms favoring tumor growth by interfering with thrombin-induced PAR-1 activation. We confirmed that exposure of tumor cells to thrombin significantly increased cell proliferation and this was coupled with downregulation of p27 and concomitant induction of cyclin D1. Dabigatran was consistently effective in antagonizing thrombin-induced proliferation as well as it restored the baseline pattern of cell cycle protein expression. Thrombin significantly upregulated the expression of proangiogenetic proteins like Twist and GRO-α in human umbilical vascular endothelial cells (HUVEC) cells and their expression was significantly brought down to control levels when dabigatran was added to culture. We also found that the chemoattractant effect of thrombin on tumor cells was lost in the presence of dabigatran, and that the thrombin antagonist was effective in dampening vascular tube formation induced by thrombin. Our data support a role of thrombin in inducing the proliferation, migration, and proangiogenetic effects of tumor cells in vitro. Dabigatran has activity in antagonizing all these effects, thereby impairing tumor growth and progression. In vivo models may help to understand the relevance of this pathway.

Topics: Antithrombins; Brain Neoplasms; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Dabigatran; Female; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Poly (ADP-Ribose) Polymerase-1; Thrombin

Limitless replicative potential is one of the hallmarks of cancer that is mainly due to the activity of telomerase. This holoenzyme maintains telomere length, adding TTAGGG repetitions at the end of chromosomes in each cell division. In addition to this function, there are extratelomeric roles of telomerase that are involved in cancer promoting events. It has been demonstrated that TERT, the catalytic component of telomerase, acts as a transcriptional modulator in many signaling pathways. Taking into account this evidence and our experience on the study of azidothymidine (AZT) as an inhibitor of telomerase activity, the present study analyzes the effect of AZT on some telomeric and extratelomeric activities. To carry out the present study, we evaluated the transcription of genes that are modulated by the Wnt/β-catenin pathway, such as c-Myc and cyclin-D1 (Cyc-D1) and cell processes related with their expression, such as, proliferation, modifications of the actin cytoskeleton, cell migration and cell cycle in a mammary carcinoma cell line (F3II). Results obtained after treatment with AZT (600 µM) for 15 passages confirmed the inhibitory effect on telomerase. Regarding extratelomeric activities, our results showed a decrease of 64, 38 and 25% in the transcription of c-Myc, Cyc-D1 and TERT, respectively (p<0.05) after AZT treatment. Furthermore, we found an effect on cell migration, reaching an inhibition of 48% (p<0.05) and a significant passage-dependent increase on cell doubling time during treatment. Finally, we evaluated the effect on cell cycle, obtaining a decline in G0/G1 in AZT-treated cells. These results allow us to postulate that AZT is not only an inhibitor of telomerase activity, but also a potential modulator of extratelomeric processes involved in cancer promotion.

Topics: Actin Cytoskeleton; Adenocarcinoma; Animals; Apoptosis; beta Catenin; Breast Neoplasms; Cell Division; Cell Movement; Cell Proliferation; Cyclin D1; Disease Models, Animal; Female; Humans; Proto-Oncogene Proteins c-myc; Telomerase; Telomere Homeostasis; Wnt Signaling Pathway; Zidovudine

Cancer stem cells (CSCs), a small fraction of cancer cells lines proved with stem cell characteristics, were regarded as "bad seeds" related to recurrence, metastasis and chemotherapy resistance of breast carcinoma in recent years. So inhibiting the growth or inducing the differentiation and apoptosis of CSCs were considered as one of the effective pathways to fight against breast cancer. Based on the recombinant protein TmSm(T34A) that was designed and prepared in our previous experiments for targeting survivin, an inhibitor of apoptosis protein(IAP), in this study, we explored the effects of TmSm(T34A) on BCSCs obtained by enriching in serum-free suspension, sorting and characterizing of MCF-7/ADM. The results showed that TmSm(T34A) could not only inhibit the proliferation and growth of BCSCs by decreasing CD44

Topics: Animals; Antigens, CD; Antineoplastic Agents; Apoptosis; Blotting, Western; Breast Neoplasms; Carcinogenesis; Cell Proliferation; Cell Separation; Culture Media, Serum-Free; Cyclin D1; Down-Regulation; Doxorubicin; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Inhibitor of Apoptosis Proteins; MCF-7 Cells; Mice, Nude; Neoplastic Stem Cells; Recombinant Proteins; Spheroids, Cellular; Survivin

Recently, the roles of sirtuins (SIRTs) in tumorigenesis have been of interest to oncologists, and protein kinase CK2 α1 (CSNK2A1) has been shown to be involved in tumorigenesis by phosphorylating various proteins, including SIRT1. Therefore, we evaluated the roles of CSNK2A1, SIRT6, and phosphorylated SIRT6 and their relationships in breast carcinoma. Nuclear expression of CSNK2A1 and SIRT6 predicted shorter overall survival and relapse-free survival by multivariate analysis. Inhibition of CSNK2A1 decreased the proliferative and invasive activity of cancer cells. In addition, CSNK2A1 was bound to SIRT6 and phosphorylated SIRT6; evidence for this is provided from immunofluorescence staining, co-immunoprecipitation of CSNK2A1 and SIRT6, a glutathione S-transferase pull-down assay, an in vitro kinase assay, and transfection of mutant CSNK2A1. Knockdown of SIRT6 decreased the proliferation and invasiveness of cancer cells. Overexpression of SIRT6 increased proliferation, but mutation at the Ser338 phosphorylation site of SIRT6 inhibited the proliferation of MCF7 cells. Moreover, both knockdown of SIRT6 and a mutation at the phosphorylation site of SIRT6 decreased expression of matrix metallopeptidase 9, β-catenin, cyclin D1, and NF-κB. Especially, SIRT6 expression was associated with the nuclear localization of β-catenin. This study demonstrates that CSNK2A1 and SIRT6 are indicators of poor prognosis for breast carcinomas and that CSNK2A1-mediated phosphorylation of SIRT6 might be involved in the progression of breast carcinoma.

Topics: beta Catenin; Breast Neoplasms; Casein Kinase II; Cell Proliferation; Cyclin D1; Disease Progression; Gene Expression; Gene Expression Regulation, Neoplastic; Humans; Mutation; NF-kappa B; Phosphorylation; Prognosis; Sirtuins

Global cancer burden increased to 14.1 million new cases in 2012; and breast cancer is the most common cancer in women worldwide, with nearly 1.7 million new cases diagnosed in 2012. Curcumin is the major bioactive ingredient extracted from the rhizome of the plant Curcuma longa (turmeric). Paclitaxel is a microtubule-stabilizing agent originally isolated from the bark of Taxus brevifolia. Curcumin and paclitaxel were evaluated with two human breast cancer cell lines as the luminal MCF-7 and the basal-like MDA-MB-231 that are either positive or negative for hormonal receptors estrogen receptor, progesterone receptor and HER2, respectively. Results indicated that curcumin combined with paclitaxel decreased c-Ha-Ras, Rho-A, p53 and Bcl-xL gene expression in comparison to control and substances alone in MCF-7 cell line. These two substances alone and combined decreased gene expression of Bcl-2 and NF-κB. However, CCND1 increased when both substances were combined in MCF-7 cells. Such substances decreased Bcl-2 and increased Bax protein expression. However, curcumin alone decreased IκBα and Stat-3 gene expression. Paclitaxel alone and combined increased IκBα and Stat-3. Curcumin alone and combined with paclitaxel increased p53, Bid, caspase-3, caspase-8 and Bax gene expression in MDA-MB-231, whereas Bcl-xL decreased such expression in MDA-MB-231 cells. When paclitaxel and curcumin were combined the expression of Bcl-2 protein was decreased. However, either substance alone and combined increased Bax protein expression corroborating the apoptotic effect of these substances. It can be concluded that curcumin may be of considerable value in synergistic therapy of breast cancer reducing the associated toxicity with use of drugs.

Topics: Antineoplastic Combined Chemotherapy Protocols; bcl-X Protein; BH3 Interacting Domain Death Agonist Protein; Breast Neoplasms; Carcinogenesis; Caspase 3; Caspase 8; Cell Line, Tumor; Curcumin; Cyclin D1; Female; Humans; MCF-7 Cells; NF-KappaB Inhibitor alpha; Paclitaxel; Proto-Oncogene Proteins p21(ras); rhoA GTP-Binding Protein; STAT3 Transcription Factor; Tumor Suppressor Protein p53

Prodigiosin, a natural red pigment produced by numerous bacterial species, has exhibited promising anticancer activity; however, the molecular mechanisms of action of prodigiosin on malignant cells remain unclear. Aberrant activation of the Wnt/β-catenin signaling cascade is associated with numerous human cancers. In this study, we identified prodigiosin as a potent inhibitor of the Wnt/β-catenin pathway. Prodigiosin blocked Wnt/β-catenin signaling by targeting multiple sites of this pathway, including the low-density lipoprotein-receptor-related protein (LRP) 6, Dishevelled (DVL), and glycogen synthase kinase-3β (GSK3β). In breast cancer MDA-MB-231 and MDA-MB-468 cells, nanomolar concentrations of prodigiosin decreased phosphorylation of LRP6, DVL2, and GSK3β and suppressed β-catenin-stimulated Wnt target gene expression, including expression of cyclin D1. In MDA-MB-231 breast cancer xenografts and MMTV-Wnt1 transgenic mice, administration of prodigiosin slowed tumor progression and reduced the expression of phosphorylated LRP6, phosphorylated and unphosphorylated DVL2, Ser9 phosphorylated GSK3β, active β-catenin, and cyclin D1. Through its ability to inhibit Wnt/β-catenin signaling and reduce cyclin D1 levels, prodigiosin could have therapeutic activity in advanced breast cancers.

Topics: Animals; Antineoplastic Agents; Apoptosis; beta Catenin; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Cyclin D1; Dishevelled Proteins; Female; HEK293 Cells; Humans; Mice, Inbred BALB C; Mice, Nude; Mice, Transgenic; Prodigiosin; Tumor Burden; Wnt Proteins; Wnt Signaling Pathway

The complexity by which cells regulate gene and protein expression is multifaceted and intricate. Regulation of 3' untranslated region (UTR) processing of mRNA has been shown to play a critical role in development and disease. However, the process by which cells select alternative mRNA forms is not well understood. We discovered that the

Topics: 3' Untranslated Regions; Breast Neoplasms; Cyclin D1; DEAD-box RNA Helicases; Female; Humans; Jumonji Domain-Containing Histone Demethylases; MCF-7 Cells; Neoplasm Proteins; Nuclear Proteins; Repressor Proteins; Retinoblastoma-Binding Protein 2; Ribonuclease III; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins

The aim of this study was to extensively analyze the signaling pathway molecules in breast cancer and to explore candidate biomarkers for clinicopathological relevance.. We assessed the expression of key factors in cell signaling, namely p-AKT, cyclin D1, P27, p-p70S6 K, p-4EBP1, and p-MAPK/ERK, within 338 invasive breast cancer patients. These factors were immunohistochemically examined in tumor tissues and assessed by staining score. Staining scores were analyzed by a clustering method to devise a new classification based on pathway activity. We investigated the relationships among staining scores, the clustering classification, and patient characteristics.. The proportion of patients displaying high expression levels were as follows: p-AKT, 75%; cyclin D1, 12%; P27, 53%; p-p70S6 K, 37%; p-4EBP1, 19%; and p-MAPK/ERK, 3%. Patients were classified into two groups on the basis of staining scores. Group 1 (39%) included more positive cases for p-4EBP1, p-MAPK/ERK, and p-p70S6 K and fewer positive cases for P27 and cyclin D1 than Group 2 (61%). The clustering classification was significantly related to subgrouping by hormone receptor and HER2 (P < 0.001), nuclear grade (P < 0.001) and histological subtype (P = 0.034). A strong positive correlation was identified between p-AKT and P27, cyclin D1 and P27, p-p70S6 K and p-4EBP1, p-p70S6 K and p-MAPK/ERK, and between p-4EBP1 and p-MAPK/ERK. Levels of p-p70S6 K were significantly related to recurrence in both univariate (RR = 0.75, P < 0.001) and multivariate (RR = 0.71, P = 0.049) analyses.. The present study helps us to understand the characteristics of signaling pathway status in breast cancers. Moreover, p-p70S6 K expression may be of use in predicting clinical outcome.

Topics: Adaptor Proteins, Signal Transducing; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Breast Neoplasms; Cell Cycle Proteins; Cyclin D1; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Middle Aged; Phosphoproteins; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins c-akt; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Young Adult

SMARCA5 partners with RSF-1 to compose the RSF complex, which belongs to the ISWI family of chromatin remodelers. Recent studies referred that SMARCA5 was overexpressed in some malignant tumors. However, expression pattern and biological roles of SMARCA5 in breast cancer have not been examined. In the present study, we found that SMARCA5 was overexpressed in breast cancer specimens by immunohistochemistry. Significant association was observed between SMARCA5 overexpression and TNM stage (p = 0.0199), tumor size (p = 0.0066), high proliferation index (p = 0.0366), and poor overall survival (p = 0.0141). SMARCA5 overexpression also correlated with Rsf-1 expression levels (p = 0.0120). Furthermore, colony formation assay and Matrigel invasion assay showed that knockdown of SMARCA5 expression in MDA-MB-231 and MDA-MB-435s cell lines with high endogenous expression decreased cell proliferation and cell invasion. Flow cytometry showed knockdown of SMARCA5-arrested cell cycle. Further analysis of cell cycle and invasion-related molecules showed that SMARCA5 downregulated cyclin A, MMP2 expression and upregulated p21 expression. In conclusion, our study demonstrated that SMARCA5 was overexpressed in human breast cancers and correlated with poor prognosis. SMARCA5 contributes to breast cancer cell proliferation and invasion.

Topics: Adenosine Triphosphatases; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Chromatin Assembly and Disassembly; Chromosomal Proteins, Non-Histone; Cyclin A; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Humans; Matrix Metalloproteinase 2; Neoplasm Invasiveness; Nuclear Proteins; Trans-Activators; Up-Regulation

Limonene is a lipophilic monoterpene found in high levels in citrus peel. Limonene demonstrates anticancer properties in preclinical models with effects on multiple cellular targets at varying potency. While of interest as a cancer chemopreventive, the biologic activity of limonene in humans is poorly understood. We conducted metabolite profiling in 39 paired (pre/postintervention) plasma samples from early-stage breast cancer patients receiving limonene treatment (2 g QD) before surgical resection of their tumor. Metabolite profiling was conducted using ultra-performance liquid chromatography coupled to a linear trap quadrupole system and gas chromatography-mass spectrometry. Metabolites were identified by comparison of ion features in samples to a standard reference library. Pathway-based interpretation was conducted using the human metabolome database and the MetaCyc database. Of the 397 named metabolites identified, 72 changed significantly with limonene intervention. Class-based changes included significant decreases in adrenal steroids (P < 0.01), and significant increases in bile acids (P ≤ 0.05) and multiple collagen breakdown products (P < 0.001). The pattern of changes also suggested alterations in glucose metabolism. There were 47 metabolites whose change with intervention was significantly correlated to a decrease in cyclin D1, a cell-cycle regulatory protein, in patient tumor tissues (P ≤ 0.05). Here, oral administration of limonene resulted in significant changes in several metabolic pathways. Furthermore, pathway-based changes were related to the change in tissue level cyclin D1 expression. Future controlled clinical trials with limonene are necessary to determine the potential role and mechanisms of limonene in the breast cancer prevention setting.

Topics: Amino Acids; Anticarcinogenic Agents; Antineoplastic Agents; Bile Acids and Salts; Breast Neoplasms; Carnitine; Cell Cycle; Collagen; Cyclin D1; Cyclohexenes; Female; Gas Chromatography-Mass Spectrometry; Glucose; Humans; Limonene; Metabolome; Metabolomics; Plasma; Terpenes

The diagnosis of intraductal papillary lesions of the breast on core biopsy remains challenging in pathology, with most patients requiring formal surgical excision for a definitive diagnosis. The aim of this study was to determine whether a representative panel of proliferative cell cycle immunohistochemical markers (cyclin A2, cyclin B1 and cyclin D1) could improve the specificity of pathological diagnosis of these lesions.. A series of 68 surgically excised intraductal papillary lesion cases were retrospectively selected, and immunohistochemistry for cyclin A2, cyclin B1 and cyclin D1 was performed.. Cyclin B1 (OR 1.80, 95% CI 1.01 to 3.2, p=0.046) and cyclin D1 (OR 1.13, 95% CI 1.05 to 1.22, p=0.002) expression was independently associated with a diagnosis of malignancy in papillary lesions, although expression was frequently heterogeneous and only focal. Cyclin A2 expression (OR 0.76, 95% CI 0.41 to 1.4, p=0.38) was not associated with a malignant diagnosis in multivariable logistic regression models. All three cyclins displayed high sensitivity (80%-95%) for a diagnosis of malignancy, although cyclin B1 showed a superior specificity of 72.7% compared with the low specificity of cyclins A2 and D1.. Our study has identified for the first time that the expression of key cell cycle markers differs between benign and malignant papillary breast lesions and identified changes to the mitotic marker, cyclin B1, as particularly significant. However, given the low level and heterogeneous nature of expression of these markers, there remains a significant risk of undersampling in core biopsies and thus they are unlikely to be useful in routine clinical practice.

Topics: Adult; Aged; Biomarkers, Tumor; Biopsy; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Papillary; Cell Cycle; Cell Proliferation; Cyclin A2; Cyclin B1; Cyclin D1; Female; Humans; Immunohistochemistry; Logistic Models; Middle Aged; Multivariate Analysis; Odds Ratio; Papilloma, Intraductal; Predictive Value of Tests; Prognosis; Retrospective Studies; Risk Factors

Ophiobolin O is a member of ophiobolin family, which has been proved to be a potent anti-tumor drug candidate for human breast cancer. However, the anti-tumor effect and the mechanism of ophiobolin O remain unclear. In this study, we further verified ophiobolin O-induced G1 phase arrest in human breast cancer MCF-7 cells, and found that ophiobolin O reduced the phosphorylation level of AKT and GSK3β, and induced down-regulation of cyclin D1. The inverse docking (INVDOCK) analysis indicated that ophiobolin O could bind to GSK3β, and GSK3β knockdown abolished cyclin D1 degradation and G1 phase arrest. Pre-treatment with phosphatase inhibitor sodium or thovanadate halted dephosphorylation of AKT and GSK3β, and blocked ophiobolin O-induced G1 phase arrest. These data suggest that ophiobolin O may induce G1 arrest in MCF-7 cells through interaction with AKT/GSK3β/cyclin D1 signaling. In vivo, ophiobolin O suppressed tumor growth and showed little toxicity in mouse xenograft models. Overall, these findings provide theoretical basis for the therapeutic use of ophiobolin O.

Topics: Animals; Aspergillus; Breast Neoplasms; Cyclin D1; Female; G1 Phase; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; MCF-7 Cells; Mice, Inbred BALB C; Neoplasm Transplantation; Oncogene Protein v-akt; Phosphorylation; Sesterterpenes; Signal Transduction

Obesity is a risk factor for breast cancer, largely due to altered expression of various adipocytokines. As it concerns adiponectin, there are not univocal results regarding its role in breast cancer occurrence and progression. Here, we demonstrate that in animals injected with human estrogen receptor (ER)-α-negative MDA-MB-231 cells pretreated with adiponectin (1 and 5 µg/ml), a significant reduction (60 and 40%, respectively) in tumor volume is observed, whereas an increased tumor growth (54 and 109%, respectively) is evidenced in the animals receiving human ER-α-positive MCF-7 cells. Moreover, cyclin D1 (CD1) mRNA and protein levels are decreased in MDA-MB-231 cells, whereas they are up-regulated in ER-α-positive cells by adiponectin. These findings fit with the opposite effects of adiponectin on CD1 promoter: 0.44- and 0.34-fold decrease in MDA-MB-231 cells and 0.63- and 0.95-fold increase in MCF-7 cells, treated with 1 and 5 µg/ml, respectively. Functional studies indicate that these effects are mediated by the specific protein 1 motif located in the CD1 promoter. In the absence of ER-α, the adiponectin-mediated down-regulation of CD1 involves the recruitment of corepressors. In the presence of ER-α, the adiponectin-induced expression of CD1 requires the involvement of an activator complex. In conclusion, we propose that a possible mechanism through which adiponectin differently affects breast cancer growth is the opposite modulation of CD1 levels accordingly to ER-α expression.

Topics: Adiponectin; Blotting, Western; Breast Neoplasms; Cell Proliferation; Chromatin Immunoprecipitation; Cyclin D1; Electrophoretic Mobility Shift Assay; Estrogen Receptor alpha; Female; Humans; Immunoenzyme Techniques; Immunoprecipitation; Mutagenesis, Site-Directed; Mutation; Promoter Regions, Genetic; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sp1 Transcription Factor; Tumor Cells, Cultured

The mammalian target of rapamycin complex 1 (mTORC1) is a critical regulator of G1 cell cycle progression. Two key substrates of mTORC1 are ribosomal subunit S6 kinase (S6K) and eukaryotic initiation factor 4E (eIF4E) binding protein-1 (4E-BP1). We reported previously that simultaneous knockdown of S6K and eIF4E causes a transforming growth factor-β (TGF-β)-dependent G1 cell cycle arrest in MDA-MB-231 human breast cancer cells. Rapamycin inhibits the phosphorylation of S6K at nano-molar concentrations in MDA-MB-231 cells; however, micro-molar concentrations of rapamycin are required to inhibit phosphorylation of 4E-BP1 - the phosphorylation of which liberates eIF4E to initiate translation. Micro-molar doses of rapamycin are required for complete G1 cell cycle arrest - indicating that 4E-BP1 is a critical target of mTOR for promoting cell cycle progression. Data are provided demonstrating that G1 cell cycle arrest induced by rapamycin is due to up-regulation of TGF-β signaling and down-regulation of Rb phosphorylation via phosphorylation of the mTORC1 substrates S6K and 4E-BP1 respectively. These findings enhance the current understanding of the cytostatic effects of mTORC1 suppression with therapeutic implications.

Topics: Antibiotics, Antineoplastic; Breast Neoplasms; Cell Line, Tumor; Cyclin D1; Dose-Response Relationship, Drug; Female; G1 Phase Cell Cycle Checkpoints; Humans; MCF-7 Cells; Phosphorylation; Retinoblastoma Protein; Sirolimus; Transforming Growth Factor beta

β-Thujaplicin, one of the major constituents in Chamaecyparis obtusa, has been demonstrated to exert different health beneficial efficacy, but the role of β-thujaplicin in regulating mammary tumorigenesis has not been investigated. In this study, we found that β-thujaplicin significantly suppressed the proliferation through arresting the cell cycle transition from G1 to S phase as well as inhibited the expression of cell cycle-related proteins, cyclin D1, and cyclin-dependent kinase 4 (CDK4) in MCF-7 and T47D luminal subtype breast cancer cells. In addition, estrogen receptor α (ER-α) was down-regulated by β-thujaplicin via enhanced proteolysis by ubiquitination, which led to cell growth inhibition. These results suggest that β-thujaplicin may be considered as a potent agent regulating the hormone sensitive mammary tumorigenesis.

Topics: Anticarcinogenic Agents; Breast Neoplasms; Carcinogenesis; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Estrogen Receptor alpha; G1 Phase Cell Cycle Checkpoints; Humans; MCF-7 Cells; Monoterpenes; Proteolysis; Resting Phase, Cell Cycle; Signal Transduction; Tropolone

MicroRNAs (miRNA) are a large family of small single-stranded RNA molecules found in all multicellular organisms. Early studies have been shown that miRNA are involved in cancer development and progression, and this role can be done by working as an oncogenes and tumor suppressor genes, so manipulation of this molecules can be a promising approach in cancer therapy, and experimental results represented that the modification in breast cancer phenotype is possible by miRNA expression alteration. miR-16, which is located in 13q14 chromosome, plays critical roles as a tumor suppressor by targeting several oncogenes which regulate cell cycle and apoptosis. Hence, in the present study, we investigated whether miR-16 could decline growth and survival of MCF-7 cell line as model of human breast cancer. MCF-7 cell line was infected with lentiviruses containing miR-16 precursor sequence. The effects of ectopic expression of miR-16 on breast cancer phenotype were examined by cell cycle analysis and apoptosis assays. miR-16 cytotoxicity effect was measured by the MTT assay. We showed that the miR-16 overexpression reduces Cyclin D1 and BCL2 at messenger RNA (mRNA) and protein levels in MCF-7 cell line. In addition, this is found that enforced expression of miR-16 decreases cell growth and proliferation and induces apoptosis in MCF-7 cells. In conclusion, our results revealed that upregulation of miR-16 would be a potential approach for breast cancer therapy.

Topics: Annexin A5; Apoptosis; Breast Neoplasms; Cell Cycle; Cell Proliferation; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Lentivirus; MCF-7 Cells; MicroRNAs; Propidium; Proto-Oncogene Proteins c-bcl-2; Recombination, Genetic; RNA, Messenger

Breast cancer stem cells (BCSCs) constitute a small fraction of the primary tumor that can self-renew and become a drug-resistant cell population, thus limiting the treatment effects of chemotherapeutic drugs. The present study evaluated the cytotoxic effects of five phytochemicals including 6-gingerol (6-G), 6-shogaol (6-S), 5-hydroxy-3,6,7,8,3',4'-hexamethoxyflavone (5-HF), nobiletin (NOL), and pterostilbene (PTE) on MCF-7 breast cancer cells and BCSCs. The results showed that 6-G, 6-S, and PTE selectively killed BCSCs and had high sensitivity for BCSCs isolated from MCF-7 cells that expressed the surface antigen CD44(+)/CD24(-). 6-S and PTE induced cell necrosis phenomena such as membrane injury and bleb formation in BCSCs and inhibited mammosphere formation. In addition, 6-S and PTE increased the sensitivity of isolated BCSCs to chemotherapeutic drugs and significantly increased the anticancer activity of paclitaxel. Analysis of the underlying mechanism showed that 6-S and PTE decreased the expression of the surface antigen CD44 on BCSCs and promoted β-catenin phosphorylation through the inhibition of hedgehog/Akt/GSK3β signaling, thus decreasing the protein expression of downstream c-Myc and cyclin D1 and reducing BCSC stemness.

Topics: Antineoplastic Agents; Breast Neoplasms; Catechols; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Humans; Neoplastic Stem Cells; Oncogene Protein p55(v-myc); Signal Transduction; Stilbenes

Let-7 miRNAs act as tumour suppressors by directly binding to the 3'UTRs of downstream gene products. The regulatory role of let-7 in downstream gene expression has gained much interest in the cancer research community, as it controls multiple biological functions and determines cell fates. For example, one target of the let-7 family is cyclin D1, which promotes G0/S cell cycle progression and oncogenesis, was correlated with endoribonuclease DICER1, another target of let-7. Down-regulated let-7 has been identified in many types of tumours, suggesting a feedback loop may exist between let-7 and cyclin D1. A potential player in the proposed feedback relationship is Dicer, a central regulator of miRNA expression through sequence-specific silencing. We first identified that DICER1 is the key downstream gene for cyclin D1-induced let-7 expression. In addition, we found that let-7 miRNAs expression decreased because of the p53-induced cell death response, with deregulated cyclin D1. Our results also showed that cyclin D1 is required for Nutlin-3 and TAX-induced let-7 expression in cancer repression and the cell death response. For the first time, we provide evidence that let-7 and cyclin D1 form a feedback loop in regulating therapy response of cancer cells and cancer stem cells, and importantly, that alteration of let-7 expression, mainly caused by cyclin D1, is a sensitive indicator for better chemotherapies response.

Topics: 3' Untranslated Regions; Apoptosis; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Survival; Cyclin D1; DEAD-box RNA Helicases; Female; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; MCF-7 Cells; MicroRNAs; Microscopy, Fluorescence; Piperazines; Ribonuclease III; RNA Interference; Signal Transduction; Spheroids, Cellular; Tumor Suppressor Protein p53

The tumor-suppressor protein fragile histidine triad (Fhit) exerts its functions in the cytoplasm, although some reports suggest that it may also act in the nucleus. We previously showed that cytosolic Fhit protein levels in cancer cell lines stimulated to proliferate were reduced by proteasomal degradation. Here, we demonstrate that Fhit is physiologically present in the nucleus of breast cancer cell lines and tissues at a low level and that proliferative stimulation increases nuclear levels. Breast cancer cells expressing the FhitY114F mutant, which do not undergo proteasomal degradation, contained mutated Fhit in the nucleus, while cells treated with a proteasome inhibitor accumulated nuclear Fhit during proliferation. Thus, Fhit nuclear shuttling and proteasome degradation phenomena occur independently. When Fhit was coupled to a nuclear localization sequence, the proliferation rate of the transfected cells increased together with levels of proliferation pathway mediators cyclin D1, phospho-MAPK, and phospho-STAT3. Fhit nuclear translocation upon mitogenic stimulation may represent a new regulatory mechanism that allows rapid restoration of Fhit cytoplasmic levels and promotes the proliferation cascade activated by mitogenic stimulation.

Topics: Acid Anhydride Hydrolases; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Cyclin D1; Cytoplasm; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Humans; Mitogen-Activated Protein Kinase Kinases; Neoplasm Proteins; Proteasome Endopeptidase Complex; STAT3 Transcription Factor

Breast carcinoma is the leading cause of cancer-associated mortality in female individuals worldwide. Previous studies have investigated the pro-apoptotic and antimetastatic effects of statins, and have demonstrated that simvastatin exhibits antitumor activity and potent chemopreventive effects. However, the mechanism underlying the effects of simvastatin in breast cancer remains to be elucidated. The present study demonstrated that simvastatin inhibited the proliferation of MDA-MB-231 human breast cancer cells in a dose-dependent manner, decreased the protein expression of B cell lymphoma 2 (Bcl-2) and increased the protein expression of Bcl-2-associated X protein in time- and dose-dependent manners. In addition, simvastatin arrested cells in the G0/G1 phase of the cell cycle, downregulated the protein expression levels of cyclin D1 and cyclin-dependent kinase (CDK)2, mediated the mitochondria-dependent caspase cascade by increasing the protein expression levels of caspase-3, -8 and -9, and downregulated the protein expression of X-linked inhibitor of apoptosis, which induced cell apoptosis. In addition, simvastatin decreased the protein expression of matrix metalloproteinase (MMP)-2 and suppressed the activation of nuclear factor (NF)-κB in the MDA-MB-231 cells. Taken together, these results demonstrated that the antitumor effect of simvastatin in the human MDA-MB-231 breast cancer cell line was via the inhibition of cell proliferation, affecting the cell cycle, downregulating the expression levels of cyclin D1 and CDKs, inducing apoptosis and decreasing the expression of MMP-2, possibly by inhibiting the activation of NF-κB. Statin treatment may provide a novel therapeutic approach for the treatment of breast cancer.

Topics: Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Cell Cycle; Cell Proliferation; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; NF-kappa B; Proto-Oncogene Proteins c-bcl-2; Simvastatin

The death-associated protein 3 (DAP3) is a highly conserved phosphoprotein involved in the regulation of autophagy. A previous clinical study by our group suggested an association between low DAP3 expression and clinicopathological parameters of human breast cancer. In the present study, we intended to determine the role of DAP3 in cancer cell behaviour in the context of human breast cancer. We developed knockdown sub-lines of MCF7 and MDA-MB-231, and performed growth, adhesion, invasion assays and electric cell-substrate impedance sensing (ECIS) studies of post-wound migration of the cells. In addition, we studied the mRNA expression of caspase 8 and 9, death ligand signal enhancer (DELE), IFN-β promoter stimulator 1 (IPS1), cyclin D1 and p21 in the control and knockdown sub-lines. The knockdown sub-lines of MCF7 and MDA-MB-231 had significantly increased adhesion and decreased growth when compared to the controls. Furthermore, invasion and migration were significantly increased in the MDA-MB-231DAP3kd cells vs. the controls. The expression of caspase 9 and IPS1, known components of the apoptosis pathway, were significantly reduced in the MCF7DAP3kd cells (p=0.05 and p=0.003, respectively). We conclude that DAP3 silencing contributes to breast carcinogenesis by increasing cell adhesion, migration and invasion. It is possible that this may be due to the activity of focal adhesion kinase further downstream of the anoikis pathway. Further research in this direction would be beneficial in increasing our understanding of the mechanisms underlying human breast cancer.

Topics: Adaptor Proteins, Signal Transducing; Apoptosis; Apoptosis Regulatory Proteins; Breast Neoplasms; Caspase 8; Caspase 9; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Female; Focal Adhesion Protein-Tyrosine Kinases; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Mitochondrial Proteins; Neoplasm Invasiveness; Ribosomal Proteins; RNA-Binding Proteins; Signal Transduction

The disulfiram/copper complex (DS/Cu) has been demonstrated to exert potent anti-tumor effects in various types of cancer. At present, whether DS/Cu has chemopreventive effects on breast cancer development remains to be fully elucidated. In the present study, using MMTV-erbB2 transgenic mice, it was identified for the first time that DS/Cu treatment was able to inhibit cell growth in breast cancer cells while sparing normal cells in vitro, in addition to delaying the development of mammary tumor development in MMTV-erbB2 transgenic mice in vivo. Morphological examination demonstrated that DS/Cu treatment resulted in cell proliferation inhibition and apoptosis activation in vitro and in vivo. Furthermore, the present study observed that DS/Cu may inhibit proliferation via inhibition of AKT and cyclin D1 signaling and promote apoptosis via c-Jun N-terminal kinase activation and suppression of nuclear factor κB signaling. These results suggested that DS/Cu treatment may be a promising therapy for the prevention of erbB2-positive breast cancer.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Chemoprevention; Copper; Cyclin D1; Disulfiram; Female; Gene Expression Regulation, Neoplastic; Humans; Mammary Neoplasms, Animal; Mice; Mice, Transgenic; Oncogene Protein v-akt; Receptor, ErbB-2; Signal Transduction

Leptin is a potent adipokine that plays an important role in the progression of breast cancer and interferes with the action of tamoxifen. We investigated the molecular mechanism underlying the effect of leptin on tamoxifen resistance in breast cancer cells that express leptin receptor (ObRb), and evaluated the impact of ObRb suppression on tamoxifen treatment in MCF-7 and tamoxifen-resistant (TAM-R) cells. Leptin-induced signaling pathway activation was examined by qRT-PCR and Western blotting. Chromatin immunoprecipitation assays were performed to further examine the binding of estrogen receptor (ER) α on the promoter of cyclin D1 (CCND1) gene. The effects of combined ObRb knockdown and tamoxifen treatment were evaluated in MCF-7 and TAM-R cells. We found that the enhanced proliferation effects induced by leptin were related to extracellular-signal-regulated kinase (ERK) 1/2 and signal transducers and activators of transcription (STAT) 3 signaling pathway activation and CCND1 upregulation. Leptin enhanced CCND1 gene transcription by inducing the binding of ERα to the promoter of CCND1 gene. ObRb knockdown significantly enhanced the inhibitory effects of tamoxifen on TAM-R cell proliferation and survival. This study suggested that long-term endocrine therapy facilitates leptin and ObRb overexpression in breast cancer cells, which attenuates the inhibitory effect of tamoxifen by activating both the ERK1/2 and STAT3 signaling pathways and upregulating CCND1 gene expression. Combination therapy involving ObRb knockdown and tamoxifen treatment may be an alternative therapeutic option for tamoxifen-resistant breast cancer.

Topics: Antineoplastic Agents, Hormonal; Breast Neoplasms; Cell Proliferation; Cyclin D1; Drug Resistance, Neoplasm; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Humans; Leptin; MCF-7 Cells; Receptors, Leptin; Signal Transduction; Tamoxifen

Breast cancer is considered the most common cancer for women worldwide and it is now the second leading cause of cancer-related deaths among females in the world. Since breast cancer is highly resistant to chemotherapy, alternative anticancer strategies have been developed. In particular, many studies have demonstrated that curcumin, a derivative of turmeric, can be used as natural agent in treatment of some types of cancer by playing antiproliferative and antioxidant effects. In our study, we assessed the antitumor activities of curcumin in ER-negative human breast cancer cell line resistant to chemotherapy, MDA.MB231 by in vitro and in vivo experiments. In vitro data allowed us to demonstrate that curcumin played a role in regulation of proliferation and apoptosis in MDA.MB231 cells. In vivo, by generation of mouse model of breast cancer, we showed that treatment of curcumin inhibited tumor growth and angiogenesis. Specifically, we showed that curcumin is able to deregulate the expression of cyclin D1, PECAM-1, and p65, which are regulated by NF-κB. Our data demonstrated that curcumin could be used as an adjuvant agent to chemotherapy in treatment of triple negative breast cancer.

Topics: Animals; Apoptosis; Breast Neoplasms; Cell Proliferation; Curcumin; Cyclin D1; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Humans; Mice; Neovascularization, Pathologic; NF-kappa B; Triple Negative Breast Neoplasms; Xenograft Model Antitumor Assays

PPP2R2A deletions were recently linked to a subgroup of luminal breast carcinoma (BC) that exhibits poor survival. This subgroup also exhibited amplification of a chromosome region containing the Cyclin D1 coding gene, CCND1. Therefore, we aimed to investigate whether a combination of PPP2R2A (B55α) and Cyclin D1 expression statuses evaluated by immunohistochemistry (IHC) could define a subgroup of luminal BC that exhibits poor survival.. First we conducted a retrospective cohort study using sequencing data from The Cancer Genome Atlas initiative to correlate PPP2R2A copy number alteration (CNA) status with its expression level and the corresponding overall survival (OS). Next, also using a retrospective cohort study design, we evaluated the PPP2R2A (B55α) expression levels by IHC in a total of 807 BC patients from two independent cohorts (discovery cohort n = 349 and validation cohort n = 458). Cyclin D1 expression was also evaluated, and the PPP2R2A (B55α)(-/low)/Cyclin D1(high) phenotype was evaluated as a predictor of disease-free survival (DFS) and OS in luminal-like BC patients.. Deletions in the PPP2R2A gene strongly correlate with lower mRNA expression and poorer OS. PPP2R2A (B55α)(-/low) carcinomas have significantly shorter DFS and OS. Furthermore, in univariate analysis, the PPP2R2A (B55α)(-/low)/Cyclin D1(high) phenotype is significantly associated with poorer DFS and OS. In a multivariate analysis, the PPP2R2A (B55α)(-/low)/Cyclin D1(high) phenotype is significantly associated with poor DFS, thus defining a group of luminal-like BC with higher risk of relapse.. We demonstrate that BCs harboring PPP2R2A deletions are associated with worse OS. Moreover, this is the first study to demonstrate that the combination of altered PPP2R2A (B55α) and high Cyclin D1 expression by IHC defines a subgroup of luminal-like BC patients with a high risk of relapse and death.

Topics: Adult; Aged; Breast Neoplasms; Cyclin D1; Female; Gene Deletion; Gene Dosage; Humans; Middle Aged; Prognosis; Protein Phosphatase 2; Retrospective Studies; Survival Analysis

We previously reported that high levels of tissue factor (TF) can induce cellular apoptosis in endothelial cells. In this study, TF-mediated mechanisms of induction of apoptosis were explored. Endothelial cells were transfected to express wild-type TF. Additionally, cells were transfected to express Asp253-substituted, or Ala253-substitued TF to enhance or prevent TF release, respectively. Alternatively, cells were pre-incubated with TF-rich and TF-poor microvesicles. Cell proliferation, apoptosis and the expression of cyclin D1, p53, bax and p21 were measured following activation of cells with PAR2-agonist peptide. Greatest levels of cell proliferation and cyclin D1 expression were observed in cells expressing wild-type or Asp253-substituted TF. In contrast, increased cellular apoptosis was observed in cells expressing Ala253-substituted TF, or cells pre-incubated with TF-rich microvesicles. The level of p53 protein, p53-phosphorylation at ser33, p53 nuclear localisation and transcriptional activity, but not p53 mRNA, were increased in cells expressing wild-type and Ala253-substituted TF, or in cells pre-incubated with TF-rich microvesicles. However, the expression of bax and p21 mRNA, and Bax protein were only increased in cells pre-incubated with TF-rich microvesicle and in cells expressing Ala253-substituted TF. Inhibition of the transcriptional activity of p53 using pifithrin-α suppressed the expression of Bax. Finally, siRNA-mediated suppression of p38α, or inhibition using SB202190 significantly reduced the p53 protein levels, p53 nuclear localisation and transcriptional activity, suppressed Bax expression and prevented cellular apoptosis. In conclusion, accumulation of TF within endothelial cells, or sequestered from the surrounding can induce cellular apoptosis through mechanisms mediated by p38, and involves the stabilisation of p53.

Topics: Amino Acid Substitution; Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Cardiovascular Diseases; Cell Line, Tumor; Cell-Derived Microparticles; Cells, Cultured; Coronary Vessels; Cyclin D1; Endothelial Cells; Enzyme Activation; Female; Gene Expression Regulation; Genes, Reporter; Humans; Imidazoles; MAP Kinase Signaling System; Oligopeptides; p38 Mitogen-Activated Protein Kinases; Pyridines; Recombinant Fusion Proteins; RNA Interference; RNA, Messenger; RNA, Small Interfering; Thromboplastin; Transfection; Tumor Suppressor Protein p53

Anxa2 is dysregulated in many types of carcinomas and implicated in several pivotal biological functions, such as angiogenesis, cell proliferation, invasion, and metastasis. We previously demonstrated that upregulation of Anxa2 enhances the proliferation and invasion of breast cancer cells. However, the detailed mechanism remains unclear. In this study, co-immunoprecipitation and LC-MS/MS-based interactome approach were employed to screen potential Anxa2 binding proteins. A total of 312 proteins were identified as candidate Anxa2 interacting partners. Using Gene Ontology, pathway annotation, and protein-protein interaction analyses, we constructed a connected network for Anxa2 interacting proteins, and Ebp1 may function as a "hub" in the Anxa2 interaction network. Moreover, Ebp1 knockdown resulted in enhanced cell proliferation and invasion, as well as increased expression of Anxa2. Furthermore, the abundance of cyclin D1 and the phosphorylation of Erk1/2 were increased in Ebp1 inhibited cells. This finding is consistent with a previous study, in which upregulation of Anxa2 results in an increased cyclin D1 expression and Erk1/2 activation. Our results suggest a novel function of Ebp1 as a binding protein and negative regulator of Anxa2. The functional association between Anxa2 and EBP1 may also participate in regulating cancer cell proliferation and invasion, thereby contributing to cancer progression.

Topics: Adaptor Proteins, Signal Transducing; Annexin A2; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Humans; Neoplasm Invasiveness; Phosphorylation; RNA-Binding Proteins; Wound Healing

Deletions of chromosome 8p occur frequently in breast cancers, but analyses of its clinical relevance have been limited to small patient cohorts and provided controversial results. A tissue microarray with 2,197 breast cancers was thus analyzed by fluorescence in-situ hybridization using an 8p21 probe in combination with a centromere 8 reference probe. 8p deletions were found in 50% of carcinomas with no special type, 67% of papillary, 28% of tubular, 37% of lobular cancers and 56% of cancers with medullary features. Deletions were always heterozygous. 8p deletion was significantly linked to advanced tumor stage (P < 0.0001), high-grade (P < 0.0001), high tumor cell proliferation (Ki67 Labeling Index; P < 0.0001), and shortened overall survival (P < 0.0001). For example, 8p deletion was seen in 32% of 290 grade 1, 43% of 438 grade 2, and 65% of 427 grade 3 cancers. In addition, 8p deletions were strongly linked to amplification of MYC (P < 0.0001), HER2 (P < 0.0001), and CCND1 (p = 0.001), but inversely associated with ER receptor expression (p = 0.0001). Remarkably, 46.5% of 8p-deleted cancers harbored amplification of at least one of the analyzed genes as compared to 27.5% amplifications in 8p-non-deleted cancers (P < 0.0001). In conclusion, 8p deletion characterizes a subset of particularly aggressive breast cancers. As 8p deletions are easy to analyze, this feature appears to be highly suited for future DNA based prognostic breast cancer panels. The strong link of 8p deletion with various gene amplifications raises the possibility of a role for regulating genomic stability.

Topics: Adult; Aged; Aged, 80 and over; Breast Neoplasms; Chromosome Deletion; Chromosomes, Human, Pair 8; Cyclin D1; Female; Gene Amplification; Genes, myc; Humans; Immunohistochemistry; In Situ Hybridization, Fluorescence; Ki-67 Antigen; Middle Aged; Prognosis; Receptor, ErbB-2; Tissue Array Analysis

17β-hydroxysteroid dehydrogenase (17β-HSD) type 1 is known as a critical target to block the final step of estrogen production in estrogen-dependent breast cancer. Recent confirmation of the role of dyhydroxytestosterone (DHT) in counteracting estrogen-induced cell growth prompted us to study the reductive 17β-HSD type 7 (17β-HSD7), which activates estrone while markedly inactivating DHT. The role of DHT in breast cancer cell proliferation is demonstrated by its independent suppression of cell growth in the presence of a physiological concentration of estradiol (E2). Moreover, an integral analysis of a large number of clinical samples in Oncomine datasets demonstrated the overexpression of 17β-HSD7 in breast carcinoma. Inhibition of 17β-HSD7 in breast cancer cells resulted in a lower level of E2 and a higher level of DHT, successively induced regulation of cyclinD1, p21, Bcl-2, and Bik, consequently arrested cell cycle in the G(0)/G(1) phase, and triggered apoptosis and auto-downregulation feedback of the enzyme. Such inhibition led to significant shrinkage of xenograft tumors with decreased cancer cell density and reduced 17β-HSD7 expression. Decreased plasma E2 and elevated plasma DHT levels were also found. Thus, the dual functional 17β-HSD7 is proposed as a novel target for estrogen-dependent breast cancer by regulating the balance of E2 and DHT. This demonstrates a conceptual advance on the general belief that the major role of this enzyme is in cholesterol metabolism.

Topics: 17-Hydroxysteroid Dehydrogenases; Androgens; Apoptosis; Apoptosis Regulatory Proteins; Breast Neoplasms; Cell Cycle Checkpoints; Cell Proliferation; Cholesterol; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Dihydrotestosterone; Estradiol; Estradiol Dehydrogenases; Estrogens; Estrone; Female; G1 Phase; Humans; MCF-7 Cells; Membrane Proteins; Mitochondrial Proteins; Multifunctional Enzymes; Proto-Oncogene Proteins c-bcl-2; Resting Phase, Cell Cycle

Polymeric nanoparticles are attractive materials that have been widely used in medicine for drug delivery, with therapeutic applications. In our study, polymeric nanoparticles and the anticancer drug, chrysin, were encapsulated into poly (D, L-lactic-co-glycolic acid) poly (ethylene glycol) (PLGA-PEG) nanoparticles for local treatment.. PLGA: PEG triblock copolymers were synthesized by ring-opening polymerization of D, L-lactide and glycolide as an initiator. The bulk properties of these copolymers were characterized using 1H nuclear magnetic resonance spectroscopy and Fourier transform infrared spectroscopy. In addition, the resulting particles were characterized by scanning electron microscopy.. The chrysin encapsulation efficiency achieved for polymeric nanoparticles was 70% control of release kinetics. The cytotoxicity of different concentration of pure chrysin and chrysin loaded in PLGA-PEG (5-640μM) on T47-D breast cancer cell line was analyzed by MTT-assay.. There is potential for use of these nanoparticles for biomedical applications. Future work should include in vivo investigation of the targeting capability and effectiveness of these nanoparticles in the treatment of breast cancer.

Topics: Apoptosis; Blotting, Western; Breast Neoplasms; Cell Cycle; Cell Proliferation; Cyclin D1; Drug Carriers; Drug Delivery Systems; Female; Flavonoids; Humans; Lactic Acid; Nanoparticles; Polyethylene Glycols; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured

PFTK1 was a cell division cycle 2-related serine/threonine protein kinase, which was up-regulated in breast cancer tissues and breast cancer lines. And up-regulated PFTK1 was highly associated with grade, axillary lymph node status, and Ki-67. Moreover, Kaplan-Meier curve showed that up-regulated PFTK1 was related to the poor breast carcinoma patients' overall survival. Here, we first discovered and confirmed that cyclin B was a new interacting protein of PFTK1, and the complex might increase the amount of DVL2, which triggers Wnt/β-catenin signaling pathway. Furthermore, knockdown of PFTK1 attenuated cell proliferation, anchorage-independent cell growth, and cell migration and invasion by inhibiting the transcriptional activation of β-catenin for cyclin D1, MMP9, and HEF1, whereas exogenous expression of PFTK1 might promote MDA-MB-231 cells proliferation, migration, and invasion via promoting PFTK1-DVL2-β-catenin axis. Our findings supported the notion that up-regulated PFTK1 might promote breast cancer progression and metastasis by activating Wnt signaling pathway through the PFTK1-DVL2-β-catenin axis.

Topics: Adaptor Proteins, Signal Transducing; beta Catenin; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinases; Dishevelled Proteins; Female; Gene Expression Regulation, Neoplastic; Humans; Matrix Metalloproteinase 9; MCF-7 Cells; Middle Aged; Neoplasm Invasiveness; Phosphoproteins; Up-Regulation; Wnt Signaling Pathway

Thyroid hormone level has been positively associated with breast cancer risk and with breast cancer cell proliferation and growth. Although breast cancer is a heterogeneous disease, this is the first study assessing pre-diagnostic levels of free triiodothyronine (fT3), free thyroxine (fT4), thyroid-stimulating hormone (TSH), and thyroid peroxidase antibodies (TPO-Ab) in relation to breast cancer subgroups and aggressiveness.. The Malmö Diet and Cancer Study collected blood samples from 17,035 women between 1991 and 1996. Free T3, free T4, TSH, and TPO-Ab were analyzed in 676 incident breast cancer cases and 680 controls. Breast tumors were classified according to tumor size, axillary lymph node involvement, histological grade, histological type, hormone receptor status (ER, PgR), as well as Ki67, cyclin D1, and p27. Odds ratios of different breast cancer subgroups were calculated using a logistic regression analysis adjusted for potential confounders.. High fT4 was associated with a statistically significant higher risk of overall breast cancer, small, grade I, ER-positive, PgR-positive, and cyclin D1 low tumors. The associations for ER and PgR were verified in a heterogeneity analysis. Low TPO-Ab was associated with a higher risk of overall breast cancer, ductal, large, ER-positive, PgR-positive, cyclin D1 low, and p27 high tumors. The heterogeneity analysis verified the association for tumor size. Free T3 was not associated with overall breast cancer risk, but in the heterogeneity analysis, high fT3 was associated with tumor size and expression of p27. There were no strong associations between TSH and overall breast cancer risk or any tumor subgroup.. High pre-diagnostic fT4 levels and low pre-diagnostic TPO-Ab levels were associated with an increased risk of breast cancer. This increase was mainly limited to a higher incidence rate of less aggressive breast cancer subgroups.

Topics: Aged; Antibodies; Breast; Breast Neoplasms; Cyclin D1; Female; Humans; Iodide Peroxidase; Ki-67 Antigen; Middle Aged; Proliferating Cell Nuclear Antigen; Receptors, Estrogen; Receptors, Progesterone; Risk; Sweden; Thyrotropin; Thyroxine; Triiodothyronine

Obesity greatly influences risk, progression and prognosis of breast cancer. As molecular effects of obesity are largely mediated by adipocytokine leptin, finding effective novel strategies to antagonize neoplastic effects of leptin is desirable to disrupt obesity-cancer axis. Present study is designed to test the efficacy of honokiol (HNK), a bioactive polyphenol from Magnolia grandiflora, against oncogenic actions of leptin and systematically elucidate the underlying mechanisms. Our results show that HNK significantly inhibits leptin-induced breast-cancer cell-growth, invasion, migration and leptin-induced breast-tumor-xenograft growth. Using a phospho-kinase screening array, we discover that HNK inhibits phosphorylation and activation of key molecules of leptin-signaling-network. Specifically, HNK inhibits leptin-induced Wnt1-MTA1-β-catenin signaling in vitro and in vivo. Finally, an integral role of miR-34a in HNK-mediated inhibition of Wnt1-MTA1-β-catenin axis was discovered. HNK inhibits Stat3 phosphorylation, abrogates its recruitment to miR-34a promoter and this release of repressor-Stat3 results in miR-34a activation leading to Wnt1-MTA1-β-catenin inhibition. Accordingly, HNK treatment inhibited breast tumor growth in diet-induced-obese mouse model (exhibiting high leptin levels) in a manner associated with activation of miR-34a and inhibition of MTA1-β-catenin. These data provide first in vitro and in vivo evidence for the leptin-antagonist potential of HNK revealing a crosstalk between HNK and miR34a and Wnt1-MTA1-β-catenin axis.

Topics: Animals; Antineoplastic Agents, Phytogenic; beta Catenin; Biphenyl Compounds; Breast Neoplasms; Cell Movement; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin D1; Drugs, Chinese Herbal; Female; Histone Deacetylases; Humans; Leptin; Lignans; Magnolia; MCF-7 Cells; Mice; Mice, Nude; Mice, Obese; MicroRNAs; Neoplasm Invasiveness; Obesity; Phosphorylation; Plant Extracts; Promoter Regions, Genetic; Repressor Proteins; RNA Interference; RNA, Small Interfering; Signal Transduction; Spheroids, Cellular; STAT3 Transcription Factor; Trans-Activators; Tumor Cells, Cultured; Wnt1 Protein; Xenograft Model Antitumor Assays

Breast cancer is one of the most common malignancies and a major cause of cancer-related mortality all over the world. A growing body of reports revealed that microRNAs play essential roles in the progression of cancers. Aberrant expression of miR-503 has been reported in several kinds of cancer. The aim of the current study was to elucidate the role of miR-503 in the pathogenesis of breast cancer. In the present study, our results suggested that miR-503 expression was markedly downregulated in breast cancer tissues and cells. Overexpression of miR-503 in breast cancer cell lines reduced cell proliferation through inducing G0/G1 cell cycle arrest by targeting CCND1. Together, our findings provide new knowledge regarding the role of miR-503 in the progression of breast cancer and indicate the role of miR-503 as a tumor suppressor microRNA (miRNA) in breast cancer.

Topics: Breast Neoplasms; Cell Proliferation; Cyclin D1; Female; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; MicroRNAs

Apelin-13 is extensively expressed in various tissues, particularly breast tissue. Apelin‑13 has been shown to promote tumor proliferation in various types of cancer, including hepatocellular, lung and ovarian cancer. However, the effect and molecular mechanism of apelin‑13 in breast cancer cells remains unclear. The present study investigated the effect of apelin‑13 on MCF‑7. Therefore, cell proliferation was determined by MTT and flow cytometry analysis. The results revealed that apelin‑13 markedly increased cell proliferation. Transwell assays demonstrated that apelin‑13 increased MCF‑7 cell invasion. Apelin‑13 also markedly increased the expression of cyclin D1, extracellular matrix metalloproteinase‑1 and amplified in breast cancer 1 (AIB1) in a dose‑dependent manner by polymerase chain reaction assays. To study the molecular mechanism, cell proliferation, invasion and cyclin D1 were inhibited by pre‑treatment with 10 µM of PD98059 (ERK(1/2) inhibitor). Western blotting results suggested that apelin‑13 significantly enhances the expression of p‑ERK(1/2) in a concentration‑dependent manner. In conclusion, the results suggest that apelin‑13 promoted MCF-7 cell proliferation and invasion via the ERK1/2/AIB1 signaling pathway.

Topics: Breast; Breast Neoplasms; Cell Proliferation; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Intercellular Signaling Peptides and Proteins; MAP Kinase Signaling System; Matrix Metalloproteinase 1; MCF-7 Cells; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neoplasm Invasiveness; Nuclear Receptor Coactivator 3; Phosphorylation

Topics: Animals; Breast Neoplasms; Cyclin D1; Female; Humans; Mice; Neoplasms; Protein Kinases; Receptor, ErbB-2

The herb Momordica cochinchinensis has been used for a variety of purposes, and been shown to have anti‑cancer properties. The present study assessed the potency and the underlying mechanisms of action of the ethyl acetate extract of seeds of Momordica cochinchinensis (ESMC2) on breast cancer cells. Therefore, the effects of ESMC2 on the cell viability, cell cycle and apoptosis of MDA‑MB‑231 cells were investigated. The results showed that ESMC2 exerted a marked growth inhibitory effect on the cells. Cell cycle arrest in G2 phase following treatment with ESMC2 was associated with a marked increase in the protein levels of cyclin B1, cyclin E and cyclin-dependent kinase 1 and a decrease in cyclin D1 expression. In addition, ESMC2 dose‑dependently induced cell apoptosis, which was mediated via upregulation of the apoptosis-associated proteins p53, B-cell lymphoma 2 (Bcl‑2)‑associated X protein, Bcl-2 homologous antagonist killer and Bcl-2-associated death promoter expression, as well as downregulation of nuclear factor kappa B, Bcl‑2 and myeloid cell leukemia‑1. Furthermore, the activation of extracellular signal-regulated kinase 1/2, p38, c-Jun N-terminal kinase (JNK) and Akt phosphorylation were decreased by ESMC2 in a dose‑dependent manner, indicating that ESMC2 exerted its effects via the mitogen-activated protein kinase/JNK pathway. Furthermore, nude mouse xenotransplant models were used to evaluate the tumor growth inhibitory effects of ESMC2. The possible chemical components of ESMC2 were analyzed by gas chromatography-mass spectrometry, and 12 compounds were detected from the major peaks based on the similarity index with entries of a compound database. The results of the present study may aid in the development of novel therapies for breast cancer.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; CDC2 Protein Kinase; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Survival; Cyclin B1; Cyclin D1; Cyclin E; Cyclin-Dependent Kinases; Dose-Response Relationship, Drug; Down-Regulation; G2 Phase; Gas Chromatography-Mass Spectrometry; Humans; JNK Mitogen-Activated Protein Kinases; Mitogen-Activated Protein Kinase 3; Momordica; Myeloid Cell Leukemia Sequence 1 Protein; NF-kappa B; Oncogene Proteins; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Plant Extracts; Seeds; Tumor Suppressor Protein p53

Although the upregulated expression of Anxa2 has been implicated in carcinogenesis, cancer progression, and poor prognosis of cancer patients, the detailed molecular mechanisms involved in these processes remain unclear. In this study, we investigated the effect of Anxa2 downregulation with small interference RNA on breast cancer proliferation. To explore molecular mechanisms underlying Anxa2-mediated cancer cell proliferation. We analyzed cell cycle distribution and signaling pathways using semi-quantitative real-time PCR and Western blotting. Anxa2 depletion in breast cancer cells significantly inhibited cell proliferation by decelerating cell cycle progression. The retarded G1-to-S phase transition in Anxa2-silenced cells was attributed to the decreased levels of cyclin D1, which is a crucial promoting factor for cell proliferation because it regulates G1-to-S phase transition during cell cycle progression. We provided evidence that Anxa2 regulates epidermal growth factor-induced phosphorylation of STAT3. The reduced expression of phosphorylated STAT3 is the main factor responsible for decreased cyclin D1 levels in Anxa2-silenced breast cancer cells. Our results revealed the direct relationship between Anxa2 and activation of STAT3, a key transcription factor that plays a pivotal role in regulating breast cancer proliferation and survival. This study provides novel insights into the functions of Anxa2 as a critical molecule in cellular signal transduction and significantly improves our understanding of the mechanism through which Anxa2 regulates cell cycle and cancer cell proliferation.

Topics: Annexin A2; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Gene Silencing; Humans; MAP Kinase Signaling System; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Ribosomal Protein S6 Kinases, 70-kDa; RNA Interference; RNA, Messenger; Signal Transduction; STAT3 Transcription Factor

Dipeptidyl peptidase 4 (DPP4) is an aminopeptidase that is widely expressed in different cell types. Recent studies suggested that DPP4 plays an important role in tumour progression in several human malignancies. Here we have examined the mechanisms by which up-regulation of DPP4 expression causes epithelial transformation and mammary tumourigenesis.. Expression of DPP4 and the peptidylprolyl cis/trans isomerase, NIMA-interacting 1 (PIN1), and the cytotoxic effects of combined treatment with sitagliptin and juglone were investigated by immunohistochemistry, immunoblotting, real-time PCR, TUNEL and soft agar assays, using MCF7 cells. The effects of sitagliptin on tumour development in vivo were studied in the syngeneic 4T1 metastatic breast cancer model.. Activity of the transcription factor E2F1 induced by EGF was enhanced by DPP4, thus increasing PIN1 expression. Furthermore, DPP4 enhanced MEK/ERK and JNK/c-Jun signalling induced by EGF, inducing AP-1 activity and epithelial cell transformation. In contrast, DPP4 silencing or DPP4 inhibition in MCF7 cells inhibited PIN1 expression via E2F1 activity induced by EGF, decreasing colony formation and inducing DNA fragmentation. In the syngeneic 4T1 metastatic breast cancer model, DPP4 overexpression increased tumour development, whereas treatment with sitagliptin and/or juglone suppressed it. Consistent with these observations, DPP4 levels were positively correlated with PIN1 expression in human breast cancer.. DPP4 promoted EGF-induced epithelial cell transformation and mammary tumourigenesis via induction of PIN1 expression, suggesting that sitagliptin targeting of DPP4 could be a treatment strategy in patients with breast cancer.

Topics: Animals; Apoptosis; Breast Neoplasms; Cell Transformation, Neoplastic; Cyclin D1; Dipeptidyl Peptidase 4; Epidermal Growth Factor; Female; Humans; MCF-7 Cells; Mice; Mice, Inbred BALB C; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; Protein Kinases; Signal Transduction; Sitagliptin Phosphate; Transcription Factor AP-1; Up-Regulation

MicroRNAs (miRNAs) have been identified as important regulators that potentially play critical roles in various biological and pathological processes of cancer cells. The aim of the present study was to investigate the expression of miR-32 in breast cancer and its biological role in tumor progression. MiR-32 expression was markedly upregulated in breast cancer tissues and breast cancer cells. Ectopic expression of miR-32 promoted cell proliferation of breast cancer, whereas miR-32-in suppressed this function. Mechanically, data from luciferase reporter assays revealed that miR-32 directly targeted to the 3'-untranslated region (3'-UTR) of PHLPP2. Overexpression of miR-32 led to downregulation of PHLPP2 protein, which resulted in the downregulation of p21 and upregulation of cyclin D1 and p-Rb. In functional assays, PHLPP2-silenced in miR-32-in-transfected ZR-75-30 cells have positive effect to promote cell proliferation, suggesting that direct PHLPP2 downregulation is required for miR-32-induced cell proliferation of breast cancer. Our findings highlighted the importance of miR-32 in promoting tumor progression, and implicate miR-32 as a potential therapeutic target in breast cancer.

Topics: 3' Untranslated Regions; Adult; Binding Sites; Breast Neoplasms; Case-Control Studies; Cell Proliferation; Cyclin D1; Down-Regulation; Female; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; MicroRNAs; Phosphoprotein Phosphatases; Phosphorylation; Retinoblastoma Protein; RNA Interference; Signal Transduction; Time Factors; Transfection

Accumulating evidence has suggested that fibroblast growth factor 3 (FGF3) is expressed in breast cancer and correlates with the stage and grade of the disease. In the present study, a specific FGF3‑binding peptide (VLWLKNR, termed FP16) was isolated from a phage display heptapeptide library with FGF3. The peptide FP16 contained four identical (WLKN) amino acids and demonstrated high homology to the peptides of the 188‑194 (TMRWLKN) site of the high‑affinity FGF3 receptor fibroblast growth factor receptor 2. Functional analyses indicated that FP16 mediated significant inhibition of FGF3‑induced cell proliferation, arrested the cell cycle at the G0/G1 phase by increasing proliferation‑associated protein 2G4, suppressing cyclin D1 and proliferating cell nuclear antigen, and inhibited the FGF3‑induced activation of extracellular signal‑regulated kinase 1/2 and Akt kinase. Taken together, these results demonstrated that the peptide FP16, acting as an FGF3 antagonist, is a promising therapeutic agent for the treatment of breast cancer.

Topics: Amino Acid Sequence; Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Drug Evaluation, Preclinical; Female; Fibroblast Growth Factor 3; G1 Phase Cell Cycle Checkpoints; Humans; Peptide Library; Peptides; Phosphorylation; Protein Binding; Proto-Oncogene Proteins c-akt; Receptors, Fibroblast Growth Factor; Recombinant Proteins; Signal Transduction

Leptin, a major adipocytokine produced by adipocytes, is emerging as a key molecule linking obesity with breast cancer therefore, it is important to find effective strategies to antagonize oncogenic effects of leptin to disrupt obesity-cancer axis. Here, we examine the potential of honokiol (HNK), a bioactive polyphenol from Magnolia grandiflora, as a leptin-antagonist and systematically elucidate the underlying mechanisms. HNK inhibits leptin-induced epithelial-mesenchymal-transition (EMT), and mammosphere-formation along with a reduction in the expression of stemness factors, Oct4 and Nanog. Investigating the downstream mediator(s), that direct leptin-antagonist actions of HNK; we discovered functional interactions between HNK, LKB1 and miR-34a. HNK increases the expression and cytoplasmic-localization of LKB1 while HNK-induced SIRT1/3 accentuates the cytoplasmic-localization of LKB1. We found that HNK increases miR-34a in LKB1-dependent manner as LKB1-silencing impedes HNK-induced miR-34a which can be rescued by LKB1-overexpression. Finally, an integral role of miR-34a is discovered as miR-34a mimic potentiates HNK-mediated inhibition of EMT, Zeb1 expression and nuclear-localization, mammosphere-formation, and expression of stemness factors. Leptin-antagonist actions of HNK are further enhanced by miR-34a mimic whereas miR-34a inhibitor results in inhibiting HNK's effect on leptin. These data provide evidence for the leptin-antagonist potential of HNK and reveal the involvement of LKB1 and miR-34a.

Topics: AMP-Activated Protein Kinase Kinases; Animals; Antineoplastic Agents, Phytogenic; Biphenyl Compounds; Blotting, Western; Breast Neoplasms; Cell Line; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Leptin; Lignans; MCF-7 Cells; Mice; MicroRNAs; Microscopy, Confocal; Protein Serine-Threonine Kinases; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Xenograft Model Antitumor Assays

To observe the effects of cyclopamine on the biological characteristics of human breast cancer MCF-7 cell line and explore its mechanism.. After human breast cancer MCF-7 cells were treated with different-concentration cyclopamine for different periods, MTT assay was used to detect the inhibitory effect of cyclopamine on MCF-7 cell proliferation, flow cytometry was used to determine the distribution of MCF-7 cell cycle and the effect of cyclopamine on MCF-7 apoptosis, and Western blot was used to measure the protein levels of cyclins D1 and p21 in MCF-7 cells.. In certain range, MCF-7 cell proliferation was inhibited by cyclopamine in a dose- and time-dependent manner, and the optimal inhibiting concentration was ten µmol/L and the optimal action time at 48 hours. With the time prolongation of cyclopamine action, the cells in G0/G1 phase were significantly increased, but the cells in S phase were significantly decreased (compared with blank control group, allp < 0.05). With the time prolongation of cyclopamine action, apoptosis rate of MCF-7 cells was also significantly increased (compared with blank control group, allp < 0.05). The level of cyclin D1 of MCF-7 cells was decreased, but cyclin p21 was increased (compared with blank control group, all p < 0.05).. Cyclopamine inhibits MCF-7 cell proliferation via arresting MCF-7 cell transformation from G1 phase to S phase. This may be associated with the expressions of Hedgehog (Hh) signaling pathway-related cyclins.

Topics: Apoptosis; Breast Neoplasms; Cell Cycle; Cell Proliferation; Cyclin D1; Dose-Response Relationship, Drug; Female; Flow Cytometry; Hedgehog Proteins; Humans; MCF-7 Cells; Veratrum Alkaloids

We previously showed the anticancer effect of crocin, a saffron carotenoid, in both breast and gastric cancers in animal models, but its mechanism of action is not clearly known, yet. In this study, the effect of crocin on cell cycle regulators is investigated. Female Wistar Albino rats were divided into two groups, with or without N-nitroso-N-methylurea (NMU) injection. After tumor formation, each group of rats was divided into two subgroups, receiving crocin or vehicle only. After 5 weeks, the rats were sacrificed and the tumors were retained for pathologic investigation and determination of the parameters. Before crocin treatment, the tumor volumes were 13.27±3.77 and 12.37±1.88, but at the end of the experiment, they were 23.66±8.82 and 11.91±2.27 in the control and crocin-treated groups, respectively. Pathologic investigation indicated the adenocarcinoma induction by NMU. Reverse transcription-polymerase chain reaction and Western blot analysis showed overexpression of cyclin D1 and p21(Cip1) in the NMU-induced breast tumors; however, the expression of both of them suppressed by crocin treatment. The previous studies indicated that crocin induces apoptosis in tumor tissue. In this study, we show that it also suppresses tumor growth and induces cell cycle arrest by downregulation of cyclin D1. In addition, crocin suppressed p21(Cip1) in a p53-dependent manner.

Topics: Animals; Antineoplastic Agents, Phytogenic; Breast Neoplasms; Carotenoids; Cell Cycle; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Female; Gene Expression Regulation, Neoplastic; Genes, p53; Mammary Neoplasms, Experimental; Methylnitrosourea; Rats, Wistar

Cancer stem cells (CSCs) play important roles in the formation, growth and recurrence of tumors, particularly following therapeutic intervention. Salinomycin has received recent attention for its ability to target breast cancer stem cells (BCSCs), but the mechanisms of action involved are not fully understood. In the present study, we sought to investigate the mechanisms responsible for salinomycin's selective targeting of BCSCs and its anti-tumor activity. Salinomycin suppressed cell viability, concomitant with the downregulation of cyclin D1 and increased p27(kip1) nuclear accumulation. Mammosphere formation assays revealed that salinomycin suppresses self-renewal of ALDH1-positive BCSCs and downregulates the transcription factors Nanog, Oct4 and Sox2. TUNEL analysis of MDA-MB-231-derived xenografts revealed that salinomycin administration elicited a significant reduction in tumor growth with a marked downregulation of ALDH1 and CD44 levels, but seemingly without the induction of apoptosis. Our findings shed further light on the mechanisms responsible for salinomycin's effects on BCSCs.

Topics: Aldehyde Dehydrogenase 1 Family; Animals; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Down-Regulation; Female; Humans; Hyaluronan Receptors; Isoenzymes; MCF-7 Cells; Mice, Inbred BALB C; Mice, Nude; Neoplastic Stem Cells; Pyrans; Retinal Dehydrogenase; Spheroids, Cellular; Transcription Factors; Xenograft Model Antitumor Assays

To investigate the preventing and treating action of Liuweidihuang pill (LP) and Jinkuishenqi pill (JP) on spontaneous breast carcinoma in mice.. A model of spontaneous breast carcinoma was derived from 11.5-month-old female Kunming breeding mice following the delivery of several litters. The mice were randomly divided into five groups: model control group (C), Liuweidihuang pill high-dose group (LH; 4.6 g · kg(-1) · d(-1)), Liuweidihuang pill low-dose group (LL; 2.3 g · kg(-1) · d(-1)), Jinkuishenqi pill high-dose group (JH; 4.6 g · kg(-1) · d(-1)) and Jinkuishenqi pill low-dose group (JL; 2.3 g · kg(-1) · d(-1)). Cancer tissue volume was measured by water immersion. Histopathology was analyzed by hematoxylin and eosin staining. Vascular endothelial growth factor (VEGF), extracellular signal-regulated kinase (ERK) and cyclin D1 protein expression in cancer tissue was assayed by western blotting.. Compared with the control group, cancer tissue volume and weight were lower in the LP and JP groups, and survival time was longer. The expression of VEGF, ERK and Cyclin D1 were inhibited in the LP and JP groups (P < 0.05), and cell differentiation was increased. Tumor weights and volumes and VEGF, ERK and Cyclin D1 expression in LL or LH were significantly lower than in JL and JH (P < 0.01).. Both LP and JP could restrain cancer growth and promote cancer cell differentiation; moreover, LP was more effective than JP The likely mechanism of action was via inhibition of VEGF, ERK and cyclin D1.

Topics: Animals; Breast Neoplasms; Cell Proliferation; Cyclin D1; Drugs, Chinese Herbal; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Mice; Vascular Endothelial Growth Factor A

CD99 is a protein initially described in the Ewing sarcoma family of tumors, but growing evidence has shown its expression in other tumors of mesenchymal, hematopoietic and even epithelial origin. Some articles report CD99 in metaplastic carcinoma of the breast, a subtype of breast carcinoma (BC) with pronounced epithelial to mesenchymal (EMT) phenotype. Our aim was to analyse the potential relationship between CD99 and selected EMT (vimentin, E-cadherin, Twist) and proliferation markers (Ki-67, c-myc, cyclin D1, topoisomerase 2), molecular subtypes of BC, as well as overall survival (OS) and progression-free survival (PFS). In a group of 122 cases CD99 membrane expression was seen in 14 (11.5%) cases: strong in 11 (9%) and moderate in 3 (2.5%). Expression of CD99 correlated with low cyclin D1 index, high level of topoisomerase 2 expression and lack of progesterone receptor (PR) but not with EMT characteristics. Additionally, strong expression of CD99 correlated with triple negative molecular BC phenotype. CD99 was prognostically irrelevant for OS and PFS. CD99 correlates with selected proliferative markers and low ER/PR receptor status but not with patients' outcome in BC. Further studies are required to explain precisely its role in molecular pathogenesis of BC.

Topics: 12E7 Antigen; Adult; Antigens, CD; Antigens, Neoplasm; Biomarkers, Tumor; Breast Neoplasms; Cell Adhesion Molecules; Cyclin D1; Disease-Free Survival; DNA Topoisomerases, Type II; DNA-Binding Proteins; Epithelial-Mesenchymal Transition; Female; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Middle Aged; Phenotype; Prognosis; Tissue Array Analysis; Triple Negative Breast Neoplasms

Gene amplification is an important mechanism in the development and progression of cancer. Currently, gene amplification status is generally determined by in situ hybridization (ISH). Multiplex ligation-dependent probe amplification (MLPA) is a PCR-based method that allows copy number detection of up to 50 nucleic acid sequences in one reaction. The aim of the present study was to compare results for HER2, CCND1, MYC and ESR1 gene amplification detected by MLPA with fluorescent in situ hybridization (FISH) and chromogenic in situ hybridization (CISH) as clinically approved methods. Tissue samples of 170 invasive breast cancers were collected. All were ER positive. Tissue samples had previously been tested for HER2 using immunohistochemistry. Amplification of the selected genes were assessed using MLPA, FISH and CISH and results were compared. HER2 MLPA and ISH results were also compared with HER2 immunohistochemistry (IHC) which detects protein overexpression. Amplification of HER2, CCND1, MYC and ESR1 by MLPA were found in 9%, 19%, 20% and 2% of samples, respectively. Amplification of HER2, CCND1, MYC and ESR1 by FISH was noted in 7%, 16%, 16% and 1% of samples, respectively. A high level of concordance was found between MLPA/ FISH (HER2: 88%, CCND1: 88%, MYC: 86%, ESR1: 92%) and MLPA/ CISH (HER2: 84%). Of all IHC 3+ cases, 91% were amplified by MLPA. In IHC 2+ group, 31% were MLPA amplified. In IHC 1+ group, 2% were MLPA amplified. None of the IHC 0 cases were amplified by MLPA. Our results indicate that there is a good correlation between MLPA, IHC and ISH results. Therefore, MLPA can serve as an alternative to ISH for detection of gene amplification.

Topics: Breast Neoplasms; Cyclin D1; Estrogen Receptor alpha; Female; Gene Amplification; Gene Dosage; Humans; Immunohistochemistry; In Situ Hybridization; Multiplex Polymerase Chain Reaction; Proto-Oncogene Proteins c-myc; Receptor, ErbB-2; Sensitivity and Specificity

Obesity is associated with a worse breast cancer prognosis, while greater breast tumor estrogen receptor beta (ERβ) expression is correlated with improved therapy response and survival. The objective of this study was to determine the impact of obesity on breast cancer cell ERβ expression, which is currently unknown. We utilized an in vitro model of obesity in which breast cancer cells were exposed to patient serum pooled by body mass index category (obese (OB): ≥30 kg/m2; normal weight (N): 18.5-24.9 kg/m2). Four human mammary tumor cell lines representing the major breast cancer subtypes (SKBR3, MCF-7, ZR75, MDA-MB-231) and mammary tumor cells from MMTV-neu mice were used. ERβ expression, assessed by qPCR and western blotting, was suppressed in the two HER2-overexpressing cell lines (SKBR3, MMTV-neu) following OB versus N sera exposure, but did not vary in the other cell lines. Expression of Bcl-2 and cyclin D1, two genes negatively regulated by ERβ, was elevated in SKBR3 cells following exposure to OB versus N sera, but this difference was eliminated when the ERβ gene was silenced with siRNA. Herceptin, a HER2 antagonist, and siRNA to HER2 were used to evaluate the role of HER2 in sera-induced ERβ modulation. SKBR3 cell treatment with OB sera plus Herceptin increased ERβ expression three-fold. Similar results were obtained when HER2 expression was silenced with siRNA. OB sera also promoted greater SKBR3 cell viability and growth, but this variance was not present when ERβ was silenced or the cells were modified to overexpress ERβ. Based on this data, we conclude that obesity-associated systemic factors suppress ERβ expression in breast cancer cells via a HER2-mediated pathway, leading to greater cell viability and growth. Elucidation of the mechanism(s) mediating this effect could provide important insights into how ERβ expression is regulated as well as how obesity promotes a more aggressive disease.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Estrogen Receptor beta; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Mice; Obesity; Proto-Oncogene Proteins c-bcl-2; Receptor, ErbB-2; Signal Transduction; Transcription, Genetic

The peptidyl-prolyl cis/trans isomerase Pin1 is overexpressed in a wide variety of cancer cells and thus considered as an important target molecule for cancer therapy. This study demonstrates that decursin, a bioactive compound from Angelica gigas, exert the anti-cancer effect against breast cancer cells via regulation of Pin1 and its related signaling molecules. We observed that decursin induced G1 arrest with decrease in cyclin D1 level in Pin1-expressing breast cancer cells MDA-MB-231, but not Pin1-non-expressing breast cancer cells MDA-MB-157. In addition, decursin significantly reduced protein expression and enzymatic activity of Pin1 in MDA-MB-231 cells. Further, we found that decursin treatment enhanced the p53 expression level and failed to down-regulate Pin1 in the cells transfected with p53 siRNA, indicating the importance of p53 in the decursin-mediated Pin1 inhibition in MDA-MB-231 cells. Decursin stimulated association between Pin1 to p53. Moreover, decursin facilitated p53 transcription in MDA-MB-231 cells. Overall, our current study suggests the potential of decursin as an attractive cancer therapeutic agent for breast cancer by targeting Pin1 protein.

Topics: Angelica; Benzopyrans; Breast Neoplasms; Butyrates; Cell Cycle Checkpoints; Cell Line, Tumor; Cyclin D1; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Humans; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; Tumor Suppressor Protein p53

The transcription factor GATA3 is a key regulator of mammary gland development and a definitive marker of luminal breast cancer. However, the molecular mechanisms underlying the role of GATA3 in breast carcinogenesis is still not fully understood. We report here that GATA3 promotes cell proliferation and tumorigenesis by facilitating the G1/S transition through its transcription regulation of the CCND1 gene in breast cancer cells. We found that GATA3 is physically associated with poly-ADP ribose polymerase-1 (PARP1), an enzyme modifying nuclear proteins by poly(ADP-ribosyl)ation. We showed that PARP1 acts as a transcription coactivator for GATA3 in breast cancer cells and demonstrated that GATA3 cooperates with PARP1 in transactivation of the CCND1 gene. We demonstrated that PARP1 competes with linker histone H1 to maintain a transcriptional competent chromatin environment for CCND1 gene. Our results unveiled a molecular basis for the coordinated regulation between GATA3 and PARP1 in transcription activation, providing a mechanism for GATA3 in breast carcinogenesis.

Topics: Animals; Apoptosis; Base Sequence; Blotting, Western; Breast Neoplasms; Cell Cycle; Cell Movement; Cell Proliferation; Cell Transformation, Neoplastic; Chromatin Immunoprecipitation; Cyclin D1; Female; Flow Cytometry; GATA3 Transcription Factor; Gene Expression Regulation, Neoplastic; Histones; Humans; Immunoprecipitation; Mice; Mice, Inbred BALB C; Mice, Nude; Molecular Sequence Data; Peptide Fragments; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sequence Homology, Nucleic Acid; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Transcription, Genetic; Tumor Cells, Cultured; Tumor Stem Cell Assay; Xenograft Model Antitumor Assays

N-cadherin and HER2/neu were found to be co-expressed in invasive breast carcinomas. To test the contribution of N-cadherin and HER2 in mammary tumor metastasis, we targeted N-cadherin expression in the mammary epithelium of the MMTV-Neu mouse. In the context of ErbB2/Neu, N-cadherin stimulated carcinoma cell invasion, proliferation and metastasis. N-cadherin caused fibroblast growth factor receptor (FGFR) upmodulation, resulting in epithelial-to-mesenchymal transition (EMT) and stem/progenitor like properties, involving Snail and Slug upregulation, mammosphere formation and aldehyde dehydrogenase activity. N-cadherin potentiation of the FGFR stimulated extracellular signal regulated kinase (ERK) and protein kinase B (AKT) phosphorylation resulting in differential effects on metastasis. Although ERK inhibition suppressed cyclin D1 expression, cell proliferation and stem/progenitor cell properties, it did not affect invasion or EMT. Conversely, AKT inhibition suppressed invasion through Akt 2 attenuation, and EMT through Snail inhibition, but had no effect on cyclin D1 expression, cell proliferation or mammosphere formation. These findings suggest N-cadherin/FGFR has a pivotal role in promoting metastasis through differential regulation of ERK and AKT, and underscore the potential for targeting the FGFR in advanced ErbB2-amplified breast tumors.

Topics: Aldehyde Dehydrogenase; Animals; Benzamides; Breast Neoplasms; Cadherins; Cell Movement; Cell Proliferation; Cyclin D1; Diphenylamine; Epithelial-Mesenchymal Transition; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Lung Neoplasms; MAP Kinase Kinase 1; Mice; Mice, Transgenic; Neoplasm Invasiveness; Neoplasm Metastasis; Phosphorylation; Proto-Oncogene Proteins c-akt; Pyrimidines; Receptor, ErbB-2; Receptors, Fibroblast Growth Factor; RNA Interference; RNA, Small Interfering; Signal Transduction; Snail Family Transcription Factors; Spheroids, Cellular; Stem Cells; Transcription Factors; Tumor Cells, Cultured

Breast Cancer is the most common malignancy among women. Family history is the strongest single predictor of breast cancer risk, and thus great attention has been focused on BRCA1 and BRCA2 genes whose mutations lead to a high risk of developing this disease. Today, only 25% of high- and moderate-risk genes are known, suggesting the importance of the discovery of new risk modifiers. Therefore, the investigation of new polygenic alterations is of great importance, especially if considered high- and moderate-risk variants. In this study, the transmission of BRCA1-2 polymorphisms in association with the transmission of polymorphisms in the genes NUMA1, CCND1, COX11, FGFR2, TNRC9 and SLC4A7 were examined in all members of a family with the BRCA2 c.6447_6448dup mutation. This is the first study about the transmission of high-risk polygenic variants in all members of a family with a strong history of breast cancer. The results about the possible polygenic variant associations that could increase and modify the risk suggested the importance to search new variants to better manage patients and their family members.

Topics: Adult; Alleles; Antigens, Nuclear; Apoptosis Regulatory Proteins; Breast Neoplasms; Cell Cycle Proteins; Copper Transport Proteins; Cyclin D1; Electron Transport Chain Complex Proteins; Electron Transport Complex IV; Female; Genes, BRCA1; Genes, BRCA2; Genetic Predisposition to Disease; Haplotypes; High Mobility Group Proteins; Humans; Inheritance Patterns; Male; Middle Aged; Mitochondrial Proteins; Mutation; Nuclear Matrix-Associated Proteins; Pedigree; Polymorphism, Single Nucleotide; Receptor, Fibroblast Growth Factor, Type 2; Receptors, Progesterone; Trans-Activators

Despite the clinical success of tamoxifen, its resistance remains a major challenge in breast cancer. Here we show that Aurora-A determines tamoxifen sensitivity by regulation of oestrogen receptor (ER)α. Ectopic expression of Aurora-A decreases and depletion of Aurora-A enhances tamoxifen sensitivity in ERα-positive breast cancer. Elevated Aurora-A was significantly associated with the recurrence of ERα-positive tumours. Notably, Aurora-A inhibitor MLN8237, which is currently in clinical trial, synergizes with tamoxifen and overcomes tamoxifen resistance. Furthermore, Aurora-A interacts with and phosphorylates ERα on serine-167 and -305, leading to increase in ERα DNA-binding and transcriptional activity. Elevated levels of Aurora-A are significantly associated with disease-free survival in ERα-positive but not ERα-negative breast cancers. These data suggest that Aurora-A has a pivotal role in tamoxifen resistance and ERα is a bona fide substrate of Aurora-A. Thus, Aurora-A represents a prognostic marker in ERα-positive tumour and a critical therapeutic target in tamoxifen-resistant breast cancer, and Aurora-A inhibitor could be used as either an independent or concurrent agent in tamoxifen-resistant tumour.

Topics: Animals; Antineoplastic Agents, Hormonal; Aurora Kinase A; Azepines; Breast Neoplasms; Cell Line, Tumor; Cyclin D1; Disease-Free Survival; Drug Resistance, Neoplasm; Drug Synergism; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Kaplan-Meier Estimate; Mice, Nude; Phosphorylation; Proportional Hazards Models; Protein Processing, Post-Translational; Pyrimidines; Tamoxifen; Transcriptional Activation; Xenograft Model Antitumor Assays

The aim of this study was to investigate the expression of β-catenin, axin, cyclin D1 and c-myc, and their correlation with various clinicopathological factors of breast carcinoma. Using immunohistochemistry, the expression of axin, β-catenin, cyclin D1 and c-myc proteins was detected in 168 breast carcinomas and 40 normal breast tissue samples, as well as in 72 breast intraductal proliferative lesions. Correlations among the expression of these proteins with the clinicopathological factors of breast carcinomas were subsequently analyzed. Gene mutations of β-catenin (exon 3) in 44 cases of breast carcinoma were analyzed using polymerase chain reaction (PCR) followed by direct sequencing. In normal tissue, the epithelial cells demonstrated a marked membranous expression of β-catenin protein at cell-cell boundaries and positive axin expression; cyclin D1 and c-myc expression, however, were negative. The abnormal rate of β-catenin expression and the overexpression of cyclin D1 and c-myc were higher in breast carcinomas compared with breast cystic hyperplasia tissues. Positive axin expression levels were lower in breast carcinomas compared with breast intraductal proliferative lesions and normal breast tissues. Axin expression correlated inversely with tumor size, histological grade, clinical tumor, node, metastasis (TNM) stage and lymph node metastasis. The abnormal expression of β-catenin and the overexpression of cyclin D1 were correlated, and the overexpression of c-myc was correlated with tumor size, histological grade, clinical TNM stage and lymph node metastasis. The abnormal expression of β-catenin was correlated with the overexpression of cyclin D1, but not with the overexpression of c-myc. Lower levels of axin expression were correlated with higher levels of nuclear β-catenin expression. Mutations in the β-catenin gene were not detected in 44 cases of breast carcinoma. The abnormal expression of β-catenin may be key in the carcinogenesis and progression of human breast carcinoma by upregulating the expression of cyclin D1. The abnormal expression of β-catenin, the reduced expression of axin, and the overexpression of cyclin D1 and c-myc may be useful markers for determining metastasis, providing a prognosis for human breast carcinoma and for guiding treatment.

Topics: Adult; Aged; Aged, 80 and over; Axin Protein; beta Catenin; Breast Neoplasms; Carcinoma; Cyclin D1; Exons; Female; Gene Expression Regulation, Neoplastic; Humans; Middle Aged; Mutation; Proto-Oncogene Proteins c-myc; Wnt Signaling Pathway

In late stages of cancer, TGF-β promotes the metastasis process by enhancing the invasiveness of cancer cells and inducing the epithelial-to-mesenchymal transition (EMT), a process that is concomitantly associated with breast cancer metastasis. Metastasis comprises of multiple steps with the regulation of complex network of signaling. Metastasis is associated with both the EMT and cell proliferation, but yet it has not been clearly distinguished how the balance between the cell proliferation and EMT is maintained together. Recently, it has been accounted that a transcription factor, NFAT has an important role for switching tumor suppressive to progressive effect of TGF-β and NFAT has a role in TGF-β mediated EMT by regulating N-cadherin. CDC 25A phosphatase, an important cell cycle regulator is overexpressed in breast cancer. Our results demonstrate that TGF-β regulating the CDC 25A in a Smad2 dependent way, translocates NFAT to nucleus and NFAT in co-operation with Smad2 promotes the tumor progression by upregulating the CDK2, CDK4, and cyclin E. This result signifies that TGF-β by regulating NFAT in different ways maintains the balance between EMT and cell proliferation mechanism concurrently during the late stage of breast cancer.

Topics: Apoptosis; Breast Neoplasms; Cadherins; cdc25 Phosphatases; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation; Humans; NFATC Transcription Factors; RNA, Small Interfering; Signal Transduction; Smad2 Protein; Transforming Growth Factor beta

Human protein arginine N-methyltransferase 2 (PRMT2, HRMT1L1) is a protein that belongs to the arginine methyltransferase family, and it has diverse roles in transcriptional regulation through different mechanisms depending on its binding partners. In this study, we provide evidences for the negative effect of PRMT2 on breast cancer cell proliferation in vitro and in vivo. Morever, cyclin D1, one of the key modulators of cell cycle, was found to be downregulated by PRMT2, and PRMT2 was further shown to suppress the estrogen receptor α-binding affinity to the activator protein-1 (AP-1) site in cyclin D1 promoter through indirect binding with AP-1 site, resulting in the inhibition of cyclin D1 promoter activity in MCF-7 cells. Furthermore, a positive correlation between the expression of PRMT2 and cyclin D1 was confirmed in the breast cancer tissues by using tissue microarray assay. In addition, PRMT2 was found to show a high absent percentage in breast caner cell nuclei and the nuclear loss ratio of PRMT2 was demonstrated to positively correlate with cyclin D1 expression and the increasing tumor grade of invasive ductal carcinoma. Those results offer an essential insight into the effect of PRMT2 on breast carcinogenesis, and PRMT2 nuclear loss might be an important biological marker for the diagnosis of breast cancer.

Topics: Adult; Aged; Animals; Biomarkers, Tumor; Blotting, Western; Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Line, Tumor; Cell Nucleus; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Heterografts; Humans; Immunohistochemistry; Intracellular Signaling Peptides and Proteins; Mice; MicroRNAs; Middle Aged; Mutagenesis, Site-Directed; Neoplasm Grading; Oligonucleotide Array Sequence Analysis; Protein-Arginine N-Methyltransferases; Real-Time Polymerase Chain Reaction; Tissue Array Analysis; Transfection

Pentacyclic triterpenes are a group of molecules with promising anticancer potential, although their precise molecular target remains elusive. The current work aims to investigate the antiproliferative and associated mechanisms of triterpenes in breast cancer cells in vitro.. Effect of triterpenes on cell cycle distribution, ROS and key regulatory proteins were analyzed in three breast cancer cells in vitro. Growth inhibition, new DNA synthesis, colony formation assays and Western blot analysis were performed to assess the EGFR inhibitory effect of triterpenes. Molecular docking was performed to study the interaction between EGFR and triterpenes.. We have demonstrated the ability of dimethyl melaleucate (DMM), a pentacyclic triterpene to exhibit cell cycle arrest at G0/G1 phase by down-regulation of cyclin D1 through PI3K/AKT inhibition. Further, to identify the upstream target of DMM, potential EGFR inhibitory activity of DMM and three structurally related pentacyclic triterpenes, ursolic acid, 18α-glycyrrhetinic acid and carbenoxolone was investigated. Interestingly, pentacyclic triterpenes limit EGF mediated breast cancer proliferation through sustained inhibition of EGFR and its downstream effectors STAT3 and cyclin D1 in breast cancer lines. We also show pentacyclic triterpenes to bind at the ATP binding pocket of tyrosine kinase domain of EGFR leading to the hypothesis that pentacyclic triterpenes could be a novel class of EGFR inhibitors. In conclusion, pentacyclic triterpenes inhibit EGFR activation through binding with tyrosine kinase domain thereby suppressing breast cancer proliferation.. Pentacyclic triterpenes may serve as a potential platform for development of novel drugs against breast cancer.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; DNA; Drug Design; ErbB Receptors; Female; Humans; Molecular Docking Simulation; Molecular Targeted Therapy; Pentacyclic Triterpenes; Reactive Oxygen Species

Insulin like growth factor (IGF)-1 and IGF-2 stimulate normal growth, development and breast cancer cell proliferation. Cyclin D1 (CCND1) promotes cell cycle by inhibiting retinoblastoma protein (RB1). The aryl hydrocarbon receptor (AHR) is a major xenobiotic receptor that also regulates cell cycle. The purpose of this study was to investigate whether IGF-2 promotes MCF-7 breast cancer proliferation by inducing AHR. Western blot and quantitative real time PCR (Q-PCR) analysis revealed that IGF-2 induced an approximately 2-fold increase (P<.001) in the expression of AHR and CCND1. Chromatin immunoprecipitation (ChIP), followed by Q-PCR indicated that IGF-2 promoted (P<.001) a 7-fold increase in AHR binding on the CCND1 promoter. AHR knockdown significantly (P<.001) inhibited IGF-2 stimulated increases in CCND1 mRNA and protein. AHR knockdown cells were less (P<.001) responsive to the proliferative effects of IGF-2 than control cells. Collectively, our findings have revealed a new regulatory mechanism by which IGF-2 induction of AHR promotes the expression of CCND1 and the proliferation of MCF-7 cells. This previously uncharacterized pathway could be important for the proliferation of IGF responsive cancer cells that also express AHR.

Topics: Basic Helix-Loop-Helix Transcription Factors; Breast Neoplasms; Cell Proliferation; Cyclin D1; Cytochrome P-450 CYP1A1; Female; Gene Knockdown Techniques; Humans; Insulin-Like Growth Factor II; MCF-7 Cells; Models, Biological; Promoter Regions, Genetic; Protein Binding; Receptors, Aryl Hydrocarbon

Resistance to antiestrogens is one of the major challenges in breast cancer treatment. Although phosphorylation of estrogen receptor α (ERα) is an important factor in endocrine resistance, the contributions of specific kinases in endocrine resistance are still not fully understood. Here, we report that an important innate immune response kinase, the IκB kinase-related TANK-binding kinase 1 (TBK1), is a crucial determinant of resistance to tamoxifen therapies. We show that TBK1 increases ERα transcriptional activity through phosphorylation modification of ERα at the Ser-305 site. Ectopic TBK1 expression impairs the responsiveness of breast cancer cells to tamoxifen. By studying the specimens from patients with breast cancer, we find a strong positive correlation of TBK1 with ERα, ERα Ser-305, and cyclin D1. Notably, patients with tumors highly expressing TBK1 respond poorly to tamoxifen treatment and show high potential for relapse. Therefore, our findings suggest that TBK1 contributes to tamoxifen resistance in breast cancer via phosphorylation modification of ERα.

Topics: Breast Neoplasms; Cell Line, Tumor; Cyclin D1; Drug Resistance, Neoplasm; Estrogen Receptor alpha; Female; Humans; Immunity, Innate; Kaplan-Meier Estimate; Phosphorylation; Phosphoserine; Protein Binding; Protein Serine-Threonine Kinases; Tamoxifen; Transcription, Genetic; Treatment Outcome

Tumor characteristics are decisive in the determination of treatment strategy for patients with breast cancer. Patients with estrogen receptor α (ERα)-positive breast cancer can benefit from long-term hormonal treatment. Nonetheless, the majority of patients will develop resistance to these therapies. Here, we investigated the role of the nuclear receptor liver receptor homolog-1 (LRH-1, NR5A2) in antiestrogen-sensitive and -resistant breast cancer cells. We identified genome-wide LRH-1-binding sites using ChIP-seq (chromatin immunoprecipitation sequencing), uncovering preferential binding to regions distal to transcriptional start sites. We further characterized these LRH-1-binding sites by integrating overlapping layers of specific chromatin marks, revealing that many LRH-1-binding sites are active and could be involved in long-range enhancer-promoter looping. Combined with transcriptome analysis of LRH-1-depleted cells, these results show that LRH-1 regulates specific subsets of genes involved in cell proliferation in antiestrogen-sensitive and antiestrogen-resistant breast cancer cells. Furthermore, the LRH-1 transcriptional program is highly associated with a signature of poor outcome and high-grade breast cancer tumors in vivo. Herein, we report the genome-wide location and molecular function of LRH-1 in breast cancer cells and reveal its therapeutic potential for the treatment of breast cancers, notably for tumors resistant to treatments currently used in therapies.

Topics: Binding Sites; Breast Neoplasms; Cell Proliferation; Chromatin; Cyclin D1; Drug Resistance, Neoplasm; Estrogen Antagonists; Estrogen Receptor alpha; Humans; MCF-7 Cells; Receptors, Cytoplasmic and Nuclear; Receptors, Estrogen; Receptors, G-Protein-Coupled; Transcription, Genetic

The two isoforms of estrogen receptor (ER) alpha and beta play opposite roles in regulating proliferation and differentiation of breast cancers, with ER-alpha mediating mitogenic effects and ER-beta acting as a tumor suppressor. Emerging data have reported that androgen receptor (AR) activation inhibits ER-positive breast cancer progression mainly by antagonizing ER-alpha signaling. However, to date no studies have specifically evaluated a potential involvement of ER-beta in the inhibitory effects of androgens.. ER-beta expression was examined in human breast cancer cell lines using real-time PCR, Western blotting and small interfering RNA (siRNA) assays. Mutagenesis studies, electromobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) analysis were performed to assess the effects of mibolerone/AR on ER-beta promoter activity and binding.. In this study, we demonstrate that mibolerone, a synthetic androgen ligand, up-regulates ER-beta mRNA and protein levels in ER-positive breast cancer cells. Transient transfection experiments, using a vector containing the human ER-beta promoter region, show that mibolerone increases basal ER-beta promoter activity. Site-directed mutagenesis and deletion analysis reveal that an androgen response element (ARE), TGTTCT motif located at positions -383 and -377, is critical for mibolerone-induced ER-beta up-regulation in breast cancer cells. This occurs through an increased recruitment of AR to the ARE site within the ER-beta promoter region, along with an enhanced occupancy of RNA polymerase II. Finally, silencing of ER-beta gene expression by RNA interference is able to partially reverse the effects of mibolerone on cell proliferation, p21 and cyclin D1 expression.. Collectively, these data provide evidence for a novel mechanism by which activated AR, through an up-regulation of ER-beta gene expression, inhibits breast cancer cell growth.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Enzyme Activation; Estrogen Receptor alpha; Estrogen Receptor beta; Female; Humans; MCF-7 Cells; Mutagenesis, Site-Directed; Nandrolone; Promoter Regions, Genetic; Protein Binding; Receptors, Androgen; rho GTP-Binding Proteins; RNA Interference; RNA Polymerase II; RNA, Messenger; RNA, Small Interfering; Testosterone Congeners; Up-Regulation

Oncogenic mutation or misregulation of small GTPases in the Ras and Rho families can promote unregulated cell cycle progression in cancer. Post-translational modification by prenylation of these GTPases allows them to signal at the cell membrane. Splice variants of SmgGDS, named SmgGDS-607 and SmgGDS-558, promote the prenylation and membrane trafficking of multiple Ras and Rho family members, which makes SmgGDS a potentially important regulator of the cell cycle. Surprisingly little is known about how SmgGDS-607 and SmgGDS-558 affect cell cycle-regulatory proteins in cancer, even though SmgGDS is overexpressed in multiple types of cancer. To examine the roles of SmgGDS splice variants in the cell cycle, we compared the effects of the RNAi-mediated depletion of SmgGDS-558 vs. SmgGDS-607 on cell cycle progression and the expression of cyclin D1, p27, and p21 in pancreatic, lung, and breast cancer cell lines. We show for the first time that SmgGDS promotes proliferation of pancreatic cancer cells, and we demonstrate that SmgGDS-558 plays a greater role than SmgGDS-607 in cell cycle progression as well as promoting cyclin D1 and suppressing p27 expression in multiple types of cancer. Silencing both splice variants of SmgGDS in the cancer cell lines produces an alternative signaling profile compared with silencing SmgGDS-558 alone. We also show that loss of both SmgGDS-607 and SmgGDS-558 simultaneously decreases tumorigenesis of NCI-H1703 non-small cell lung carcinoma (NSCLC) xenografts in mice. These findings indicate that SmgGDS promotes cell cycle progression in multiple types of cancer, making SmgGDS a valuable target for cancer therapeutics.

Topics: Animals; Breast Neoplasms; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Guanine Nucleotide Exchange Factors; Heterografts; Humans; Lung Neoplasms; Mice; Mice, SCID; Pancreatic Neoplasms; Proliferating Cell Nuclear Antigen; Protein Isoforms; rho GTP-Binding Proteins

Fangchinoline (Fan) inhibits cell proliferation and induces apoptosis in several cancer cell lines. The effects of Fan on cell growth and proliferation in breast cancer cells remain to be elucidated. Here, we show that Fan inhibited cell proliferation in the MDA-MB-231 breast cancer cell line through suppression of the AKT/Gsk- 3beta/cyclin D1 signaling pathway. Furthermore, Fan induced apoptosis by increasing the expression of Bax (relative to Bcl-2), active caspase 3 and cytochrome-c. Fan significantly inhibited cell proliferation of MDA- MB-231 cells in a concentration and time dependent manner as determined by MTT assay. Flow cytometry analysis demonstrated that Fan treatment of MDA-MB-231 cells resulted in cell cycle arrest at the G1 phase, which correlated with apparent downregulation of both mRNA and protein levels of both PCNA and cyclin D1. Further analysis demonstrated that Fan decreased the phosphorylation of AKT and GSK-3beta. In addition, Fan up-regulated active caspase3, cytochrome-c protein levels and the ratio of Bax/Bcl-2, accompanied by apoptosis. Taken together, these results suggest that Fan is a potential natural product for the treatment of breast cancer.

Topics: Apoptosis; Benzylisoquinolines; Blotting, Western; Breast Neoplasms; Cell Cycle; Cell Proliferation; Cyclin D1; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Phosphorylation; Proto-Oncogene Proteins c-akt; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured

Breast cancer is a leading cause of morbidity and mortality in women worldwide. About 70 % of breast cancers are estrogen receptor (ER) positive. Blocking estrogen action by tamoxifen has been the treatment of choice in ER positive breast cancers for more than 30 years. In the past, several studies have revealed associations between gene copy number alterations and responsiveness to tamoxifen therapy, but so far no single gene copy number alteration could completely explain the response variation observed between individual breast cancer patients. Here, we set out to perform a simultaneous analysis of copy number alterations of several genes involved in the prognosis and response to therapy by multiplex ligation-dependent probe amplification (MLPA).. A case-control study was designed encompassing 170 non-metastatic ER positive breast cancer patients (case group = 85, control group = 85). All patients in the control group had received standard adjuvant tamoxifen treatment for 5 years without any evidence of recurrence. Patients in the case group had experienced early recurrences while receiving tamoxifen treatment. 76 % of the patients of the case group and 73 % of the patients of the control group had received anthracycline-based adjuvant chemotherapy. Gene copy number alterations detected by MLPA in both groups were compared.. Amplification of CCND1 (OR = 3.13; 95 % CI = 1.35 to 7.26; p = 0.006) and TOP2A (OR = 3.05; 95 % CI = 1.13 to 8.24; p = 0.022) were significantly more prevalent in the case group, compared to the control group. In a multivariate analysis CCND1 (p = 0.01) and TOP2A (p = 0.041) amplifications remained significant predictors of recurrence.. Our results indicate that CCND1 amplification may serve as a useful biomarker for hormone responsiveness, and that TOP2A amplification may serve as a useful prognostic biomarker.

Topics: Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; Breast Neoplasms; Case-Control Studies; Cyclin D1; DNA Copy Number Variations; DNA Topoisomerases, Type II; DNA-Binding Proteins; Female; Gene Amplification; Humans; Middle Aged; Multiplex Polymerase Chain Reaction; Multivariate Analysis; Neoplasm Invasiveness; Poly-ADP-Ribose Binding Proteins; Predictive Value of Tests; Prognosis; Receptor, ErbB-2; Tamoxifen; Treatment Outcome

Polymorphisms in DNA repair and cell cycle genes contribute to increased breast cancer (BC) risk. Their association and interaction in relation to betel quid and tobacco chewing habits need exhaustive multi-analytical investigation to explain BC predisposition due to DNA damage. Polymorphism in TP53-72Arg>Pro, RAD51-135G>C, BRCA2, and CCND1-G870A were examined in 204 BC cases and 217 controls from Northeast Indian population. Multifaceted analytic approaches were used to explore relationships between polymorphisms, tobacco history, and BC susceptibility. Betel quid chewing was identified as the predominant risk factor. CCND-AA and dominant model showed protection towards BC in betel quid chewer (BQC) [(0.28 (0.10-0.77), 0.01 and 0.32 (0.12-0.81), 0.01)] and non-betel quid chewers (NBQC) [(0.26 (0.09-0.78), 0.01 and 0.37 (0.16-0.87), 0.02)]. TP53-Pro/Pro genotype showed protection towards BC in NBQC (0.29 (0.10-0.81), p=0.01) and (0.51 (0.32-0.80), p=0.003, respectively). RAD51-C allele was associated with BC risk (2.03 (1.26-3.30) 0.002) in BQC. Two BQC cases had BRCA2 8415G>T:K2729N mutation in Exon18. MDR analysis showed best four locus model with TBA 0.6765 (0.005) and CVC of 10/10 in NBQC. Interaction diagram concurred the interactions between TP53 and RAD51 (1.32 %) with independent effect (1.89 %) of CCND1in NBQC. In CART analysis, BQC with CCND1 GG genotype were at risk (OR=33.0; 95 % CI=6.08-179.07), p<0.001) followed by combination of BQC, CCND1, No-Smk, and Alc (OR=42.00; 95 % CI=5.11-345.11, p<0.001). Risk was also observed in BQC, CCND1, No-Smk, Non-Alc, and TP53 combination (OR=14.84; 95 % CI=3.13-70.34, p<0.001) and BQC, CCND1, No-Smk, Non-Alc, TP53 (OR=9.40; 95 % CI=1.99-44.34, p<0.001). NBQC group showed risk with combination of NBQC and TP53 (OR=5.54; 95 % CI=1.11-27.42, p=0.03). Genetic variants in DNA repair and cell cycle genes contribute to BC risk through gene-gene and gene-environmental interactions.

Topics: Adult; Aged; Areca; Breast Neoplasms; Cell Cycle; Cyclin D1; DNA Repair; Entropy; Female; Genes, BRCA2; Genes, p53; Genetic Predisposition to Disease; Genetic Variation; Humans; Middle Aged; Polymorphism, Single Nucleotide; Rad51 Recombinase; Risk

The progesterone receptor (PR) and its coactivators are direct targets of activated cyclin-dependent kinases (CDKs) in response to peptide growth factors, progesterone, and deregulation of cell cycle inhibitors. Herein, using the T47D breast cancer model, we probed mechanisms of cell cycle-dependent PR action. In the absence of exogenous progestin, the PR is specifically phosphorylated during the G2/M phase. Accordingly, numerous PR target genes are cell cycle regulated, including HSPB8, a heat-shock protein whose high expression is associated with tamoxifen resistance. Progestin-induced HSPB8 expression required cyclin D1 and was insensitive to antiestrogens but blocked by antiprogestins or inhibition of specificity factor 1 (SP1). HSPB8 expression increased with or without ligand when cells were G2/M synchronized or contained high levels of cyclin D1. Knockdown of PRs abrogated ligand-independent HSPB8 expression in synchronized cells. Notably, PRs and cyclin D1 copurified in whole-cell lysates of transiently transfected COS-1 cells and in PR-positive T47D breast cancer cells expressing endogenous cyclin D1. PRs, cyclin D1, and SP1 were recruited to the HSPB8 promoter in progestin-treated T47D breast cancer cells. Mutation of PR Ser345 to Ala (S345A) or inhibition of CDK2 activity using roscovitine disrupted PR/cyclin D1 interactions with DNA and blocked HSPB8 mRNA expression. Interaction of phosphorylated PRs with SP1 and cyclin D1 provides a mechanism for targeting transcriptionally active PRs to selected gene promoters relevant to breast cancer progression. Understanding the functional linkage between PRs and cell cycle regulatory proteins will provide keys to targeting novel PR/cyclin D1 cross talk in both hormone-responsive disease and HSPB8-high refractory disease with high HSPB8 expression.

Topics: Animals; Breast Neoplasms; Cell Cycle; Cell Extracts; Cell Line, Tumor; Chlorocebus aethiops; COS Cells; Cyclin D1; Cyclin-Dependent Kinase 2; Female; Gene Expression Regulation, Neoplastic; Heat-Shock Proteins; Humans; Molecular Chaperones; Phosphorylation; Phosphoserine; Protein Serine-Threonine Kinases; Receptors, Progesterone; Transcription, Genetic

Activation of Wnt/β-catenin signaling can result in up-regulation of mTORC1 signaling in cancer cells. The low density lipoprotein receptor-related protein-6 (LRP6) is an essential Wnt co-receptor for Wnt/β-catenin signaling. We found that rottlerin, a natural plant polyphenol, suppressed LRP6 expression and phosphorylation, and inhibited Wnt/β-catenin signaling in HEK293 cells. Furthermore, the inhibitory effects of rottlerin on LRP6 expression/phosphorylation and Wnt/β-catenin signaling were confirmed in human prostate cancer PC-3 and DU145 cells and breast cancer MDA-MB-231 and T-47D cells. Mechanistically, rottlerin promoted LRP6 degradation, but had no effects on LRP6 transcriptional activity. In addition, rottlerin-mediated LRP6 down-regulation was unrelated to activation of 5'-AMP-activated protein kinase (AMPK). Importantly, we also found that rottlerin inhibited mTORC1 signaling in prostate and breast cancer cells. Finally, we demonstrated that rottlerin was able to suppress the expression of cyclin D1 and survivin, two targets of both Wnt/β-catenin and mTORC1 signaling, in prostate and breast cancer cells, and displayed remarkable anticancer activity with IC(50) values between 0.7 and 1.7 μM for prostate cancer PC-3 and DU145 cells and breast cancer MDA-MB-231 and T-47D cells. The IC(50) values are comparable to those shown to suppress the activities of Wnt/β-catenin and mTORC1 signaling in prostate and breast cancer cells. Our data indicate that rottlerin is a novel LRP6 inhibitor and suppresses both Wnt/β-catenin and mTORC1 signaling in prostate and breast cancer cells, and that LRP6 represents a potential therapeutic target for cancers.

Topics: Acetophenones; Antineoplastic Agents; Benzopyrans; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Drug Screening Assays, Antitumor; Female; HEK293 Cells; Humans; Inhibitor of Apoptosis Proteins; Inhibitory Concentration 50; Low Density Lipoprotein Receptor-Related Protein-6; Male; Mechanistic Target of Rapamycin Complex 1; Multiprotein Complexes; Promoter Regions, Genetic; Prostatic Neoplasms; Proteolysis; Survivin; TOR Serine-Threonine Kinases; Transcription, Genetic; Wnt Signaling Pathway; Wnt3A Protein

Breast cancer is a disease of cell cycle, and the dysfunction of cell cycle checkpoints plays a vital role in the occurrence and development of breast cancer. We employed multi-gene fluorescence in situ hybridization (M-FISH) to investigate gene copy number aberrations (CNAs) of 4 genes (Rb1, CHEK2, c-Myc, CCND1) that are involved in the regulation of cell cycle, in order to analyze the impact of gene aberrations on prognosis in the young breast cancer patients. Gene copy number aberrations of these 4 genes were more frequently observed in young breast cancer patients when compared with the older group. Further, these CNAs were more frequently seen in Luminal B type, Her2 overexpression, and tiple-negative breast cancer (TNBC) type in young breast cancer patients. The variations of CCND1, Rb1, and CHEK2 were significantly correlated with poor survival in the young breast cancer patient group, while the amplification of c-Myc was not obviously correlated with poor survival in young breast cancer patients. Thus, gene copy number aberrations (CNAs) of cell cycle-regulated genes can serve as an important tool for prognosis in young breast cancer patients.

Topics: Adult; Age Factors; Aged; Breast Neoplasms; Checkpoint Kinase 2; Cyclin D1; Female; Gene Dosage; Genes, cdc; Genes, myc; Humans; In Situ Hybridization, Fluorescence; Prognosis; Receptor, ErbB-2; Retinoblastoma Protein

To study the mechanism of tea polyphenols (TP)-induced apoptosis of breast cancer cells. Proliferation of MCF-7 and SK-BR-3 cells was evaluated by MTT assays. Cellular ultrastructure was examined by electron microscopy. Apoptosis was detected by TUNEL. PCNA、 Cyclin D1、 Cyclin E and Survivin expression was measured by Western blot. Cell proliferation was significantly inhibited by TP. Spindle and round cells were loosely distributed with increased particles after TP treatment. Increased cell size, frequent nuclear atypia and a collapse of apoptosis were observed. The nucleus was pushed towards one side, while the cytoplasm was rich in free ribosome. The membrane of mitochondria was thickening, and the cell apoptotic body was observed. TP treated cells experienced significantly enhanced apoptosis compared with 5-Fu treated or control groups. The expression of survivin was downregulated by TP. To conclude, TP can inhibit cell growth and induce apoptosis through downregulating the expression of survivin in breast cancer.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Cyclin D1; Cyclin E; Dose-Response Relationship, Drug; Female; Gene Expression Regulation, Neoplastic; Humans; Inhibitor of Apoptosis Proteins; MCF-7 Cells; Mice; Mice, Nude; Mitochondria; Polyphenols; Proliferating Cell Nuclear Antigen; Signal Transduction; Survivin; Tea; Tumor Burden; Xenograft Model Antitumor Assays

Lithocarpus polystachyus leaves have been used as tea beverage and folk medicine for healthy care in the Southwest of China. The purpose of this study is to investigate the anticancer activity of Lithocarpus polystachyus Rehd leaf aqueous extract (LPAE) and to explore the possible mechanism of its activity. Growth inhibition effects of LPAE breast cancer were tested in vitro and in vivo. The possible mechanism of its activity was analyzed with cell biological and molecular biological assays. After LPAE treatment, the proliferation and colony formation of cancer cells decreased; apoptotic cells increased; DNA fragmentations were evident; mRNA and protein expressions of PPARγ, Bax, and caspase-3 genes increased and expressions of cyclin D1 and Bcl-2 genes decreased; in vivo experiment, LPAE inhibited human beast cancer growth. The findings in this experimental study suggested that LPAE has potential cytotoxic and apoptotic effects on human breast cancer cells in vitro and inhibits the cancer growth in vivo, and its mechanism of activity might be associated with apoptosis induction of cancer cells through upregulation of the mRNA and protein expressions of PPARγ, Bax, and capase-3 genes and downregulation of the expressions of cyclin D1 and Bcl-2 genes.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Caspase 3; China; Cyclin D1; DNA Fragmentation; Fagaceae; Female; Humans; MCF-7 Cells; Mice, Inbred BALB C; Mice, Nude; Plant Extracts; Plant Leaves; PPAR gamma; RNA, Messenger; Xenograft Model Antitumor Assays

ARHI is a maternally imprinted tumor suppressor gene that is expressed in normal breast epithelial cells but not in most breast cancer cells. Aberrant methylation and hypernomic histone deacetylation have been implicated in the silencing of ARHI. To investigate the mechanism of ARHI induction, MDA-MB-231 breast cancer cells were either transfected with the eukaryotic expression vector, pcDNA3.1(+)-ARHI, or were simultaneously treated with a histone deacetylase inhibitor, [trichostatin A, (TSA)] and the methyltransferase inhibitor, 5-aza-2'-deoxycytidine (DAC). The latter treatment group also included the targeting of ARHI by small interfering RNA (siRNA) to further examine interactions between ARHI and the drugs applied. Levels of ARHI were detected by western blotting, MTT assays were used to evaluate cell proliferation, and both cell cycle progression and apoptosis were detected using flow cytometry. Both the transfection of pcDNA3.1(+)‑ARHI and the application of TSA+DAC induced the expression of ARHI. Furthermore, reduced cell proliferation, cell cycle arrest and enhanced apoptosis were observed for both groups compared to controls. However, a G1/S cell cycle arrest was observed for the pcDNA3.1(+)-ARHI group, while a G2 cell cycle arrest was observed for the TSA+DAC group. The latter effect was reversed with the introduction of ARHI-targeted siRNA in combination with TSA+DAC treatment. To further clarify these observations, expression levels of several key cell cycle regulators were analyzed by western blotting. The pcDNA3.1(+)-ARHI group exhibited higher expression levels of p53, p21 and p27, and lower levels of cyclin D1, CDK4 and CDK6 when compared to the control group (P<0.05). For the TSA+DAC group, higher levels of p53, p21, cyclin B1 and Chk1 were detected, concomitant with lower levels of CDK1, when compared to the control group. Taken together, these results suggest that ARHI acts as a tumor suppressor gene in MDA-MB-231 cells and, although TSA+DAC can block the cells at different cell cycle phage, the antitumor effect is ARHI-dependent.

Topics: Apoptosis; Azacitidine; Breast Neoplasms; CDC2 Protein Kinase; Cell Line, Tumor; Cell Proliferation; Checkpoint Kinase 1; Cyclin B1; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Decitabine; Female; G2 Phase Cell Cycle Checkpoints; Genetic Vectors; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Methyltransferases; Protein Kinases; rho GTP-Binding Proteins; RNA Interference; RNA, Small Interfering; S Phase Cell Cycle Checkpoints; Transfection; Tumor Suppressor Protein p53

Approximately 30% of breast tumors do not express the estrogen receptor (ER) α, which is necessary for endocrine therapy approaches. Studies are ongoing in order to restore ERα expression in ERα-negative breast cancer. The aim of the present study was to determine if calcitriol induces ERα expression in ER-negative breast cancer cells, thus restoring antiestrogen responses.. Cultured cells derived from ERα-negative breast tumors and an ERα-negative breast cancer cell line (SUM-229PE) were treated with calcitriol and ERα expression was assessed by real time PCR and western blots. The ERα functionality was evaluated by prolactin gene expression analysis. In addition, the effects of antiestrogens were assessed by growth assay using the XTT method. Gene expression of cyclin D1 (CCND1), and Ether-à-go-go 1 (EAG1) was also evaluated in cells treated with calcitriol alone or in combination with estradiol or ICI-182,780. Statistical analyses were determined by one-way ANOVA.. Calcitriol was able to induce the expression of a functional ERα in ER-negative breast cancer cells. This effect was mediated through the vitamin D receptor (VDR), since it was abrogated by a VDR antagonist. Interestingly, the calcitriol-induced ERα restored the response to antiestrogens by inhibiting cell proliferation. In addition, calcitriol-treated cells in the presence of ICI-182,780 resulted in a significant reduction of two important cell proliferation regulators CCND1 and EAG1.. Calcitriol induced the expression of ERα and restored the response to antiestrogens in ERα-negative breast cancer cells. The combined treatment with calcitriol and antiestrogens could represent a new therapeutic strategy in ERα-negative breast cancer patients.

Topics: Breast Neoplasms; Calcitriol; Cell Line, Tumor; Cyclin D1; Estradiol; Estrogen Receptor alpha; Estrogen Receptor Modulators; Female; Fulvestrant; Gene Expression Regulation, Neoplastic; Humans; Receptors, Calcitriol

The purpose of the present study is to evaluate the potent growth inhibitory effects of aqueous wheatgrass extract (AWE) alone and in combination with cisplatin on human breast and cervical cancer cells.. The cytotoxic potential of AWE alone and in combination with cisplatin was evaluated on human breast and cervical cancer cells (MCF-7 and HeLa) by cell viability assay. Further, the mode of cell death induced by AWE was determined by nuclear morphological examination and cell cycle analysis. These effects were then correlated with the expression of genes involved in apoptosis and proliferation (cyclin D1 and Bax) by RT-PCR.. AWE showed dose- and time dependent selective cytotoxicity towards the cancer highlighting its safe profile. Lower dose combinations of AWE and cisplatin induced increased growth inhibition compared with the individual drugs on both cell lines (combination index < 1) indicating strong synergistic interactions. AWE was found to induce apoptosis and arrested the cells at G0-G1 phase of the cell cycle which correlated with the modulation of expression of bax and cyclin D1 in a time-dependent manner in MCF-7 and HeLa cells.. These results suggest that the anti-cancer potential of AWE may be due to apoptosis induction and its anti-proliferative properties. This study also provides the first evidence demonstrating synergism between AWE and cisplatin, which may enhance the therapeutic index of prevention and/or treatment of human breast and cervical cancer.

Topics: Adjuvants, Immunologic; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Cell Proliferation; Cisplatin; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; HeLa Cells; Humans; MCF-7 Cells; Plant Extracts; Triticum; Uterine Cervical Neoplasms

The aim of this present study was to examine duration of breastfeeding in relation to the risk of different subgroups of breast cancer. A prospective cohort, The Malmö Diet and Cancer study, including 14092 parous women, were followed during a mean of 10.2 years and a total of 424 incident breast cancers were diagnosed.. Tumours were classified regarding invasiveness, tumour size, axillary lymph node status, Nottingham grade, tumour proliferation (Ki67), HER2, cyclin D1 and p27, WHO histological type and hormone receptor status. Duration of breastfeeding was measured using total time of breastfeeding, categorized in quartiles using the lowest as the reference group (<4.0, ≥4.0- < 8.0, ≥8.0- < 13.0 and ≥13.0 months). Average duration of breastfeeding per child and breastfeeding duration of the first child were also used as exposures in separate analyses. Relative risks, with 95% confidence intervals, were obtained using a Cox's proportional hazards analysis adjusted for potential confounders.. Overall risk for breast cancer was similar in all quartiles of breastfeeding. No strong results regarding breastfeeding duration and breast cancer subgroups were seen. A few results indicated an association between a relatively long duration of breastfeeding and tumours with high proliferation (Ki67) and grade III histological grade.. Breastfeeding duration was not associated with breast cancer risk and no strong results were seen with regard to breast cancer subgroups.

Topics: Adult; Aged; Biomarkers, Tumor; Breast Feeding; Breast Neoplasms; Cyclin D1; Female; Gene Expression; Humans; Ki-67 Antigen; Lymphatic Metastasis; Middle Aged; Neoplasm Grading; Proliferating Cell Nuclear Antigen; Proportional Hazards Models; Prospective Studies; Receptor, ErbB-2; Risk Factors; Sweden; Time Factors

Protocatechualdehyde (PCA) is a natural polyphenol compound isolated from the root of the herb S. miltiorrhiza and barley tea plants. PCA possesses antiproliferative and pro-apoptotic properties in human colorectal cancer cells. However, the cellular mechanism has not been fully understood. β-catenin and cyclin D1 are proto-oncogene that is overexpressed in many types of cancers and leads to cancer development. The present study was performed to elucidate the molecular mechanism by which PCA stimulates cell growth arrest and apoptosis in human breast cancer cells. PCA repressed cell proliferation and induced apoptosis in dose-dependent manner. PCA suppressed the expression of β-catenin and cyclin D1 with no changes in mRNA levels. Inhibition of proteosomal degradation using MG-132 and Ada-(Ahx)3-(Leu)3-vinyl sulfone ameliorates PCA-induced downregulation of β-catenin and cyclin D1. PCA treatment decreased the half-life of β-catenin and cyclin D1. PCA-mediated β-catenin downregulation depends on GSK3β. We further provide the evidence that PCA increased nuclear translocation of nuclear factor kappa-B (NF-κB) and the blockage of NF-κB using Bay11-7082 inhibited PCA-mediated β-catenin downregulation. The current study demonstrates that PCA suppress β-catenin expression through GSK3β- and NF-κB-mediated proteosomal degradation. In addition, PCA decreased cyclin D1 expression independent to β-catenin through proteosomal degradation.

Topics: Antineoplastic Agents; Apoptosis; Benzaldehydes; beta Catenin; Breast Neoplasms; Catechols; Cell Cycle; Cell Line, Tumor; Cyclin D1; Down-Regulation; Female; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; NF-kappa B; Proto-Oncogene Mas

Fluorescent nucleoside analogs replacing natural DNA bases in an oligonucleotide have been widely used for the detection of genetic material. Previously, we have described 2-((4-(trifluoromethyl) phenyl)-trans-vinyl)-2'-deoxy-adenosine, 6, a nucleoside analog with intrinsic fluorescence (NIF). Analog 6 exhibits a quantum yield 3115-fold higher than that of adenosine (φ 0.81) and maximum emission which is 120 nm red shifted (λem 439 nm). Here, we incorporated this analog in one or several positions of cyclin D1-targeting 15-mer oligonucleotides (ONs). The fluorescence of 6 was quenched upon incorporation into an oligonucleotide (ca. 1.5-22 fold), and was further reduced upon duplex formation. Specifically, ON7 exhibited a fluorescence decrease of ca. 2- or 3-fold upon duplex formation with complementary DNA or RNA strand, respectively. We determined the kinetics of dehybridization/rehybridization process in the presence of ssDNA or ssRNA targets to optimize our probes length and established the probes' selectivity towards a specific target. Furthermore, we proved specificity of our probe to the target vs. singly mismatched targets. Our most promising ds-NIF-probe, ON7:RNA, was used for the detection of cyclin D1 mRNA marker in cancerous cells total RNA extracts. The ds-probe specifically recognized the target as observed by a 2-fold fluorescence increase within 30 s at RT. These findings illustrate the potential of ds-NIF-probes for the diagnosis of breast cancer.

Topics: Adenosine; Biomarkers, Tumor; Breast Neoplasms; Cinnamates; Cyclin D1; Female; Fluorescent Dyes; Humans; Kinetics; Temperature; Tumor Cells, Cultured

The cyclin D1 gene encodes the regulatory subunit of a holoenyzme that phosphorylates the retinoblastoma protein (pRb) and nuclear respiratory factor (NRF1) proteins. The abundance of cyclin D1 determines estrogen-dependent gene expression in the mammary gland of mice. Using estradiol (E2) and an E2-dendrimer conjugate that is excluded from the nucleus, we demonstrate that E2 delays the DNA damage response (DDR) via an extranuclear mechanism. The E2-induced DDR required extranuclear cyclin D1, which bound ERα at the cytoplasmic membrane and augmented AKT phosphorylation (Ser473) and γH2AX foci formation. In the nucleus, E2 inhibited, whereas cyclin D1 enhanced homology-directed DNA repair. Cyclin D1 was recruited to γH2AX foci by E2 and induced Rad51 expression. Cyclin D1 governs an essential role in the E2-dependent DNA damage response via a novel extranuclear function. The dissociable cytoplasmic function to delay the E2-mediated DDR together with the nuclear enhancement of DNA repair uncovers a novel extranuclear function of cyclin D1 that may contribute to the role of E2 in breast tumorigenesis.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Chromatin; Cyclin D1; DNA Damage; DNA Repair; Estradiol; Estrogen Receptor alpha; Estrogens; Female; Humans; Mice; Protein Binding; Rad51 Recombinase; RNA Interference; Signal Transduction

Sox2, a transcription factor and an embryonic stem cell marker, has been implicated in the pathogenesis of breast cancer (BC). YB-1 is another transcription factor that has been shown to promote stemness in BC cells.. Western blotting, quantitative PCR, and siRNAs were used to query the regulatory relationships between YB-1, Sox2, and their downstream targets. Chromatin immunoprecipitation was used to detect YB-1 interactions at the Sox2 promoter. Mammosphere and soft agar assays were used to assess the phenotypic consequences of YB-1 knockdown.. Here, we report that YB-1 regulates Sox2. YB-1 was found to bind to the SOX2 promoter and down-regulate its expression in MCF7 and ZR751. The regulatory interaction between YB-1 and Sox2 was drastically different between the two phenotypically distinct cell subsets, purified based on their differential response to a Sox2 reporter. They are referred to as the reporter unresponsive (RU) cells and the reporter responsive (RR) cells. Upon siRNA knockdown of YB-1, RU cells showed an increase in Sox2 expression but no change in Sox2 reporter activity; in contrast, RR cells exhibited increased expression and reporter activity of Sox2. Correlating with these findings, YB-1 knockdown induced a differential response in the expression of genes known to be regulated by both Sox2 and YB-1 (e.g. CCND1 and ITGA6). For instance, in response to YB-1 knockdown, CCND1 and ITGA6 expression were decreased or unchanged in RU cells but paradoxically increased in RR cells. Compared to RU cells, RR cells were significantly more resistant to the suppression of mammosphere formation due to YB-1 knockdown. Importantly, mammospheres derived from parental MCF7 cells treated with YB-1 siRNA knockdown exhibited higher expression levels of SOX2 and its downstream targets.. To conclude, in a subset of BC cells, namely RR cells, YB-1 regulates Sox2 to coordinately maintain stemness and tumorigenic properties.

Topics: Binding Sites; Breast Neoplasms; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Genes, Reporter; Homeodomain Proteins; Humans; Integrin alpha6; MCF-7 Cells; Nanog Homeobox Protein; Neoplastic Stem Cells; Phenotype; Promoter Regions, Genetic; RNA Interference; SOXB1 Transcription Factors; Transfection; Y-Box-Binding Protein 1

Acylglycerol kinase (AGK) is reported to be overexpressed in multiple cancers. The clinical significance and biological role of AGK in breast cancer, however, remain to be established.. AGK expression in breast cancer cell lines, paired patient tissues were determined using immunoblotting and Real-time PCR. 203 human breast cancer tissue samples were analyzed by immunochemistry (IHC) to investigate the relationship between AGK expression and the clinicopathological features of breast cancer. Functional assays, such as colony formation, anchorage-independent growth and BrdU assay, and a xenograft tumor model were used to determine the oncogenic role of AGK in human breast cancer progression. The effect of AGK on FOXO1 transactivity was further investigated using the luciferase reporter assays, and by detection of the FOXO1 downstream genes.. Herein, we report that AGK was markedly overexpressed in breast cancer cells and clinical tissues. Immunohistochemical analysis showed that the expression of AGK significantly correlated with patients' clinicopathologic characteristics, including clinical stage and tumor-nodule-metastasis (TNM) classification. Breast cancer patients with higher levels of AGK expression had shorter overall survival compared to patients with lower AGK levels. We gained valuable insights into the mechanism of AGK expression in breast cancer cells by demonstrating that overexpressing AGK significantly enhanced, whereas silencing endogenous AGK inhibited, the proliferation and tumorigenicity of breast cancer cells both in vitro and in vivo. Furthermore, overexpression of AGK enhanced G1-S phase transition in breast cancer cells, which was associated with activation of AKT, suppression of FOXO1 transactivity, downregulation of cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1 and upregulation of the cell cycle regulator cyclin D1.. Taken together, these findings provide new evidence that AGK plays an important role in promoting proliferation and tumorigenesis in human breast cancer and may serve as a novel prognostic biomarker and therapeutic target in this disease.

Topics: Adenocarcinoma; Animals; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Female; Forkhead Box Protein O1; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Nude; Neoplasm Invasiveness; Phosphotransferases (Alcohol Group Acceptor); Proto-Oncogene Proteins c-akt; RNA, Small Interfering; Signal Transduction; Xenograft Model Antitumor Assays

A needle biopsy of a mass in the right breast of a 36-year-old woman revealed invasive ductal carcinoma (IDC), and approximately 20% of cancer cells showed unequivocal membranous staining with the HercepTest. After systemic therapy with trastuzumab and paclitaxel followed by FEC (fluorouracil + epirubicin + cyclophosphamide), a right mastectomy was performed. By histological and immunohistochemical examinations, the resected tumor consisted mainly of E-cadherin-negative invasive lobular carcinoma (ILC), and the rest was ERBB2-positive IDC; thus, the diagnosis was mixed ductal and lobular carcinoma. Multiplex ligation-dependent probe amplification and fluorescence in situ hybridization (FISH) analyses revealed that ILC and IDC shared high-level amplification of CCND1 in homogeneously staining regions (HSR) and that IDC had an additional HSR-type amplicon of ERBB2. These findings strongly indicate that IDC and ILC had a common precursor cell with CCND1 amplification. Review of the biopsy specimen with FISH showed IDC with gene amplifications of CCND1 and ERBB2 as a minor component, IDC without amplification of CCND1 or ERBB2 as a major component, and a minute portion of ILC with CCND1 amplification. We speculate that chemotherapy and trastuzumab caused a marked reduction in IDC; however, ILC with CCND1 amplification was resistant to chemotherapy and grew.

Topics: Adult; Antineoplastic Agents; Biomarkers, Tumor; Biopsy, Needle; Breast; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Lobular; Cyclin D1; DNA Fingerprinting; DNA, Neoplasm; Female; Humans; In Situ Hybridization, Fluorescence; Mastectomy; Multiplex Polymerase Chain Reaction; Receptor, ErbB-2; Treatment Outcome

Emerging evidence indicates that activation of Wnt/β-catenin signaling at the cell surface results in inhibition of glycogen synthase kinase 3β (GSK3β), leading to activation of mTORC1 signaling in cancer cells. The low density lipoprotein receptor-related protein-6 (LRP6) is an essential Wnt co-receptor for Wnt/β-catenin signaling. Salinomycin is a novel small molecule inhibitor of LRP6. In the present study, we found that LRP6 overexpression induced mTORC1 signaling activation in cancer cells, and that salinomycin was not only a potent Wnt/β-catenin signaling inhibitor, but also a strong mTORC1 signaling antagonist in breast and prostate cancer cells. Mechanistically, salinomycin activated GSK3β in cancer cells. Moreover, salinomycin was able to suppress the expression of cyclin D1 and survivin, two targets of both Wnt/β-catenin and mTORC1 signaling, in prostate and breast cancer cells, and displayed remarkable anticancer activity. Our results present novel mechanisms underlying salinomycin-mediated cancer cell death.

Topics: Anti-Bacterial Agents; Apoptosis; beta Catenin; Breast; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Enzyme Activation; Female; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; HEK293 Cells; Humans; Inhibitor of Apoptosis Proteins; Low Density Lipoprotein Receptor-Related Protein-6; Male; MCF-7 Cells; Mechanistic Target of Rapamycin Complex 1; Multiprotein Complexes; Prostate; Prostatic Neoplasms; Pyrans; Survivin; TOR Serine-Threonine Kinases; Wnt Proteins; Wnt Signaling Pathway

Protein arginine methyltransferases (PRMTs) methylate arginine residues on histones and target transcription factors that play critical roles in many cellular processes, including gene transcription, mRNA splicing, proliferation, and differentiation. Recent studies have linked PRMT-dependent epigenetic marks and modifications to carcinogenesis and metastasis in cancer. However, the role of PRMT2-dependent signaling in breast cancer remains obscure. We demonstrate PRMT2 mRNA expression was significantly decreased in breast cancer relative to normal breast. Gene expression profiling, Ingenuity and protein-protein interaction network analysis after PRMT2-short interfering RNA transfection into MCF-7 cells, revealed that PRMT2-dependent gene expression is involved in cell-cycle regulation and checkpoint control, chromosomal instability, DNA repair, and carcinogenesis. For example, PRMT2 depletion achieved the following: 1) increased p21 and decreased cyclinD1 expression in (several) breast cancer cell lines, 2) decreased cell migration, 3) induced an increase in nucleotide excision repair and homologous recombination DNA repair, and 4) increased the probability of distance metastasis free survival (DMFS). The expression of PRMT2 and retinoid-related orphan receptor-γ (RORγ) is inversely correlated in estrogen receptor-positive breast cancer and increased RORγ expression increases DMFS. Furthermore, we found decreased expression of the PRMT2-dependent signature is significantly associated with increased probability of DMFS. Finally, weighted gene coexpression network analysis demonstrated a significant correlation between PRMT2-dependent genes and cell-cycle checkpoint, kinetochore, and DNA repair circuits. Strikingly, these PRMT2-dependent circuits are correlated with pan-cancer metagene signatures associated with epithelial-mesenchymal transition and chromosomal instability. This study demonstrates the role and significant correlation between a histone methyltransferase (PRMT2)-dependent signature, RORγ, the cell-cycle regulation, DNA repair circuits, and breast cancer survival outcomes.

Topics: Breast Neoplasms; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Chromosomal Instability; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; DNA Breaks, Double-Stranded; DNA Repair; Epithelial-Mesenchymal Transition; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Intracellular Signaling Peptides and Proteins; Kinetochores; MCF-7 Cells; Nuclear Receptor Subfamily 1, Group F, Member 3; Protein Interaction Maps; Protein-Arginine N-Methyltransferases; Receptors, Estrogen; RNA Interference; RNA, Messenger; RNA, Small Interfering; Signal Transduction

Both cyclin D1 and the transcription factor C/EBPβ are required for mammary epithelial cell differentiation; however, the pathway in which they operate is uncertain. Previous analyses of the patterns of gene expression in human tumors suggested a connection between cyclin D1 overexpression and C/EBPβ, but whether this represents a cancer-specific gain of function for cyclin D1 is unknown. C/EBPβ is an intronless gene encoding three protein isoforms--LAP1, LAP2, and LIP. Here, we provide evidence that cyclin D1 engages C/EBPβ in an isoform-specific manner. Cyclin D1 binds to LAP1, an event that activates the transcriptional function of LAP1 by relieving its autoinhibited state effected by intramolecular interactions. Reexpression of LAP1 but not LAP2 or LIP restores the ability of C/EBPβ-deficient mammary epithelial cells to differentiate and does so in a manner dependent on cyclin D1. And cyclin D1-mediated activation of LAP1 participates in mammary epithelial cell differentiation. Our findings indicate that cyclin D1 and C/EBPβ LAP1 operate in a common pathway to promote mammary epithelial cell differentiation.

Topics: Animals; Breast Neoplasms; CCAAT-Enhancer-Binding Protein-beta; Cell Differentiation; Cell Line, Tumor; Cyclin D1; Female; HEK293 Cells; Humans; Mammary Glands, Human; MCF-7 Cells; Mice; Mice, Inbred C57BL; Mice, Knockout; Protein Binding; Protein Isoforms

Chemokine (C-C motif) receptor 7 (CCR7) is involved in lymph-node homing of naive and regulatory T cells and lymphatic metastasis of cancer cells. Sialic acids comprise a group of monosaccharide units that are added to the terminal position of the oligosaccharide chain of glycoproteins by sialyation. Recent studies suggest that aberrant sialylation of receptor proteins contributes to proliferation, motility, and drug resistance of cancer cells. In this study, we addressed whether CCR7 is a sialylated receptor protein and tried to elucidate the effect of sialylation in the regulation of signal transduction and biological function of CCR7. Our results demonstrated that α-2, 3-sialyltransferase which catalyze sialylation reaction in vivo was overexpressed in breast tumor tissues and cell lines. Lectin blot analysis clearly demonstrated that CCR7 receptor was sialyated in breast cancer cells. Chemokine (C-C motif) ligand 19 (CCL19), the cognate ligand for CCR7, induced the activation of extracellular signal-regulated kinase (ERK) and AKT signaling and increased the expression of cell cycle regulatory proteins and proliferation of breast cancer cells. When cells were pre-treated with a sialyltransferase inhibitor AL10 or sialidase, CCL19-induced cell growth was significantly suppressed. CCL19 also increased invasion and prevented anoikis by up-regulating pro-survival proteins Bcl-2 and Bcl-xL. Inhibition of sialylation by AL10 totally abolished these effects. Finally, we showed that AL10 inhibited tumorigenicity of breast cancer in experimental animals. Taken together, we demonstrate for the first time that CCR7 receptor is a sialylated protein and sialylation is important for the paracrine stimulation by its endogenous ligand CCL19. In addition, inhibition of aberrant sialylation of CCR7 suppresses proliferation and invasion and triggers anoikis in breast cancer cells. Targeting of sialylation enzymes may be a novel strategy for breast cancer treatment.

Topics: Animals; Anoikis; beta-Galactoside alpha-2,3-Sialyltransferase; Breast Neoplasms; Cell Line; Cell Movement; Cell Proliferation; Cell Transformation, Neoplastic; Chemokine CCL19; Cyclin D1; Disease Models, Animal; Epithelial Cells; Female; Gene Expression; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Heterografts; Humans; Receptors, CCR7; RNA Processing, Post-Transcriptional; Sialyltransferases; Signal Transduction; Tumor Burden

The acetyltransferase inhibitor garcinol, a polyisoprenylated benzophenone, is extracted from the rind of the fruit of Garcinia indica, a plant found extensively in tropical regions. Anti-cancer activity has been suggested but there is no report on its action via inhibiting acetylation against cell proliferation, cell cycle progression, and apoptosis-inhibtion induced by estradiol (E2) in human breast cancer MCF-7 cells. The main purposes of this study were to investigate the effects of the acetyltransferase inhibitor garcinol on cell proliferation, cell cycle progression and apoptosis inhibition in human breast cancer MCF-7 cells treated with estrogen, and to explore the significance of changes in acetylation levels in this process. We used a variety of techniques such as CCK-8 analysis of cell proliferation, FCM analysis of cell cycling and apoptosis, immunofluorescence analysis of NF-κB/ p65 localization, and RT-PCR and Western blotting analysis of ac-H3, ac-H4, ac-p65, cyclin D1, Bcl-2 and Bcl- xl. We found that on treatment with garcinol in MCF-7 cells, E2-induced proliferation was inhibited, cell cycle progression was arrested at G0/G1 phase, and the cell apoptosis rate was increased. Expression of ac-H3, ac-H4 and NF-κB/ac-p65 proteins in E2-treated MCF-7 cells was increased, this being inhibited by garcinol but not ac- H4.The nuclear translocation of NF-κB/p65 in E2-treated MCF-7 cells was also inhibited, along with cyclin D1, Bcl-2 and Bcl-xl in mRNA and protein expression levels. These results suggest that the effect of E2 on promoting proliferation and inhibiting apoptosis is linked to hyperacetylation levels of histones and nonhistone NF-κB/ p65 in MCF-7 cells. The acetyltransferase inhibitor garcinol plays an inhibitive role in MCF-7 cell proliferation promoted by E2. Mechanisms are probably associated with decreasing ac-p65 protein expression level in the NF-κB pathway, thus down-regulating the expression of cyclin D1, Bcl-2 and Bcl-xl.

Topics: Acetyltransferases; Apoptosis; Biomarkers, Tumor; Blotting, Western; Breast Neoplasms; Cell Cycle; Cell Proliferation; Cyclin D1; Estradiol; Estrogens; Female; Flow Cytometry; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; Humans; Immunoenzyme Techniques; NF-kappa B; Proto-Oncogene Proteins c-bcl-2; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Terpenes; Tumor Cells, Cultured

Cyclin D1/CDK4 activity is upregulated in up to 50% of breast cancers and CDK4-mediated phosphorylation negatively regulates the TGFβ superfamily member Smad3. We sought to determine if CDK4 inhibition and doxorubicin chemotherapy could impact Smad3-mediated cell/colony growth and apoptosis in breast cancer cells. Parental and cyclin D1-overexpressing MCF7 cells were treated with CDK4 inhibitor, doxorubicin, or combination therapy and cell proliferation, apoptosis, colony formation, and expression of apoptotic proteins were evaluated using an MTS assay, TUNEL staining, 3D Matrigel assay, and apoptosis array/immunoblotting. Study cells were also transduced with WT Smad3 or a Smad3 construct resistant to CDK4 phosphorylation (5M) and colony formation and expression of apoptotic proteins were assessed. Treatment with CDK4 inhibitor/doxorubicin combination therapy, or transduction with 5M Smad3, resulted in a similar decrease in colony formation. Treating cyclin D overexpressing breast cancer cells with combination therapy also resulted in the greatest increase in apoptosis, resulted in decreased expression of anti-apoptotic proteins survivin and XIAP, and impacted subcellular localization of pro-apoptotic Smac/DIABLO. Additionally, transduction of 5M Smad3 and doxorubicin treatment resulted in the greatest change in apoptotic protein expression. Collectively, this work showed the impact of CDK4 inhibitor-mediated, Smad3-regulated tumor suppression, which was augmented in doxorubicin-treated cyclin D-overexpressing study cells.

Topics: Antibiotics, Antineoplastic; Apoptosis; Apoptosis Regulatory Proteins; Breast Neoplasms; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Doxorubicin; Drug Synergism; Humans; Inhibitor of Apoptosis Proteins; MCF-7 Cells; Mutation; Phosphorylation; Protein Kinase Inhibitors; Signal Transduction; Smad3 Protein; Survivin; X-Linked Inhibitor of Apoptosis Protein

The Tongshu Capsule (TSC) is a prevalent form of traditional Chinese medicine widely used for its purported effects in treating mammary gland hyperplasia and inflammation. Though successful in several clinical studies, there is no clear evidence as to why TSC has a positive treatment effect, and little known about underlying mechanism that may account for it. In this study, we examined the effects of TSC and found that it has a comparatively strong growth inhibition on ERα positive breast cancer cells. TSC seems to cause G1 cell cycle arrest instead of apoptosis. Interestingly, TSC also down-regulated the expression of ERα and Cyclin D1. Consistently, TSC suppressed E2 mediated ERα downstream gene expression and cell proliferation in ERα positive breast cancer cell lines MCF7 and T47D. Depletion of ERα partially abolished the effects of TSC on the decrease of Cyclin D1 and cell viability. Our findings suggest that TSC may have therapeutic effects on ERα positive breast cancers and moreover that TSC may suppress breast epithelial cell proliferation by inhibiting the estrogen pathway.

Topics: Breast Neoplasms; Cyclin D1; Estrogen Receptor alpha; Estrogens; Female; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation; Humans; Medicine, Chinese Traditional; Signal Transduction

Kruppel-like-factor 17 (KLF17) is a negative regulator of metastasis and epithelial-mesenchymal-transition (EMT). However, its expression is downregulated in metastatic breast cancer that contains p53 mutations. Here, we show that mutant-p53 plays a key role to suppress KLF17 and thereby enhances cancer progression, which defines novel gain-of-function (GOF) of mutant-p53. Mutant-p53 interacts with KLF17 and antagonizes KLF17 mediated EMT genes transcription. Depletion of KLF17 promotes cell viability, decreases apoptosis and induces drug resistance in metastatic breast cancer cells. KLF17 suppresses cell migration and invasion by decreasing CD44, PAI-1 and Cyclin-D1 expressions. Taken together, our results show that KLF17 is important for the suppression of metastasis and could be a potential therapeutic target during chemotherapy.

Topics: Apoptosis; Breast Neoplasms; Carcinoma; Cell Line, Tumor; Cell Movement; Cell Survival; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Hyaluronan Receptors; MCF-7 Cells; Mutation; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; Plasminogen Activator Inhibitor 1; RNA Interference; Transcription Factors; Tumor Suppressor Protein p53

The aim of the present study was to investigate the involvement of emodin on the growth of human breast cancer MCF-7 and MDA-MB-231 cells and the estrogen (E2) signal pathway in vitro.. MTT assays were used to detect the effects of emodin on E2 induced proliferation of MCF-7 and MDA-MB-231 cells. Flow cytometry (FCM) was applied to determine the effect of emodin on E2-induced apoptosis of MCF-7 cells. Western blotting allowed detection of the effects of emodin on the expression of estrogen receptor α, cyclin D1 and B-cell lymphoma-2 (Bcl-2), mitogen-activated protein kinases (MAPK) and phosphatidylinostiol 3-kinases (PI3K). Luciferase assays were emplyed to assess transcriptional activity of ERα.. Emodin could inhibit E2-induced MCF-7 cell proliferation and anti-apoptosis effects, and arrest the cell cycle in G0/G1 phase, further blocking the effect of E2 on expression and transcriptional activity of ERα. Moreover, Emodin influenced the ER α genomic pathway via downregulation of cyclin D1 and Bcl-2 protein expression, and influenced the non-genomic pathway via decreased PI3K/Akt protein expression.. These findings indicate that emodin exerts inhibitory effects on MCF-7 cell proliferation via inhibiting both non-genomic and genomic pathways.

Topics: Apoptosis; Blotting, Western; Breast Neoplasms; Cell Cycle; Cell Proliferation; Cyclin D1; Emodin; Estrogen Receptor alpha; Estrogens; Female; Flow Cytometry; Humans; Mitogen-Activated Protein Kinases; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Tumor Cells, Cultured

ω-3-17,18-Epoxyeicosapentaenoic acid decreases cell proliferation and activates apoptosis, whereas its regioisomers stimulate growth. We evaluated synthetic ω-3 epoxides of saturated fatty acids as antiproliferative and pro-apoptotic agents in MDA-MB-231 breast cancer cells. The epoxides, but not their urea, amide, or carbamate isosteres, impaired ATP production, enhanced caspase-3 activity, and activated c-jun-N-terminal-kinase signaling, leading to cyclin D1 down-regulation and cell cycle arrest in G1-phase. Fatty acid ω-3 monoepoxides may represent a novel class of antitumor agents.

Topics: Adenosine Triphosphate; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Caspase 3; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Enzyme Activation; Epoxy Compounds; Fatty Acids, Omega-3; Female; Humans; Immunoblotting; JNK Mitogen-Activated Protein Kinases; Models, Chemical; Molecular Structure; Signal Transduction

The aim of this study was to investigate the effect of a Lipofectamine2000 (Life2000) Transfection Reagent transfected psiRNA-STAT3 plasmid on 4T1 breast cancer cells.. MTT was used to detect the cell proliferation of breast cancer 4T1 cells at different periods (0h, 6h, 8h, 10h); the cell cycle was assessed by flow cytometry; variation of apoptosis and mitochondrial membrane potential was observed under a fluorescence microscope; immunohistochemical staining was used to determine the expression of caspase-3 and cyclin-D1 protein.. An obvious effect of inhibition to 4T1 cancer cells could be observed at 8h after the psiRNA-STAT3 was transfected. Typical alterations of apoptotic morphological features were visible in the psiRNA-STAT3 treatment group. Mitochondrial membrane potential decreased significantly, the number of cells was increased in G0/G1 phase, and the number of cells was decreased in S phase, and the data were statistically significant (p<0.05), compared with the Scramble and Mock groups. Expression of caspase-3 protein was increased significantly, while that of cyclin D1 was significantly decreased.. Life2000 transfected psiRNA-STAT3 plasmid can inhibit 4T1 tumor cell proliferation and promote apoptosis of 4T1 tumor cells, which process depends on the regulation of expression of cyclin D1 and caspase-3 protein.

Topics: Animals; Apoptosis; Breast Neoplasms; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; G1 Phase Cell Cycle Checkpoints; Lipids; Membrane Potential, Mitochondrial; Mice; RNA, Small Interfering; S Phase Cell Cycle Checkpoints; STAT3 Transcription Factor; Transfection

Cyclin D1b, a splice variant of the cell cycle regulator cyclin D1, holds oncogenic functions in human cancer. However, the mechanisms underlying cyclin D1b function remain poorly understood. Here we introduced wild-type cyclin D1a or cyclin D1b variant into non-metastatic MCF-7 cells. Our results show that ectopic expression of cyclin D1b promotes invasiveness of the cancer cells in a cyclin D1a independent manner. Specifically, cyclin D1b is found to modulate the expression of αvβ3, which characterizes the metastatic phenotype, and enhance tumor cell invasive potential in cooperating with HoxD3. Notably, cyclin D1b promotes αvβ3-mediated adhesion and invasive migration, which are associated with invasive potential of breast cancer cells. Further exploration indicates that cyclin D1b makes breast cancer cells more sensitive to toll-like receptor 4 ligand released from damaged tumor cells. These findings reveal a role of cyclin D1b as a possible mediator of αvβ3 transcription to promote tumor metastasis.

Topics: Animals; Breast Neoplasms; Cell Adhesion; Cell Movement; Cyclin D1; Female; Homeodomain Proteins; Humans; Integrin alphaVbeta3; Integrin beta3; Ligands; Lung Neoplasms; Lymphatic Metastasis; MCF-7 Cells; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Phenotype; Protein Isoforms; Time Factors; Toll-Like Receptor 4; Transcription Factors; Transfection

Estrogen and estrogen receptor (ER)-mediated signaling pathways play important roles in the etiology and progression of human breast, endometrial, and ovarian cancers. Attenuating ER activities by natural products and their derivatives is a relatively practical strategy to control and reduce breast, endometrial, and ovarian cancer risk. Here, we found 3-butoxy-1,8,9-trihydroxy-6H-benzofuro[3,2-c]benzopyran-6-one (BTB), a new derivative of wedelolactone, could effectively inhibit the 17-estradiol (E2)-induced ER transactivation and suppress the growth of breast cancer as well as endometrial and ovarian cancer cells. Our results indicate that 2.5 μM BTB effectively suppresses ER-positive, but not ER-negative, breast, endometrial, and ovarian cancer cells. Furthermore, our data indicate that BTB can modulate ER transactivation and suppress the expression of E2-mediated ER target genes (Cyclin D1, E2F1, and TERT) in the ER-positive MCF-7, Ishikawa, and SKOV-3 cells. Importantly, this BTB mediated inhibition of ER activity is selective since BTB does not suppress the activities of other nuclear receptors, including glucocorticoid receptor and progesterone receptor, suggesting that BTB functions as a selective ER signaling inhibitor with the potential to treat breast, endometrial, and ovarian cancers.

Topics: Breast Neoplasms; Cell Proliferation; Coumarins; Cyclin D1; Endometrial Neoplasms; Estradiol; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Ovarian Neoplasms; Signal Transduction

Breast cancer (BC) is the most frequently malignancy in women. Therefore, establishment of an animal model for the development of preventative measures and effective treatment for tumors is required. A novel heterogeneous spontaneous mammary tumor animal model of Kunming mice was generated. The purpose of this study was to characterize the spontaneous mammary tumor model. Histopathologically, invasive nodular masses of pleomorphic tubular neoplastic epithelial cells invaded fibro-vascular stroma, adjacent dermis and muscle tissue. Metastatic spread through blood vessel into liver and lungs was observed by hematoxylin eosin staining. No estrogen receptor (ER) or progesterone receptor (PR) immunoreactivity was detected in their associated malignant tumors, human epidermal growth factor receptor-2 (HER-2) protein weak expression was found by immunohistochemistry. High expression of vascular endothelial growth factor (VEGF), moderate or high expression of c-Myc and cyclin D1 were observed in tumor sections at different stages (2, 4, 6 and 8 weeks after cancer being found) when compared with that of the normal mammary glands. The result showed that the model is of an invasive ductal carcinoma. Remarkably in the mouse model, ER and PR-negative and HER2 weak positivity are observed. The high or moderate expressions of breast cancer markers (VEGF, c-Myc and cyclin D1) in mammary cancer tissue change at different stages. To our knowledge, this is the first report of a spontaneous mammary model displaying colony-strain, outbred mice. This model will be an attractive tool to understand the biology of anti-hormonal breast cancer in women.

Topics: Animals; Biomarkers, Tumor; Breast Neoplasms; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Mammary Neoplasms, Animal; Mice; Proto-Oncogene Proteins c-myc; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone; Vascular Endothelial Growth Factor A

Circulating tumor cells (CTCs) are promising surrogate markers for systemic disease, and their molecular characterization might be relevant to guide more individualized cancer therapies. To enable fast and efficient purification of individual CTCs, we developed a work flow from CellSearch(TM) cartridges enabling high-resolution genomic profiling on the single-cell level.. Single CTCs were sorted from 40 CellSearch samples from patients with metastatic breast cancer using a MoFlo XDP cell sorter. Genomes of sorted single cells were amplified using an adapter-linker PCR. Amplification products were analyzed by array-based comparative genomic hybridization, a gene-specific quantitative PCR (qPCR) assay for cyclin D1 (CCND1) locus amplification, and genomic sequencing to screen for mutations in exons 1, 9, and 20 of the phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) gene and exons 5, 7, and 8 of the tumor protein p53 (TP53) gene.. One common flow-sorting protocol was appropriate for 90% of the analyzed CellSearch cartridges, and the detected CTC numbers correlated positively with those originally detected with the CellSearch system (R(2) = 0.9257). Whole genome amplification was successful in 72.9% of the sorted single CTCs. Over 95% of the cells displayed chromosomal aberrations typical for metastatic breast cancers, and amplifications at the CCND1 locus were validated by qPCR. Aberrant CTCs from 2 patients harbored mutations in exon 20 of the PIK3CA gene.. This work flow enabled effective CTC isolation and provided insights into genomic alterations of CTCs in metastatic breast cancer. This approach might facilitate further molecular characterization of rare CTCs to increase understanding of their biology and as a basis for their molecular screening in the clinical setting.

Topics: Biomarkers, Tumor; Breast Neoplasms; Class I Phosphatidylinositol 3-Kinases; Comparative Genomic Hybridization; Cyclin D1; DNA Copy Number Variations; Exons; Female; Flow Cytometry; Humans; Leukocyte Common Antigens; Mutation; Neoplasm Metastasis; Neoplastic Cells, Circulating; Phosphatidylinositol 3-Kinases; Phycocyanin; Phycoerythrin; Single-Cell Analysis; Tumor Suppressor Protein p53

Transducin (β)-like 1 X-linked receptor 1(TBLR1) is an F-box-like and WD repeat-containing protein which functions as a switch in transcriptional activation, However, the clinical significance and biological role of TBLR1 in breast cancer remains largely unknown.. Western blotting, immunocytochemistry and real-time PCR were used to evaluate TBLR1 expression in normal breast epithelial cells and breast cancer cell lines, clinical tissue samples and adjacent nontumor tissues, and in 214 paraffin-embedded specimens. Statistical analyses were used to test for the prognostic and diagnostic associations. The biological role of TBLR1 -induced proliferation and tumorigenicity in breast cancer cells was explored in vitro and in vivo. The effect of TBLR1 on the expression of cyclin D1 and β-catenin signaling was examined by Western blotting, luciferase reporter assay and by several immunoprecipitation techniques.. TBLR1 was significantly upregulated in breast cancer cells and tissues compared to normal control samples. Immunohistochemical analysis revealed high expression of TBLR1 in 113 of 214 (52.8%) paraffin-embedded archival breast cancer. The overall expression level of TBLR1 was significantly correlated with clinical stage (P <0.001), the tumor classification (P <0.001), node classification (P =0.024), and metastasis classification (P = 0.004), histological grade (P = 0.044), as well as with the expression level of c-erbB2 (P = 0.036) and Ki-67 (P <0.001). Patients with higher TBLR1 expression had shorter overall survival time, whereas patients with lower TBLR1 expression had better survival. Multivariate analysis suggested that TBLR1 expression might be an independent prognostic indicator for the survival of breast cancer patients. TBLR1 overexpression promoted, whereas TBLR1 silencing inhibited, proliferation and tumorigenicity in breast cancer cells both in vitro and in vivo. We found that TBLR1 expression was implicated in the upregulation of cyclin D1, phosphorylation of cell-cycle control protein Rb (pRb) and activation of β-catenin signaling in breast cancer.. TBLR1 plays a key role in the development and progression of breast cancer cells via cyclin D1-transactivation and activation of the β-catenin signaling pathway. TBLR1 may be a novel prognostic marker and a potential therapeutic target in the treatment human breast cancer.

Topics: Animals; beta Catenin; Breast Neoplasms; Carcinogenesis; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Gene Expression; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Kaplan-Meier Estimate; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Multivariate Analysis; Neoplasm Transplantation; Nuclear Proteins; Prognosis; Proportional Hazards Models; Receptors, Cytoplasmic and Nuclear; Repressor Proteins; Transcriptional Activation; Tumor Burden; Wnt Signaling Pathway

To observe the inhibition effects of the Buthus matensii Karsch (BmK) scorpion venom extracts on the growth of human breast cancer MCF-7 cells, and to explore its mechanisms.. Two common tumor cells (SMMC7721, MCF-7) were examined for the one which wasmore sensitivity to scorpion venom by MTT method. Cell cycle was determined by flow cytometry. Immunocytochemistry was applied to detect apoptosis-related protein Caspase-3 and Bcl-2 levels, while the expression of cell cycle-related protein Cyclin D1 was shown by Western blotting.. Our data indicated that MCF-7 was the more sensitive cell line to scorpion venom. The extracts of scorpion venom could inhibit the growth and proliferation of MCF-7 cells. Furthermore, the extract of scorpion venom induced apoptosis through Caspase-3 up-regulation while Bcl-2 down-regulation in MCF-7 cells. In addition, the extracts of scorpion venom blocked the cells from G0/G1 phase to S phase and decreased cell cycle-related protein Cyclin D1 level after drug intervention compared with the negative control group.. These results showed that the BmK scorpion venom extracts could inhibit the growth of MCF-7 cells by inducing apoptosis and blocking cell cycle in G0/G1 phase. The BmK scorpion venom extracts will be very valuable for the treatment of breast cancer.

Topics: Animals; Antineoplastic Agents; Apoptosis; Biological Products; Breast Neoplasms; Caspase 3; Cell Cycle; Cell Proliferation; Cyclin D1; Down-Regulation; Female; Humans; MCF-7 Cells; Proto-Oncogene Proteins c-bcl-2; Scorpion Venoms; Scorpions

Human Rad 9 (hRad9), part of the Rad9-Hus1-Rad1 complex plays an important role in DNA damage repair as an up-stream regulator of checkpoint signaling, however little is known about its role in response to chemotherapy of breast cancer and whether hRad9 inhibition can potentiate the cytotoxic effects of chemotherapy on breast cancer cells remains to be elucidated. Fifty cases of breast cancer receiving neoadjuvant therapy were collected. All these cases were revised and classified into chemotherapy sensitive (CS) or chemotherapy resistant (CR) group according to the Miller and Payne (MP) grading system. Immunohistochemically, hRad9 positive tumours showed nuclear and/or cytoplasmic staining. hRad9 over-expression was associated with an impaired neoadjuvant chemotherapy response. A significant correlation was found between expression of hRad9 and Cyclin D1. In vitro, hRad9 was knocked down using siRNA in breast cancer cell line MCF-7 and MDA-MB-231. Deregulated expression of Rad9 accompanied by down expression of chk1 enhanced the sensitivity of human breast cancer cells to doxorubicin. Our work suggests that hRad9 might be a potential predictor for the response to chemotherapy in patients with breast cancer and its clinical value as a target for improving chemosensitivity needs further exploration.

Topics: Breast Neoplasms; Cell Cycle Proteins; Cell Line, Tumor; Cell Nucleus; Checkpoint Kinase 1; Chemotherapy, Adjuvant; Cyclin D1; Cytoplasm; DNA Damage; DNA Repair; Doxorubicin; Drug Resistance, Neoplasm; Female; Gene Expression; Humans; MCF-7 Cells; Middle Aged; Neoadjuvant Therapy; Protein Kinases

Eleutherococcus trifoliatus has been used as a folk medicine since ancient times, especially as refreshing qi medicines. In our current study, taiwanin E, which possesses strong cytotoxicity, was isolated from the branches of E. trifoliatus by using a bioactivity guided fractionation procedure. Taiwanin E presented a potent anti-proliferation activity on the growth of a human breast adenocarcinoma cell line (MCF-7), with an IC50 value for cytotoxicity of 1.47 μM. Cell cycle analysis revealed that the proportion of cells in the G0/G1 phase increased in a dose-dependent manner (from 79.4% to 90.2%) after 48 h exposure to taiwanin E at a dosage range from 0.5 to 4μM. After treatment with taiwanin E, phosphorylation of retinoblastoma protein (pRb) in MCF-7 cells was inhibited, accompanied by a decrease in the levels of cyclin D1, cyclin D3 and cyclin-dependent kinase 4 (cdk4) and cdk6; in addition, there was an increase in the expression of cyclin-dependent kinase inhibitors p21(WAF-1/Cip) and p27(Kip1). The results suggest that taiwanin E inhibits cell cycle progression of MCF-7 at the G0/G1 transition.

Topics: Breast Neoplasms; Cell Cycle; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Down-Regulation; Eleutherococcus; Female; Growth Inhibitors; Humans; MCF-7 Cells; Phosphorylation; Plant Extracts; Retinoblastoma Protein

The purposes of this study were to investigate the effects of B cell translocation gene 2 (BTG2) on the proliferation, apoptosis, and invasion of triple-negative breast cancer and to provide an experimental basis for the future treatment of human triple-negative breast cancer. A pcDNA3.1-BTG2 eukaryotic expression vector was constructed and transfected into the MDA-MB-231 human triple-negative breast cancer cell line using lipofection. Then, relevant changes in the biological characteristics of the BTG2-expressing cell line were analyzed using MTT (tetrazolium blue), flow cytometry, and Transwell invasion chamber assays. Additionally, the effects of BTG2 expression on cyclin D1, caspase 3, and matrix metalloproteinases 1/2 (MMP-1/-2) expression were analyzed. Cell proliferation was significantly lower in the pcDNA3.1-BTG2-transfected group compared to the empty vector and blank control groups (p<0.05). There was no significant difference between the empty vector and blank control groups. FCM results demonstrated that there were significantly more cells in the G1 phase of the cell cycle and fewer S phase cells in the pcDNA3.1-BTG2 group than in the empty vector and blank control groups (p<0.05). Additionally, the proportion of cells that migrated across the membrane was significantly lower in the pcDNA3.1-BTG2 group than in the empty vector and blank control groups (p<0.05). Cyclin D1 and MMP-1/-2 expression were significantly lower in MDA-MB-231 cells transfected with pcDNA3.1-BTG2 as compared to the empty vector and blank control groups (p<0.05). Caspase 3 expression was significantly higher in MDA-MB-231 cells from the pcDNA3.1-BTG2 group compared to the empty vector and blank control groups (p<0.05). In conclusion, BTG2 may inhibit MDA-MB-231 proliferation and promote apoptosis. Additionally, BTG2 may also inhibit the invasion of MDA-MB-231 human triple-negative breast cancer cells.

Topics: Apoptosis; Blotting, Western; Breast Neoplasms; Caspase 3; Cell Adhesion; Cell Movement; Cell Proliferation; Cyclin D1; Female; Flow Cytometry; Humans; Immediate-Early Proteins; Matrix Metalloproteinase 1; Matrix Metalloproteinase 2; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone; Tumor Cells, Cultured; Tumor Suppressor Proteins

The oncogenic role of estrogen receptor (ER)α and its correlation with let-7 microRNAs (miRNAs) have been studied and confirmed in breast tumors; however, this correlation has not been investigated in breast cancer stem cells (BCSCs). In the present study, we detected the expression of let-7 and ERα in ER-positive breast tumor tissues. Furthermore, we used a FACSAria cell sorter to separate side population (SP) cells from the MCF-7 and T47-D cell lines by Hoechst 33342 staining. The expression of let-7 miRNAs, ERα and its downstream genes in SP and non-SP (NSP) cells were analyzed. In additional experiments, we transfected a plasmid expressing let-7a into SP cells isolated from the MCF-7 and T47-D cell lines in order to observe changes in the expression of downstream genes (cyclin D1 and pS2). The correlation among let-7, ERα and ERα downstream genes suggested that let-7 acts as a tumor suppressor by inhibiting ERα-mediated cellular malignant growth in ER-positive breast cancer stem cells. The suppression of ERα by the upregulation of let-7 expression may be a promising strategy for the inhibition of the ER signaling pathway and for the elimination of cancer stem cells, thus aiding in the treatment of breast cancer.

Topics: Apoptosis; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cyclin D1; Estrogen Receptor alpha; Female; Humans; MCF-7 Cells; MicroRNAs; Middle Aged; Neoplastic Stem Cells; Presenilin-2; Side-Population Cells; Signal Transduction; Up-Regulation

Transforming growth factor-β (TGF-β) is a potent inhibitor of the growth of normal mammary epithelial cells, and has a pleiotropic, context-dependent, concentration-dependent action. We found attenuation of TGF-β signalling in mammary adenoma carcinoma cells. Phosphorylation at the linker site of Smad2 occurred in a cooperative way during the attenuation of TGF-β signalling, and was associated with upregulation of CDK2 and cyclin D1. CDK2 inhibitor restored the anti-proliferative effect of TGF-β by upregulating p21, with inhibition of linker phosphorylation of Smad2. CDK2-mediated linker phosphorylation of Smad2 may be a plausible mechanism for the attenuation of TGF-β signalling in breast cancer.

Topics: Adenocarcinoma; Breast Neoplasms; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p21; Female; G1 Phase Cell Cycle Checkpoints; Humans; MCF-7 Cells; Phosphorylation; RNA Interference; RNA, Small Interfering; Signal Transduction; Smad2 Protein; Transforming Growth Factor beta; Up-Regulation

BRG1, a core component of the SWI/SNF chromatin-remodeling complex, has been implicated in cancer development; however, the biological significance of BRG1 in breast cancer remains unknown. We explored the role of BRG1 in human breast cancer pathogenesis. Using tissue microarray and immunohistochemistry, we evaluated BRG1 staining in 437 breast cancer specimens and investigated its role in breast cancer cell proliferation, migration and invasion. Our Kaplan-Meier survival curves showed that high BRG1 expression is inversely correlated with both overall (P = 0.000) and disease-specific (P = 0.000) 5-year patient survival. Furthermore, we found that knockdown of BRG1 by RNA interference markedly inhibits cell proliferation and causes cessation of cell cycle. This reduced cell proliferation is due to G1 phase arrest as cyclin D1 and cyclin E are diminished whereas p27 is upregulated. Moreover, BRG1 depletion induces the expression of TIMP-2 but reduces MMP-2, thereby inhibiting the ability of cells to migrate and to invade. These results highlight the importance of BRG1 in breast cancer pathogenesis and BRG1 may serve as a prognostic marker as well as a potentially selective therapeutic target.

Topics: Breast Neoplasms; Cell Cycle; Cyclin D1; Cyclin E; DNA Helicases; Female; Humans; Immunohistochemistry; In Vitro Techniques; Kaplan-Meier Estimate; Nuclear Proteins; Prognosis; Transcription Factors

Analysis of 4,405 variants in 89,050 European subjects from 41 case-control studies identified three independent association signals for estrogen-receptor-positive tumors at 11q13. The strongest signal maps to a transcriptional enhancer element in which the G allele of the best candidate causative variant rs554219 increases risk of breast cancer, reduces both binding of ELK4 transcription factor and luciferase activity in reporter assays, and may be associated with low cyclin D1 protein levels in tumors. Another candidate variant, rs78540526, lies in the same enhancer element. Risk association signal 2, rs75915166, creates a GATA3 binding site within a silencer element. Chromatin conformation studies demonstrate that these enhancer and silencer elements interact with each other and with their likely target gene, CCND1.

Topics: Binding Sites; Breast Neoplasms; Case-Control Studies; Cell Line, Tumor; Chromatin; Chromatin Immunoprecipitation; Chromosomes, Human, Pair 11; Cyclin D1; Electrophoretic Mobility Shift Assay; Enhancer Elements, Genetic; ets-Domain Protein Elk-4; Female; GATA3 Transcription Factor; Gene Expression Regulation, Neoplastic; Humans; Luciferases; Polymorphism, Single Nucleotide; Promoter Regions, Genetic; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Silencer Elements, Transcriptional

Glycogen synthase kinase 3α/β (GSK3α/β) is a constitutively active serine/threonine kinase involved in multiple physiological processes, such as protein synthesis, stem cell maintenance and apoptosis, and acts as a key suppressor of the Wnt-β-catenin pathway. In the present study, we examined the therapeutic potential of a novel GSK3 inhibitor, CG0009, in the breast cancer cell lines, BT549, HS578T, MDA-MB-231, NCI/ADR-RES, T47D, MCF7 and MDA-MB-435, from the NCI-60 cancer cell line panel. Assessment of cytotoxicity, apoptosis and changes in estrogen-signaling proteins was performed using cell viability assays, Western blotting and quantitative real-time PCR. CG0009 enhanced the inactivating phosphorylation of GSK3α at Ser21 and GSK3β at Ser9 and simultaneously decreased activating phosphorylation of GSK3β at Tyr216, and induced caspase-dependent apoptosis independently of estrogen receptor α (ERα) expression status, which was not observed with the other GSK3 inhibitors examined, including SB216763, kenpaullone and LiCl. CG0009 treatment (1 µmol/L) completely ablated cyclin D1 expression in a time-dependent manner in all the cell lines examined, except T47D. CG0009 alone significantly activated p53, leading to relocation of p53 and Bax to the mitochondria. GSK3 inhibition by CG0009 led to slight upregulation of the β-catenin target genes, c-Jun and c-Myc, but not cyclin D1, indicating that CG0009-mediated cyclin D1 depletion overwhelms the pro-survival signal of β-catenin, resulting in cell death. Our findings suggest that the novel GSK3 inhibitor, CG0009, inhibits breast cancer cell growth through cyclin D1 depletion and p53 activation, and may thus offer an innovative therapeutic approach for breast cancers resistant to hormone-based therapy.

Topics: Antineoplastic Agents; Apoptosis; Benzazepines; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Cyclin D1; Female; Glycogen Synthase Kinase 3; Humans; Indoles; Lithium Chloride; Maleimides; Real-Time Polymerase Chain Reaction

Biallelic inactivation of LKB1, a serine/threonine kinase, has been detected in 30% of lung adenocarcinomas, and inhibition of breast tumor growth has been demonstrated. We have identified the tumor suppressor, Nischarin, as a novel binding partner of LKB1. Our mapping analysis shows that the N terminus of Nischarin interacts with amino acids 44-436 of LKB1. Time lapse microscopy and Transwell migration data show that the absence of both Nischarin and LKB1 from an invasive breast cancer cell line (MDA-MB-231) enhances migration as measured by increased distance and speed of migrating cells. Our data suggest that this is a result of elevated PAK1 and LIMK1 phosphorylation. Moreover, the absence of Nischarin and LKB1 increased tumor growth in vivo. Consistent with this, the percentage of S phase cells was increased, as demonstrated by flow cytometry and enhanced cyclin D1. The absence of Nischarin and LKB1 also led to a dramatic increase in the formation of lung metastases. Our studies, for the first time, demonstrate functional interaction between LKB1 and Nischarin to inhibit cell migration and breast tumor progression. Mechanistically, we show that these two proteins together regulate PAK-LIMK-Cofilin and cyclin D1/CDK4 pathways.

Topics: AMP-Activated Protein Kinase Kinases; Animals; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cyclin D1; Cyclin-Dependent Kinase 4; Epithelial Cells; Female; Humans; Imidazoline Receptors; Intracellular Signaling Peptides and Proteins; Lim Kinases; Mammary Glands, Human; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Neoplasm Transplantation; Protein Serine-Threonine Kinases; Transplantation, Heterologous; Tumor Suppressor Proteins

Receptor for Advanced Glycation End Products (RAGE) is an oncogenic trans-membranous receptor overexpressed in various human cancers. However, the role of RAGE in breast cancer development and proliferation is still unclear. In this study, we demonstrated that RAGE expression levels are correlated to the degree of severity of breast cancer. Furthermore, there is a decrease in the proliferation of all sub-types of breast cancer, MCF-7, SK-Br-3 and MDA-MB-231, as a result of the effect of RAGE siRNA. RAGE siRNA arrested cells in the G1 phase and inhibited DNA synthesis (p < 0.05). Moreover, qRT-PCR and Western Blot results demonstrated that RAGE siRNA decreases the expression of transcriptional factor NF-κB p65 as well as the expression of cell proliferation markers PCNA and cyclinD1. RAGE and RAGE ligands can thus be considered as possible targets for breast cancer management and therapy.

Topics: Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; DNA, Neoplasm; Female; G1 Phase Cell Cycle Checkpoints; Gene Expression; Gene Knockdown Techniques; Humans; MCF-7 Cells; Proliferating Cell Nuclear Antigen; Receptor for Advanced Glycation End Products; Receptors, Immunologic; RNA, Messenger; RNA, Neoplasm; RNA, Small Interfering; Transcription Factor RelA; Triple Negative Breast Neoplasms

Metastasis is one of the greatest challenges in cancer treatment. In this study, a bioreducible polymer, Tween 85-s-s-polyethyleneimine 2K (TSP), was synthesized and used as a non-viral gene vector for p65 shRNA to block NF-κB signaling pathway, thereby inhibiting the growth and metastasis of breast cancer. The TSP/p65 shRNA complex nanoparticles (TSNs) could significantly down-regulate p65 expression in breast cancer cells due to the rapid degradation of TSP with prompt shRNA release, and consequently not only inhibit cell proliferation and invasion, but also induce cell apoptosis and disrupt the tube formation. Most importantly, TSNs showed high accumulation in tumor and almost completely inhibited the growth and metastasis of the breast cancer xenograft in nude mice induced by MDA-MB-435 cells. All these results indicated the promising of TSP as a non-viral gene vector to knock down p65 expression and inhibit the growth and metastasis of breast cancer.

Topics: Animals; Biocompatible Materials; Breast Neoplasms; Cell Line; Cell Nucleus; Cell Proliferation; Cell Shape; Cyclin D1; Female; Humans; Lymph Nodes; Magnetic Resonance Spectroscopy; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Nanoparticles; Neoplasm Invasiveness; Neoplasm Metastasis; Neovascularization, Pathologic; Polyethyleneimine; Polysorbates; RNA, Small Interfering; Signal Transduction; Tissue Distribution; Transcription Factor RelA

Histone deacetylases (HDACs) are overexpressed in various types of primary human cancer and have become attractive targets for cancer therapy. We designed and synthesized a series of new class of HDAC inhibitors (HDACi). Among these, S-(E)-3-(1-(1-(benzo[d]oxazol-2-yl)-2-methylpropyl)-1H-1,2,3-triazol-4-yl)-N-hydroxyacrylamide (NK-HDAC-1) showed potent antitumor activity. In the present study, we examined the antitumor effects of NK-HDAC-1 on breast cancer in vitro and in vivo. The inhibitory effects of NK-HDAC-1 on HDAC enzyme activity and cell growth were more potent compared to suberoylanilide hydroxamic acid (SAHA). NK-HDAC-1 caused G1 cell cycle arrest at concentrations below 0.2 µM and G2/M arrest at concentrations above 0.4 µM through p21 upregulation and cyclin D1 downregulation. NK-HADC-1 induced hyperacetylation of histone H3 and H4 around the promoter region of p21. NK-HDAC-1 promoted apoptosis in MDA-MB-231 breast cancer cells by activating both the intrinsic and the extrinsic pathway NK-HDAC-1 at doses of 3, 10 and 30 mg/kg reduced the tumor volume in MDA-MB-231 xenografts by 25.9, 48.8 and 63.6%, respectively. The results suggested that NK-HDAC-1 may be a promising therapeutic candidate in treating human breast cancer.

Topics: Animals; Apoptosis; Benzoxazoles; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; Female; G1 Phase Cell Cycle Checkpoints; G2 Phase Cell Cycle Checkpoints; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Male; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; Up-Regulation; Vorinostat; Xenograft Model Antitumor Assays

Angiogenesis and cell cycle control play critical roles in breast cancer susceptibility and clinical outcome and are mainly controlled by vascular endothelial growth factor (VEGF) and cyclin-dependent kinases, respectively. Functional germline polymorphisms in these genes alter the function, thereby causing inter-individual differences in breast cancer risk and clinical outcome. In this study, we investigated the influence of the functional polymorphisms VEGF-A rs3025039 C > T and CCND1 rs9344 G > A on risk and clinical outcome in early-stage breast cancer. DNA of 539 female patients with histologically confirmed early-stage breast cancer and 804 control subjects was genotyped for these polymorphisms. Genotypes were tested for associations with breast cancer risk and clinical outcome. There was no significant association between the polymorphisms and breast cancer risk. However, the minor allele of VEGF-A rs3025039 C > T was significantly associated with decreased recurrence-free survival (HR 1.845; 95% confidence interval [CI] 1.035-3.290; P = 0.038) and remained significant in multivariate analysis (HR 1.880; 95% CI 1.020-3.465; P = 0.043). Patients carrying at least one A-allele in CCND1 rs9344 G > A showed a trend towards decreased recurrence-free survival in univariate analysis (HR 2.379; 95% CI 0.841-6.728; P = 0.068). This study provides evidence that the functional VEGF-A rs3025039 C > T polymorphism influences recurrence-free survival in early-stage breast cancer.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Breast Neoplasms; Cyclin D1; Female; Follow-Up Studies; Genotype; Humans; Male; Middle Aged; Neoplasm Grading; Neoplasm Recurrence, Local; Neoplasm Staging; Polymerase Chain Reaction; Polymorphism, Single Nucleotide; Prognosis; Retrospective Studies; Risk Factors; Survival Rate; Vascular Endothelial Growth Factor A

The Pin1 prolyl isomerase regulates phosphorylation signaling by controlling protein conformation after phosphorylation, and its upregulation promotes oncogenesis via acting on numerous oncogenic molecules. SUMOylation and deSUMOylation are dynamic mechanisms regulating a spectrum of protein activities. The SUMO proteases (SENP) remove SUMO conjugate from proteins, and their expression is deregulated in cancers. However, nothing is known about the role of SUMOylation in regulating Pin1 function. Here, we show that Pin1 is SUMOylated on Lys6 in the WW domain and on Lys63 in the PPIase domain. Pin1 SUMOylation inhibits its protein activity and oncogenic function. We further identify that SENP1 binds to and deSUMOylates Pin1. Importantly, either overexpression of SENP1 or disruption of Pin1 SUMOylation promotes the ability of Pin1 to induce centrosome amplification and cell transformation. Moreover, SENP1 also increases Pin1 protein stability in cell cultures, and Pin1 levels are positively correlated with SENP1 levels in human breast cancer specimens. These results not only uncover Pin1 SUMOylation on Lys6/63 as a novel mechanism to inhibit its activity and function but also identify a critical role for SENP1-mediated deSUMOylation in promoting Pin1 function during tumorigenesis.

Topics: Amino Acid Sequence; Amino Acid Substitution; Animals; Breast Neoplasms; Cell Line; Cell Transformation, Neoplastic; Centrosome; Chromosomal Instability; Cyclin D1; Cysteine Endopeptidases; Endopeptidases; Female; Gene Knockdown Techniques; Humans; Mice; NIMA-Interacting Peptidylprolyl Isomerase; Oxidative Stress; Peptidylprolyl Isomerase; Protein Binding; Protein Interaction Domains and Motifs; Signal Transduction; Sumoylation

The Drosophila Eyes Absent Homologue 1 (EYA1) is a component of the retinal determination gene network and serves as an H2AX phosphatase. The cyclin D1 gene encodes the regulatory subunits of a holoenzyme that phosphorylates and inactivates the pRb protein. Herein, comparison with normal breast showed that EYA1 is overexpressed with cyclin D1 in luminal B breast cancer subtype. EYA1 enhanced breast tumor growth in mice in vivo, requiring the phosphatase domain. EYA1 enhanced cellular proliferation, inhibited apoptosis, and induced contact-independent growth and cyclin D1 abundance. The induction of cellular proliferation and cyclin D1 abundance, but not apoptosis, was dependent upon the EYA1 phosphatase domain. The EYA1-mediated transcriptional induction of cyclin D1 occurred via the AP-1-binding site at -953 and required the EYA1 phosphatase function. The AP-1 mutation did not affect SIX1-dependent activation of cyclin D1. EYA1 was recruited in the context of local chromatin to the cyclin D1 AP-1 site. The EYA1 phosphatase function determined the recruitment of CBP, RNA polymerase II, and acetylation of H3K9 at the cyclin D1 gene AP-1 site regulatory region in the context of local chromatin. The EYA1 phosphatase regulates cell-cycle control via transcriptional complex formation at the cyclin D1 promoter.

Topics: Animals; Apoptosis; Binding Sites; Breast Neoplasms; Cell Growth Processes; Cyclin D1; Female; HEK293 Cells; Homeodomain Proteins; Humans; Intracellular Signaling Peptides and Proteins; MCF-7 Cells; Mice; Mutation; Nuclear Proteins; Promoter Regions, Genetic; Protein Tyrosine Phosphatases; Transcription Factor AP-1

Kallistatin, a plasma protein, exerts pleiotropic effects in inhibiting angiogenesis, inflammation and tumor growth. Canonical Wnt signaling is the primary pathway for oncogenesis in the mammary gland. In this study, we demonstrate that kallistatin bound to the Wnt coreceptor low-density lipoprotein receptor-related protein 6 (LRP6), thus, blocking Wnt/β-catenin signaling and Wnt-mediated growth and migration in MDA-MB-231 breast cancer cells. Kallistatin inhibited Wnt3a-induced proliferation, migration, and invasion of cultured breast cancer cells. Moreover, kallistatin was bound to LRP6 in breast cancer cells, as identified by immunoprecipitation followed by western blot. Kallistatin suppressed Wnt3a-mediated phosphorylation of LRP6 and glycogen synthase kinase-3β, and the elevation of cytosolic β-catenin levels. Furthermore, kallistatin antagonized Wnt3a-induced expression of c-Myc, cyclin D1, and vascular endothelial growth factor. These findings indicate a novel role of kallistatin in preventing breast tumor growth and mobility by direct interaction with LRP6, leading to blockade of the canonical Wnt signaling pathway.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Female; Gene Expression; Gene Expression Regulation, Neoplastic; Humans; Low Density Lipoprotein Receptor-Related Protein-6; Protein Binding; Proto-Oncogene Proteins c-myc; Serpins; Vascular Endothelial Growth Factor A; Wnt Signaling Pathway; Wnt3A Protein

Crk-like (CRKL) is an adapter protein that has crucial roles in multiple biological processes, including cell proliferation, adhesion, and migration. However, the expression pattern of CRKL protein and its clinical significance in human breast cancers have not been well characterized. In this study, expression of CRKL was evaluated in 108 human invasive ductal carcinoma (IDC) tissues by immunohistochemistry. CRKL protein was upregulated in the cancer tissues compared with adjacent normal mammary glands. Overexpression of CRKL was found in 40 of 108 (37.03 %) breast cancer samples and correlated with advanced p-tumor-node-metastasis stage (p = 0.002), nodal metastasis (p = 0.0323), and tumor size (p = 0.0075). In addition, overexpression of CRKL in the MDA-MB-435 cell line promoted cell proliferation, and small interfering RNA knockdown of CRKL in the MDA-MB-453 cell line inhibited proliferation. Further analysis of cell cycle-related molecules showed that CRKL induced cyclin D1 and phosphorylated extracellular signal-regulated kinase expression. In conclusion, this study demonstrated that overexpression of CRKL correlated with progression and malignant proliferation of human breast cancers.

Topics: Adaptor Proteins, Signal Transducing; Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression; Gene Expression Regulation, Neoplastic; Humans; Middle Aged; Nuclear Proteins; Phosphorylation; Protein Processing, Post-Translational

Previously, we have described 5-((4-methoxy-phenyl)-trans-vinyl)-2'-deoxy-uridine, 6, as a fluorescent uridine analogue exhibiting a 3000-fold higher quantum yield (Φ 0.12) and maximum emission (478 nm) which is 170 nm red-shifted as compared to uridine. Here, we utilized 6 for preparation of labeled oligodeoxynucleotide (ODN) probes based on MS2 and cyclin D1 (a known breast cancer mRNA marker) sequences. Cyclin D1-derived labeled-ssODN showed a 9.5-fold decrease of quantum yield upon duplex formation. On the basis of this finding, we developed the ds-NIF (nucleoside with intrinsic fluorescence)-probe methodology for detection of cyclin D1 mRNA, by which the fluorescent probe is released upon recognition of target mRNA by the relatively dark NIF-duplex-probe. Indeed, we successfully detected, a ss-deoxynucleic acid (DNA) variant of cyclin D1 mRNA using a dark NIF-labeled duplex-probe, and monitoring the recognition process by fluorescence spectroscopy and gel electrophoresis. Furthermore, we successfully detected cyclin D1 mRNA in RNA extracted from cancerous human cells, using ds-NIF methodology.

Topics: Base Sequence; Biomarkers, Tumor; Breast Neoplasms; Cell Line, Tumor; Cyclin D1; DNA Probes; Humans; Molecular Probe Techniques; Oligodeoxyribonucleotides; RNA, Messenger; Spectrometry, Fluorescence; Temperature

Various preclinical and clinical studies have linked diabetes and breast cancer, but little is known regarding the molecular mechanism involved. This study aimed to investigate the effect of high glucose and insulin in breast cancer cells (MCF-7: non-invasive, hormone dependent, and MDA-MB-231: invasive, hormone independent). In contrast to MCF-7 cells, high glucose augmented proliferation of MDA-MB-231 cells as observed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and bromodeoxyuridine assays. The high-glucose condition led to increased expression of cyclin D1, de-phosphorylation of p38, and increased phosphorylation of ERK in MDA-MB-231 cells but not in MCF-7 cells. Interestingly, we observed increased phosphorylation of GSK-3β, NF-κB, and ERα only in MCF-7 cells, highlighting their role as potential targets in prevention of progression of breast cancer under a high-glucose and insulin condition. Furthermore, insulin treatment under a high-glucose condition resulted in increased histone H3 phosphorylation and de-acetylation only in MDA-MB-231 cells. Taken together, we provide the first evidence that high glucose and insulin promotes proliferation of MDA-MB-231 cells by differential alteration of GSK-3β, NF-κB, and ERα expression and histone H3 modifications, which may directly or indirectly modulate the expression of genes involved in its proliferation.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Estrogen Receptor alpha; Extracellular Signal-Regulated MAP Kinases; Female; Glucose; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Histones; Humans; Insulin; MCF-7 Cells; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Processing, Post-Translational; Reactive Oxygen Species

Breast cancer remains the leading cause of cancer-related deaths among women. Owing to high efficiency and low toxic effects, further exploration of natural compounds from Chinese herbal medicine may be an efficient approach for breast cancer drug discovery. In this study, we investigated the effects of evodiamine on the growth and metastasis of MDA-MB-231 human breast cancer cells in vitro and in vivo. In vitro, evodiamine inhibited cell migration and invasion abilities through downregulation of MMP-9, urokinase-type plasminogen activator (uPA) and uPAR expression. Evodiamine-induced G0/G1 arrest and apoptosis were associated with a decrease in Bcl-2, cyclin D1 and cyclin-dependent kinase 6 (CDK6) expression and an increase in Bax and p27Kip1 expression. Moreover, evodiamine regulated p-ERK and p-p38 MAPK expression. Evodiamine-induced apoptosis was enhanced by its combination with the extracellular signal-regulated kinase (ERK) inhibitor PD98059 or the p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB203580. Evodiamine-inhibited metastasis was partly blocked by combination with PD98059 or SB203580. In vivo, the administration of evodiamine (10 mg/kg) significantly reduced tumor growth and pulmonary metastasis. These results demonstrate that evodiamine possesses antitumor activities via inhibition of cell migration and invasion, arrest of the cell cycle and induction of cell apoptosis in MDA-MB-231 cells.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinase Inhibitor p27; Down-Regulation; Extracellular Signal-Regulated MAP Kinases; Female; G1 Phase; Humans; Matrix Metalloproteinase 9; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; p38 Mitogen-Activated Protein Kinases; Proto-Oncogene Proteins c-bcl-2; Quinazolines; Receptors, Urokinase Plasminogen Activator; Resting Phase, Cell Cycle; Urokinase-Type Plasminogen Activator

CARMA3 was recently reported to be overexpressed in several cancers and associated with malignant behavior of cancer cells. However, the expression pattern and biological roles of CARMA3 in breast cancer have not been reported. In the present study, we found that CARMA3 was overexpressed in 41.9 % of breast cancer specimens. Significant association was observed between CARMA3 overexpression and TNM stage (p = 0.0223), tumor size (p = 0.0227), and ErbB-2 status (p = 0.0049). Furthermore, knockdown of CARMA3 expression in MDA-MB-435 cells with high endogenous expression decreased cell proliferation and sensitized cell to paclitaxel-induced apoptosis, while overexpression of CARMA3 in MDA-MB-231 cell line promoted cell proliferation and inhibited apoptosis. Further analysis showed that CARMA3 depletion downregulated, and its overexpression upregulated cyclin D1, Bcl-2, and p-IκB levels. In conclusion, our study demonstrated that CARMA3 is overexpressed in breast cancers. CARMA3 facilitates proliferation and inhibits apoptosis through nuclear factor-kappaB signaling.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Breast Neoplasms; CARD Signaling Adaptor Proteins; Case-Control Studies; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Gene Expression; Humans; Mammary Glands, Human; Middle Aged; NF-kappa B; Paclitaxel; Proto-Oncogene Proteins c-bcl-2; Signal Transduction

The poor prognostic outcome of breast cancer is largely due to its resistance to cancer therapies. Development of therapeutic agents that can inhibit growth and induce apoptosis in breast cancer cells can help solve the problem. Emodin is an active anthraquinone that has been reported to have diverse biological effects.. In this study, the anticancer effects of emodin on growth inhibition, apoptosis induction and the expression of apoptosis-related genes in MCF-7 cells were investigated.. Growth inhibition induced by emodin was investigated by the MTS assay and the colony formation assay; while emodin-induced apoptosis was determined by the COMET assay and DNA fragmentation detection. Emodin (35 μM)-induced alterations in the expression of apoptotic-related genes were detected by using real-time PCR.. Emodin had significant growth inhibitory effects on MCF-7 cells with IC₅₀ = 7.22 µg/ml (∼30 μM). It also exerted a concentration-dependant inhibitory effect on the colony-forming ability of MCF-7 cells with IC₅₀ = 7.60 µg/ml (∼30 µM). Hallmarks of apoptosis, such as single-strand DNA breakage and DNA fragmentation, were observed in emodin-treated MCF-7 cells. The gene expression of Fas ligand (FASL) was up-regulated (p < 0.01) but those of MCL1, CCND1 and C-MYC were down-regulated (p < 0.05) in emodin-treated MCF-7 cells.. This study indicated that emodin could induce growth inhibition and apoptosis in MCF-7 cells through the modulation of the expression of apoptosis-related genes. The growth inhibitory effects of emodin might involve both the intrinsic and the extrinsic apoptotic pathways and cell cycle arrest.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Regulatory Proteins; Breast Neoplasms; Carcinoma; Cathartics; Cell Proliferation; Cyclin D1; DNA Breaks, Single-Stranded; DNA Fragmentation; Emodin; Fas Ligand Protein; Female; Gene Expression Regulation, Neoplastic; Humans; Inhibitory Concentration 50; MCF-7 Cells; Myeloid Cell Leukemia Sequence 1 Protein; Neoplastic Stem Cells; Proto-Oncogene Proteins c-myc

Phosphoethanolamine (Pho-s) is a compound involved in phospholipid turnover, acting as a substrate for many phospholipids of the cell membranes. In a recent study, we showed that Pho-s has antitumor effect in the several tumor cells. In this study we evaluated the antitumor activity of synthetic Pho-s on MCF-7 breast cancer cells. Here we demonstrate that Pho-s is cytotoxic to MCF-7 cells in a dose-dependent manner, while it is cytotoxic to MCF10 only at higher concentrations. In addition, Pho-s induces a disruption in mitochondrial membrane potential (Δψm). Furthermore, Pho-s induces mitochondria aggregates in the cytoplasm and DNA fragmentation of MCF-7 cells visualized by confocal microscopy. In agreement with the reduction on Δψm, we showed that Pho-s induces apoptosis followed by an increase in cytochrome c expression and capase-3-like activity in MCF-7 cells. Our results demonstrate that Pho-s induces a cell cycle arrest in the G1 phase through an inhibition of cyclin D1 and stimulates p53. An additional highlight of this study is the finding that Pho-s inhibits Bcl-2, inducing apoptosis through the mitochondrial pathway. Taken together, these results show that Pho-s is a promising compound in the fight against cancer.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Caspase 3; Cell Cycle Checkpoints; Cell Death; Cell Line, Tumor; Cyclin D1; Cytochromes c; Cytoplasm; DNA Fragmentation; Ethanolamines; Female; G1 Phase; Humans; MCF-7 Cells; Membrane Potential, Mitochondrial; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Tumor Suppressor Protein p53

Estrogen signaling pathways play an important role in the regulation of the physiological function of breast cancer cell proliferation and apoptosis. The article used MTT assay, flow cytometer analysis and Western blot to detect the inhibition of fraxetin on MCF-7 cell cycle distribution and apoptosis, ERα, cyclin D1 and Bcl-2 expression levels, MAPK and PI3K signaling pathway to investigate the mechanism of anti-breast cancer of fraxetin. The results showed fraxetin inhibited E2β-stimulated MCF-7 cell proliferation in a dose- and time-dependent manner, reversed E2β-induced anti-apoptosis and promoted G0/G1 phase arrest. After treatment with fraxetin, the expression of ERα in MCF-7 cell was decreased, and estrogen genomic signaling pathway was inhibited by down-regulating the expression of cyclin D1 and Bcl-2 proteins. After MCF-7 cells were treated with fraxetin, the expressions of MAPK/Erk1/2 protein were reduced, which affected estrogen non-genomic signaling pathway. The results suggest fraxetin plays a part in anti-breast cancer function through E2β-mediated genomic and non-genomic signaling pathways.

Topics: Apoptosis; Breast Neoplasms; Cell Proliferation; Coumarins; Cyclin D1; Estrogen Receptor alpha; Estrogens; Humans; MCF-7 Cells; Proto-Oncogene Proteins c-bcl-2; Signal Transduction

K(+)-Cl(-) cotransporter (KCC) has been shown to be involved in cell proliferation as well as cell volume regulation. A regulatory role of KCC in cell cycle progression of breast cancer MDA-MB-231 cells was explored by using synchronized MDA-MB-231 cells and dihydro-indenyloxy-alkanoic acid (DIOA), a potent inhibitor of KCC. MDA-MB-231 cells cultured in the presence of DIOA exhibited an increase in cell volume, a decrease in intracellular Cl(-) concentration, and reduction in cell proliferation with the G0/G1 phase arrest, which was accompanied with down-regulation of cyclin D1 and cyclin E2, and up-regulation of p21. Among these molecules, the expression of cyclin E2, a molecule essential for the transition from G1 to S phase, was markedly suppressed by DIOA treatment. DIOA-mediated up- or down-regulation of these molecules occurred at the transcriptional level. These findings suggest that KCC plays an important role in the early phase of cell cycle progression by regulating the expression of cyclin D1, cyclin E2, and p21, the molecules essential for the cell cycle progression.

Topics: Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Gene Expression Regulation, Neoplastic; Humans; K Cl- Cotransporters; Symporters

Cyclin D1 and its binding partners CDK4/6 are essential regulators of cell cycle progression and are implicated in cancer progression. Our aim was to investigate a potential regulatory role of these proteins in other essential tumor biological characteristics. Using a panel of breast cancer cell lines and primary human breast cancer samples, we have demonstrated the importance of these cell cycle regulators in both migration and stem-like cell activity. siRNA was used to target cyclin D1 and CDK4/6 expression, having opposing effects on both migration and stem-like cell activity dependent upon estrogen receptor (ER) expression. Inhibition of cyclin D1 or CDK4/6 increases or decreases migration and stem-like cell activity in ER-ve (ER-negative) and ER+ve (ER-positive) breast cancer, respectively. Furthermore, overexpressed cyclin D1 caused decreased migration and stem-like cell activity in ER-ve cells while increasing activity in ER+ve breast cancer cells. Treatment of breast cancer cells with inhibitors of cyclin D1 and CDK4/6 (Flavopiridol/PD0332991), currently in clinical trials, mimicked the effects observed with siRNA treatment. Re-expression of ER in two ER-ve cell lines was sufficient to overcome the effects of either siRNA or clinical inhibitors of cyclin D1 and CDK4/6.   In conclusion, cyclin D1 and CDK4/6 have alternate roles in regulation of migration and stem-like cell activity. Furthermore, these effects are highly dependent upon expression of ER. The significance of these results adds to our general understanding of cancer biology but, most importantly, could be used diagnostically to predict treatment response to cell cycle inhibition in breast cancer.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Female; Flavonoids; Gene Knockdown Techniques; Humans; Neoplastic Stem Cells; Piperidines; Receptors, Estrogen; RNA, Small Interfering; Spheroids, Cellular

It has long been hypothesized that body tissue uptake of aluminum may have biological implications in breast cancer. In vitro and in vivo studies have shown that aluminum may trigger genomic instability by interfering with DNA strands. The objective of this study was to examine the relationship between aluminum concentrations in the peripheral and central areas of breast tumors with the instability of three key genes in breast cancer, ERBB2, C-MYC, and CCND1 and aneuploidy of the chromosomes harboring these genes. Tissue samples of 118 women treated for breast cancer were obtained. Evaluation of aluminum content was carried out using graphite furnace atomic absorption spectrometry. A tissue microarray slide containing the tumor samples was used in FISH assays to assess ERBB2, C-MYC, and CCND1 expressions as well as the statuses of their respective chromosomes 17, 8, and 11. Clinicopathological data were obtained from patient's records. Aluminum levels of >2.0 mg/kg were found in 20.3 and 22.1% of the central and peripheral breast tumor areas, respectively. Amplification and/or aneuploid-positive statuses for ERBB2/CEP17, C-MYC/CEP8, and CCND1/CEP11 were detected in 24, 36.7, and 29.3% of the tumors, respectively. We found that aluminum concentration was not related to these altered gene statuses. Our findings suggest that aluminum concentration does not affect genomic stability in breast tissues. Tissue microenvironment modifications, due to the presence of aluminum compounds, seem more appealing as a possible target for future studies to determine the implications of aluminum in breast carcinogenesis.

Topics: Adult; Aluminum; Aneuploidy; Breast; Breast Neoplasms; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 17; Chromosomes, Human, Pair 8; Cyclin D1; Female; Genomic Instability; Humans; In Situ Hybridization, Fluorescence; Middle Aged; Proto-Oncogene Proteins c-myc; Receptor, ErbB-2; Spectrophotometry, Atomic

In an endeavor to develop novel and improved selective estrogen receptor modulators as anti-breast cancer agents, the benzopyran compounds have been synthesized and identified which act as potent anti-estrogen at uterine level. The present study evaluates the anti-tumor activity of 2-[piperidinoethoxyphenyl]-3-phenyl-2H-benzo(b)pyran (CDRI-85/287) and explores the mechanism of action with a view to describe its potential to inhibit proliferation in ER-positive breast cancer cells MCF-7 and T47D. The compound decreased the expression of ERα while increased the expression of ERβ thereby altering ERα/ERβ ratio in both cell lines. Although the compound showed low binding affinity to ERs, it acted as ERα antagonist and ERβ agonist in decreasing ERE- or AP-1-mediated transcriptional activation in these cells. Transactivation studies in ERα/β-transfected MDA-MB231 cells suggested that at cyclin D1 promoter, compound antagonized the action of ERα-mediated E2 response while acted as estrogen agonist via ERβ. Further, the compound led to decreased expression of ERα-dependent proliferation markers and ERβ-dependent cell cycle progression markers. The expression of cell cycle inhibitory protein p21 was increased leading to G2/M phase arrest. In parallel, compound also interfered with EGFR activation, caused inhibition of PI-3-K/Akt pathway and subsequent induction of apoptosis via intrinsic pathway. A significant reduction in tumor mass and volume was observed in 85/287-treated mice bearing MCF-7 xenograft. We conclude that compound 85/287 exhibits significant anti-tumor activity via modulation of genomic as well as non-genomic mechanisms involved in cellular growth and arrested the cells in G2 phase in both MCF-7 and T47D breast cancer cells. Study suggests that CDRI-85/287 may have therapeutic potential in ER-positive breast cancer.

Topics: Animals; Antineoplastic Agents; Apoptosis; Benzopyrans; Binding, Competitive; Biomarkers, Tumor; Breast Neoplasms; Cell Cycle Checkpoints; Cell Proliferation; Cyclin D1; Epidermal Growth Factor; ErbB Receptors; Estrogen Receptor alpha; Estrogen Receptor beta; G2 Phase Cell Cycle Checkpoints; Humans; M Phase Cell Cycle Checkpoints; MCF-7 Cells; Mice; Phosphatidylinositol 3-Kinases; Piperidines; Promoter Regions, Genetic; Proto-Oncogene Proteins c-akt; Receptors, Estrogen; Signal Transduction; Transcription Factor AP-1; Transcriptional Activation; Xenograft Model Antitumor Assays

Cyclin D1 is a component of the core cell-cycle machinery and is frequently overexpressed in breast cancer. It physically interacts with the tumor suppressor Dmp1 that attenuates the oncogenic signals from Ras and HER2 by inducing Arf/p53-dependent cell-cycle arrest. Currently, the biological significance of Dmp1-cyclin D1 interplay in breast cancer has not been determined. Here, we show that cyclin D1 bound to Dmp1 to activate both Arf and Ink4a promoters and, consequently, induced apoptosis or G2/M cell-cycle delay in normal cells to protect them from neoplastic transformation. The cyclin D1-induced Ink4a/Arf gene expression was dependent on Dmp1 because the induction was not detected in Dmp1-deficient or DMP1-depleted cells. Arf/Ink4a expression was increased in pre-malignant mammary glands from Dmp1(+/+);MMTV-cyclin D1 and Dmp1(+/+);MMTV-D1T286A mice but significantly down-regulated in those from Dmp1-deficient mice. Selective Dmp1 deletion was found in 21% of the MMTV-D1 and D1T286A mammary carcinomas, and the Dmp1 heterozygous status significantly accelerated mouse mammary tumorigenesis with reduced apoptosis and increased metastasis. Overall, our study reveals a pivotal role of combined Dmp1 loss and cyclin D1 overexpression in breast cancer.

Topics: Animals; Apoptosis; Breast Neoplasms; Carcinogenesis; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Female; Fibroblasts; G2 Phase; Gene Expression Regulation, Neoplastic; Humans; Mammary Neoplasms, Experimental; Mammary Tumor Virus, Mouse; Mice; Mice, Transgenic; Mitosis; Mutation; Neoplasm Metastasis; Promoter Regions, Genetic; Transcription Factors

Industrialisation, the proximity of factories to cities, and human work activities have led to a disproportionate use of substances containing heavy metals, such as cadmium (Cd), which may have deleterious effects on human health. Carcinogenic effects of Cd and its relationship with breast cancer, among other tumours, have been reported. 5-Fluorouracil (5-FU) is a fluoropyrimidine anticancer drug used to treat solid tumours of the colon, breast, stomach, liver, and pancreas. The purpose of this work was to study the effects of Cd on cell cycle, apoptosis, and gene and protein expression in MCF-7 breast cancer cells treated with 5-FU. Cd altered the cell cycle profile, and its effects were greater when used either alone or in combination with 5-FU compared with 5-FU alone. Cd significantly suppressed apoptosis of MCF-7 cells pre-treated with 5-FU. Regarding gene and protein expression, bcl2 expression was mainly upregulated by all treatments involving Cd. The expression of caspase 8 and caspase 9 was decreased by most of the treatments and at all times evaluated. C-myc expression was increased by all treatments involving Cd, especially 5-FU plus Cd at the half time of treatment. Cd plus 5-FU decreased cyclin D1 and increased cyclin A1 expression. In conclusion, our results indicate that exposure to Cd blocks the anticancer effects of 5-FU in MCF-7 cells. These results could have important clinical implications in patients treated with 5-FU-based therapies and who are exposed to high levels of Cd.

Topics: Antimetabolites; Apoptosis; Breast Neoplasms; Cadmium Chloride; Caspase 8; Caspase 9; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin A1; Cyclin D1; Drug Interactions; Female; Fluorouracil; G1 Phase Cell Cycle Checkpoints; Gene Expression; Humans; MCF-7 Cells; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc

Caesalpinia sappan Linn. has long been used in traditional medicine in China. Here, the anticancer activity of brazilein, a compound isolated from C. sappan Linn. was investigated. MTT assay showed that the IC50 value of brazilein against human breast cancer MCF-7 cells was 7.23 ± 0.24 μmol/L. PI staining and flow cytometry analysis indicated that brazilein caused cell cycle arrest in G1 phase. Western blot and RT-PCR assay demonstrated that cyclin D1, a key factor of the G1 to S phase progression, was downregulated in a concentration-dependent manner by brazilein treatment. Further Western blot and RNA interference assay showed that brazilein treatment activated GSK-3β and following reduced β-Catenin protein, which accounted for the downregulation of cyclin D1 and blockage of cell cycle at G1 phase. Together, all these results illustrated that brazilein induced growth inhibition of breast cancer cells and downregulation of GSK-3β/β-Catenin pathway was involved in its action mechanism.

Topics: Antineoplastic Agents, Phytogenic; Benzopyrans; beta Catenin; Breast Neoplasms; Caesalpinia; Cell Proliferation; Cyclin D1; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Female; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Indenes; MCF-7 Cells; Molecular Structure; Structure-Activity Relationship; Tumor Cells, Cultured

Euphorbia tirucalli is a long‑established treatment for a wide variety of cancers. However, the mechanism of its anticancer effect is yet to be elucidated. In the present study, we examined the anticancer effect of euphol, a tetracyclic triterpene alcohol isolated from the sap of Euphorbia tirucalli, in T47D human breast cancer cells. Following the treatment of cells with different doses of euphol for 24, 48 and 72 h, the cell proliferation, cell cycle, and mRNA and protein levels of cell cycle regulatory molecules were analyzed, respectively. Treatment of the cells with euphol resulted in decreased cell viability, which was accompanied by an accumulation of cells in the G1 phase. Further studies demonstrated that euphol treatment downregulated cyclin D1 expression and the hypophosphorylation of Rb. Furthermore, this effect was correlated with the downregulation of cyclin‑dependent kinase 2 (CDK2) expression and the upregulation of the CDK inhibitors p21 and p27. Reduced expression levels of cyclin A and B1 were also observed, corresponding to the decreased distribution of cells in the S and G2/M phases, respectively. These findings indicated that euphol is an active agent in Euphorbia tirucalli that exerts anticancer activity by arresting the cell cycle of cancer cells.

Topics: Antineoplastic Agents, Phytogenic; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Drug Screening Assays, Antitumor; Female; G1 Phase Cell Cycle Checkpoints; Gene Expression; Gene Expression Regulation, Neoplastic; Humans; Lanosterol; Retinoblastoma; Transcription, Genetic

This study examined the effects of parathyroid hormone-related protein (PTHrP) derived from human MDA-MB-231 breast cancer cells on the tumor growth and osteoblast inhibition. Results revealed that knocking down PTHrP expression in the breast cancer cells strikingly inhibited the formation of subcutaneous tumors in nude mice. PTHrP knockdown dramatically decreased the levels of cyclins D1 and A1 proteins and arrested the cell cycle progression at the G1 stage. PTHrP knockdown led to the cleavage of Caspase 8 and induced apoptosis of the tumor cells. Interestingly, knocking down PTHrP increased the levels of Beclin1 and LC3-II and promoted the formation of autophagosomes. Knocking down PTHrP expression significantly reduced the abilities of the breast cancer cells to inhibit osteoblast differentiation and bone formation in vitro and in vivo. Finally, we found that PTHrP activated its own expression through an autocrine mechanism in MDA-MB-231 cells. Collectively, these studies suggest that targeting PTHrP expression in the tumor cells could be a potential therapeutic strategy for breast cancers, especially those with skeletal metastases.

Topics: Analysis of Variance; Animals; Apoptosis; Apoptosis Regulatory Proteins; Autocrine Communication; Beclin-1; Breast Neoplasms; Caspase 8; Cell Differentiation; Cell Line, Tumor; Cyclin A1; Cyclin D1; DNA Primers; Enzyme-Linked Immunosorbent Assay; Female; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Immunohistochemistry; Mice; Mice, Nude; Osteoblasts; Parathyroid Hormone-Related Protein; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction

Thymoquinone (TQ), the predominant bioactive constituent of black seed oil (Nigella Sativa), has been shown to possess antineoplastic activity against multifarious tumors. However, the meticulous mechanism of TQ on Akt mediated survival pathway is still unrevealed in breast cancer. Here, we investigated TQ's mechanism of action against PI3K/Akt signaling and its downstream targets by modulating proteins translational machinery, leading to apoptosis in cancer cells.. MDA-MB-468 and T-47D cells were treated with TQ and evaluated for its anticancer activity through phase distribution and western blot. Modulatory effects of TQ on Akt were affirmed through kinase and drug potential studies.. Studies revealed G1 phase arrest till 24h incubation with TQ while extended exposure showed phase shift to subG1 indicating apoptosis, supported by suppression of cyclin D1, cyclin E and cyclin dependent kinase inhibitor p27 expression. Immunoblot and membrane potential studies revealed mitochondrial impairment behind apoptotic process with upregulation of Bax, cytoplasmic cytochrome c and procaspase-3, PARP cleavage along with Bcl-2, Bcl-xL and survivin downregulation. Moreover, we construed the rationale behind mitochondrial dysfunction by examining the phosphorylation status of PDK1, PTEN, Akt, c-raf, GSK-3β and Bad in TQ treated cells, thus ratifying the involvement of Akt in apoptosis. Further, the consequential effect of Akt inhibition by TQ is proven by translational repression through deregulated phosphorylation of 4E-BP1, eIF4E, S6R and p70S6K.. Our observations for the first time may provide a new insight for the development of novel therapies for Akt overexpressed breast cancer by TQ.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Regulatory Proteins; Benzoquinones; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Dose-Response Relationship, Drug; Female; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Membrane Potential, Mitochondrial; Molecular Targeted Therapy; Protein Biosynthesis; Proto-Oncogene Proteins c-akt; Signal Transduction

Fucoidan is an active component of seaweed, which inhibits proliferation and induces apoptosis of several tumor cells while the detailed mechanisms underlying this process are still not clear. In this study, the effect of Fucoidan on the proliferation and apoptosis of human breast cancer MCF-7 cells and the molecular mechanism of Fucoidan action were investigated. Viable cell number of MCF-7 cells was decreased by Fucoidan treatment in a dose-dependent manner as measured by MTT assay. Fucoidan treatment resulted in G1 phase arrest of MCF-7 cells as revealed by flow cytometry, which was associated with the decrease in the gene expression of cyclin D1 and CDK-4. Annexin V/PI staining results showed that the number of apoptotic cells was associated with regulation of cytochrome C, caspase-8, Bax and Bcl-2 at transcriptional and translational levels. Both morphologic observation and Hoechst 33258 assay results confirmed the pro-apoptotic effect of Fucoidan. Meanwhile, the ROS production was also increased by Fucoidan treatment, which suggested that Fucoidan induced oxidative damage in MCF-7 cells. The results of present study demonstrated that Fucoidan could induce G1 phase arrest and apoptosis in MCF-7 cells through regulating the cell cycle and apoptosis-related genes or proteins expression, and ROS generation is also involved in these processes.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Breast Neoplasms; Caspase 8; Caspases; Cell Proliferation; Cell Size; Cyclin D1; Cyclin-Dependent Kinase 4; Cytochromes c; Dose-Response Relationship, Drug; Fucus; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Microscopy, Fluorescence; Molecular Structure; Polysaccharides; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction

Bisphenol A (BPA) with its estrogenic properties is intensively studied since its presence in the environment and human body. Besides other adverse effects, the compound is suspected of contributing to hormone-related cancers. The present study was aimed to investigate short time (24 h) effects of BPA on the important genes/proteins involved in apoptosis and the cell cycle progression in the breast cancer cells MCF7. The experimental design covered cell treatment with a broad BPA concentration scale: 10-12M corresponding to ubiquitous exposure, 10-9M relevant to human levels, and 10-6M as experimentally usual. We further investigated the combined effects of low BPA dose (10-12M) with physiological concentration of estradiol (E2) (10-9M).. The expression of particular proteins and genes was studied by Western blotting and real time RT-PCR, respectively.. Estrogenic effect of BPA was confirmed in the following checkpoints: mRNA expression of estrogen receptor α, expression of cyclin D1 and A2 proteins and CCNA2 gene, Bax and Bcl2 protein levels. For both cyclins protein levels, the maximum stimulation was present at 10-9M BPA and the effects resembled the "inverted U"-shape, a nonmonotonic dose-response curve reported for the action of xenoestrogens. The combined effect of low BPA dose with physiological E2 concentration differ from those of individual compounds, the character of stimulatory response is neither additive nor synergistic.. The results obtained strongly support the evidence of BPA and BPA+E2 proliferation-promoting effects in human breast carcinoma cells, even after short time exposure, partially via reduced rate of apoptosis by the action of BPA+E2.

Topics: Adenocarcinoma; Apoptosis; bcl-2-Associated X Protein; Benzhydryl Compounds; Breast Neoplasms; Cell Cycle; Cell Proliferation; Cyclin A2; Cyclin D1; Dose-Response Relationship, Drug; Drug Synergism; Estradiol; Estrogens; Estrogens, Non-Steroidal; Female; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Phenols; Proto-Oncogene Proteins c-bcl-2

It is suggested that dietary phytosterols, such as β-sitosterol (ST), have cancer chemopreventive effects; however, studies are limited to support such claims. Here, we evaluated the efficacy of ST on three different human cancer cell lines including skin epidermoid carcinoma A431 cells, lung epithelial carcinoma A549 cells and breast adenocarcinoma MDA-MB-231.. Cell growth assay, cell cycle analysis, FACS, JC-1 staining, annexin V staining and immunoblotting were used to study the efficacy of ST on cancer cells.. ST (30-90 μM) treatments for 48 h and 72 h did not show any significant effect on cell growth and death in A431 cells. Whereas similar ST treatments moderately inhibited the growth of A549 cells by up to 13% (p ≤ 0.05) in 48 h and 14% (p ≤ 0.05-0.0001) in 72 h. In MDA-MB-231 cells, ST caused a significant dose-dependent cell growth inhibition by 31- 63% (p ≤ 0.0001) in 48 h and 40-50% (p ≤ 0.0001) in 72 h. While exploring the molecular changes associated with strong ST efficacy in breast cancer cells, we observed that ST induced cell cycle arrest as well as cell death. ST caused G0/G1 cell cycle arrest which was accompanied by a decrease in CDK4 and cyclin D1, and an increase in p21/Cip1and p27/Kip1 protein levels. Further, cell death effect of ST was associated with induction of apoptosis. ST also caused the depolarization of mitochondrial membrane potential and increased Bax/Bcl-2 protein ratio.. These results suggest prominent in vitro anti-proliferative and pro-apoptotic effects of ST in MDA-MB-231 cells. This study provides valuable insight into the chemopreventive efficacy and associated molecular alterations of ST in breast cancer cells whereas it had only moderate efficacy on lung cancer cells and did not show any considerable effect on skin cancer cells. These findings would form the basis for further studies to understand the mechanisms and assess the potential utility of ST as a cancer chemopreventive agent against breast cancer.

Topics: Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Carcinoma; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; G1 Phase Cell Cycle Checkpoints; Humans; Membrane Potential, Mitochondrial; Sitosterols

Cyclin D1 encodes the regulatory subunit of a holoenzyme that phosphorylates the pRB protein and promotes G1/S cell-cycle progression and oncogenesis. Dicer is a central regulator of miRNA maturation, encoding an enzyme that cleaves double-stranded RNA or stem-loop-stem RNA into 20-25 nucleotide long small RNA, governing sequence-specific gene silencing and heterochromatin methylation. The mechanism by which the cell cycle directly controls the non-coding genome is poorly understood. Here we show that cyclin D1(-/-) cells are defective in pre-miRNA processing which is restored by cyclin D1a rescue. Cyclin D1 induces Dicer expression in vitro and in vivo. Dicer is transcriptionally targeted by cyclin D1, via a cdk-independent mechanism. Cyclin D1 and Dicer expression significantly correlates in luminal A and basal-like subtypes of human breast cancer. Cyclin D1 and Dicer maintain heterochromatic histone modification (Tri-m-H3K9). Cyclin D1-mediated cellular proliferation and migration is Dicer-dependent. We conclude that cyclin D1 induction of Dicer coordinates microRNA biogenesis.

Topics: Animals; Breast Neoplasms; Cell Movement; Cell Proliferation; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; HCT116 Cells; Histones; Humans; Mammary Neoplasms, Experimental; MCF-7 Cells; Mice; Mice, Inbred C57BL; Mice, Transgenic; MicroRNAs; Protein Processing, Post-Translational; Ribonuclease III

Antiproliferative gene B-cell translocation gene, member 2 (BTG2) is a member of the BTG/TOB antiproliferative gene family. In this study, we investigated the effect of BTG2 gene overexpression on the radiosensitivity of breast cancer cells in vitro and in vivo. Results show that in human breast cancer cell line MCF-7 stably overexpressing BTG2 gene, cell sensitivity to ionizing radiation increased. The MCF-7-BTG2 cells were more susceptible to radiation-caused apoptosis with decreased cyclin B1, cyclin D1, Ku70, FEN-1, and XRCC1 protein expression as well as increased BAX protein expression. The findings indicate for the first time that BTG2 can improve the radiosensitivity of breast cancer cells by affecting cell cycle distribution, enhancing radiation-induced apoptosis, and inhibiting DNA repair-related protein expression.

Topics: Animals; Antigens, Nuclear; Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Cell Cycle; Cyclin B1; Cyclin D1; DNA Damage; DNA-Binding Proteins; Dose-Response Relationship, Radiation; Female; Flap Endonucleases; Gene Expression Regulation, Neoplastic; Humans; Immediate-Early Proteins; Ku Autoantigen; MCF-7 Cells; Mice; Mice, Inbred BALB C; Mice, Nude; Radiation Tolerance; Time Factors; Transfection; Tumor Suppressor Proteins; Up-Regulation; X-ray Repair Cross Complementing Protein 1; Xenograft Model Antitumor Assays

The role of the progesterone receptor (PR) in breast cancer remains a major clinical challenge. Although PR induces mammary tumor growth, its presence in breast tumors is a marker of good prognosis. We investigated coordinated PR rapid and nonclassical transcriptional effects governing breast cancer growth and endocrine therapy resistance.. We used breast cancer cell lines expressing wild-type and mutant PRs, cells sensitive and resistant to endocrine therapy, a variety of molecular and cellular biology approaches, in vitro proliferation studies and preclinical models to explore PR regulation of cyclin D1 expression, tumor growth, and response to endocrine therapy. We investigated the clinical significance of activator protein 1 (AP-1) and PR interaction in a cohort of 99 PR-positive breast tumors by an immunofluorescence protocol we developed. The prognostic value of AP-1/PR nuclear colocalization in overall survival (OS) was evaluated using Kaplan-Meier method, and Cox model was used to explore said colocalization as an independent prognostic factor for OS.. We demonstrated that at the cyclin D1 promoter and through coordinated rapid and transcriptional effects, progestin induces the assembly of a transcriptional complex among AP-1, Stat3, PR, and ErbB-2 which functions as an enhanceosome to drive breast cancer growth. Our studies in a cohort of human breast tumors identified PR and AP-1 nuclear interaction as a marker of good prognosis and better OS in patients treated with tamoxifen (Tam), an anti-estrogen receptor therapy. Rationale for this finding was provided by our demonstration that Tam inhibits rapid and genomic PR effects, rendering breast cancer cells sensitive to its antiproliferative effects.. We here provided novel insight into the paradox of PR action as well as new tools to identify the subgroup of ER+/PR + patients unlikely to respond to ER-targeted therapies.

Topics: Animals; Breast Neoplasms; Cell Nucleus; Cyclin D1; Female; Follow-Up Studies; Humans; Medroxyprogesterone Acetate; Mice, Inbred BALB C; Phosphorylation; Promoter Regions, Genetic; Receptor, ErbB-2; Receptors, Progesterone; Retrospective Studies; Selective Estrogen Receptor Modulators; STAT3 Transcription Factor; Tamoxifen; Transcription Factor AP-1; Treatment Outcome

Signal copy number (SCN) and signal intensity (SI) of subtelomeres (ST) are investigated in auxiliary lymph node (ALN) and buccal (BUC) cells by fluorescence in situ hybridization. The extracted total cell of 38256 and 2309 was, respectively, analyzed from the benign ALN- and BUC-cells of an affected breast cancer patient. The Periodic model was based on ST behavior including normal-, down-, and upregulated clones with diverse SCN. The arm-p/q ratio based signature, as a subtelomeric array, reflects discordance and concordance of ST-behavior within individual chromosomes as a concept of "Individualization of Cells" rather than "Global Insight of Cells". The Periodic charts could be considered as a reliable and refreshable platform through which the cellular evolution could be patterned and characterized. Signature of ST-profile in the BUC and ALN cells and the nature of diverse SCN and SI as quantitative and qualitative value led to modeling the real personalized perspective of cellular evolution. Protein expression of Ki67, Cyclin D1, and Cyclin E was assayed, as a complementary panel. These targets could be applied as the predictive and preventive markers for an early detection at BUC and ALN levels to plan the required managements in the breast cancer patients.

Topics: Aged; Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Transformation, Neoplastic; Chromosomes, Human; Cyclin D1; Cyclin E; Female; Humans; In Situ Hybridization, Fluorescence; Ki-67 Antigen; Lymph Nodes; Models, Biological; Mouth Mucosa; Telomere

Breast cancer patients with HER-2 positive or estrogen receptor negative tumors have a poor prognosis because these tumors are aggressive and respond poorly to standard therapies. Histone deacetylase (HDAC) inhibitors have been shown to decreased cell survival, which suggests that HDAC inhibitors may be developed for preventing and treating breast cancer. Curcumin has anti-inflammatory and proapoptotic effects in cancer cells. We determined whether the HDAC inhibitor, Tricostatin A (TSA) in combination with curcumin would produce greater antiproliferative and apoptotic effects than either agent alone. Increasing the concentration of curcumin from 10 to 20 µM enhanced the growth inhibitory effects of the combination in SkBr3 and 435eB breast cancer cells, which was accompanied by decreased viability along with decreased phosphorylation of ERK and Akt. The decreased cell viability observed in SkBr3 cells when curcumin was combined with TSA led to a G0/G1 cell cycle arrest and increased p21 and p27, and decreased Cyclin D1 protein expression. The combination induced cleavage of caspase 3 and poly(ADP-ribose) polymerase-1, suggesting that cell death occurred by apoptosis. There were no changes in protein expression of Bcl2, Bax, or Bcl-xL and decreased expression of p53. The combination increased protein expression of phosphorylated JNK and phosphorylated p38. Pharmacological inhibition of JNK, but not p38, attenuated the decreased viability induced by the curcumin and TSA combination. We conclude that p53 independent apoptosis induced by combining curcumin and TSA involves JNK activation. These findings provide a rationale for exploring the potential benefits of the combination of curcumin with TSA for treatment of breast cancer.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Breast Neoplasms; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Survival; Curcumin; Cyclin D1; Enzyme Inhibitors; Female; Humans; Hydroxamic Acids; Imidazoles; MAP Kinase Kinase 4; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Pyridines

Fatty acids are endogenous ligands of peroxisome proliferator-activated receptor-alpha (PPARα), which is linked to the regulation of fatty acid uptake, lipid metabolism and breast cancer cell growth. This study was designed to screen candidate fatty acids from breast cancer tissue and to investigate the effects of these candidate fatty acids on PPARα expression, cell growth and cell cycle progression in breast cancer cell lines. One breast cancer tissue and one reference tissue were each taken from 30 individual breasts to examine for fatty acid composition and PPARα expression. The cancer cell lines MDA-MB-231 (ER-), MCF-7 (ER++++) and BT-474 (ER++) were used to explore the mechanisms regulating cell proliferation. We found that arachidonic acid (AA) and PPARα were highly expressed in the breast cancer tissues. AA stimulated the growth of all three breast cancer cells in a time- and dose-dependent manner. The growth stimulatory effect of AA was associated with PPARα activation, and the most potent effect was found in MCF-7 cells. The stimulation of cell proliferation by AA was accompanied by the increased expression of cyclin E, a reduced population of G1 phase cells, and a faster G1/S phase transition. In contrast, AA had no effects on the levels of CDK2, CDK4, cyclin D1, p27, Bcl-2 and Bax. Our results demonstrate that high levels of AA and PPARα expression in human breast cancer tissues are associated with ER-overexpressed breast cancer cell proliferation, which is involved in activating PPARα, stimulating cyclin E expression, and promoting faster G1/S transition.

Topics: Adult; Arachidonic Acid; bcl-2-Associated X Protein; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Dose-Response Relationship, Drug; Fatty Acids; Female; G1 Phase; Humans; Indoles; Middle Aged; PPAR alpha

The tumor-associated calcium signal transducer 2 (TACSTD2) gene has been reported to be highly expressed in many types of human epithelial cancers, and is associated with tumor metastasis and poor prognosis. The aims of the present investigation were to analyze the TACSTD2 and Cyclin D1 expression at the mRNA and protein levels and to assess its prognostic significance in invasive ductal breast cancer (IDC). The expressions of TACSTD2 and Cyclin D1 in IDC tissues were consistently higher than those in the tumor-adjacent non-malignant tissues by a one-step real-time polymerase chain reaction and immunohistochemistry (P<0.001 and P=0.023, respectively). The statistical analysis of clinicopathologic characteristics and immunohistochemistry by the χ(2) test showed that the high expression of TACSTD2 in IDC was correlated to histological grade (P=0.023), P53 status (P=0.042), Cyclin D1 status (P<0.001), lymph node metastasis (P<0.001), distant metastasis (P=0.004) and TNM staging (P<0.001). Kaplan-Meier survival and Cox regression analyses were performed to evaluate the prognosis of IDC. These analyses also showed that a high TACSTD2 expression (P=0.003), a high Cyclin D1 expression (P=0.041), and lymph node metastasis (P=0.006) were independent prognosis factors. Collectively, our studies demonstrated that the high expression of TACSTD2 correlates with a poor prognosis in IDC.

Topics: Antigens, Neoplasm; Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Adhesion Molecules; Cyclin D1; Female; Humans; Lymphatic Metastasis; Neoplasm Metastasis; Neoplasm Staging; Prognosis; RNA, Messenger; Up-Regulation

Recently an increased interest on Elk1 protein and its role in breast cancer evolution has been noted. This protein is an element of the Ets family of transcription factors and it has been involved in a number of important cell processes through the activation of different genes, in a number of normal tissues as well as in many malignancies.. One hundred and seventy (n = 170) cases of operable breast cancer (invasive ductal, lobular and mixed type breast carcinomas) were randomly selected and investigated for the expression of pElk-1, Ki-67 and Cyclin D1 using immunohistochemistry. Our findings were correlated with tumors' clinicopathologic data and biologic profile.. Activated Elk1 is positively associated with ER (p-value: 0.018) and also shows a positive association of with Cyclin D1 (p-value: <0.001). No relationship was noted between pElk1 and Ki67 (p-value: 0.213). Luminal A and B Her-2 negative breast cancer subtypes were showing greater pElk-1 immunoreactivity compared to Her-2 and Basal breast cancer subtypes, and also a higher staining intensity. No association of the molecule with other clinicopathologic characteristics (tumor size, stage, histological type or lymph node metastases) or disease adverse events (local recurrence, metastasis or death) was evidenced.. Our findings offer a new perspective for the role of pElk-1 in breast neoplasia suggesting a direct relation of this molecule to tumor biology and a putative target of personalized breast cancer therapies, although its prognostic/discriminant role is not supported.

Topics: Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Lobular; Cyclin D1; ets-Domain Protein Elk-1; Female; Humans; Immunohistochemistry; Ki-67 Antigen; Phosphorylation; Prognosis; Receptor, ErbB-2; Receptors, Estrogen

We previously reported that quinacrine (QC) has anticancer activity against breast cancer cells. Here, we examine the mechanism of action of QC and its ability to inhibit Wnt-TCF signaling in two independent breast cancer cell lines. QC altered Wnt-TCF signaling components by increasing the levels of adenomatous polyposis coli (APC), DAB2, GSK-3β and axin and decreasing the levels of β-catenin, p-GSK3β (ser 9) and CK1. QC also reduced the activity of the Wnt transcription factor TCF/LEF and its downstream targets cyclin D1 and c-MYC. Using a luciferase-based Wnt-TCF transcription factor assay, it was shown that APC levels were inversely associated with TCF/LEF activity. Induction of apoptosis and DNA damage was observed after treatment with QC, which was associated with increased expression of APC. The effects induced by QC depend on APC because the inhibition of Wnt-TCF signaling by QC is lost in APC-knockdown cells, and consequently, the extent of apoptosis and DNA damage caused by QC is reduced compared with parental cells. Because we previously showed that QC inhibits topoisomerase, we examined the effect of another topoisomerase inhibitor, etoposide, on Wnt signaling. Interestingly, etoposide treatment also reduced TCF/LEF activity, β-catenin and cyclin D1 levels commensurate with induction of DNA damage and apoptosis. Lycopene, a plant-derived antioxidant, synergistically increased QC activity and inhibited Wnt-TCF signaling in cancer cells without affecting the MCF-10A normal breast cell line. Collectively, the data suggest that QC-mediated Wnt-TCF signal inhibition depends on APC and that the addition of lycopene synergistically increases QC anticancer activity.

Topics: Adenomatous Polyposis Coli Protein; Anticarcinogenic Agents; Antineoplastic Agents, Phytogenic; Apoptosis; beta Catenin; Blotting, Western; Breast Neoplasms; Carotenoids; Cell Cycle; Cell Proliferation; Comet Assay; Cyclin D1; Drug Synergism; Etoposide; Female; Flow Cytometry; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Lycopene; Promoter Regions, Genetic; Quinacrine; RNA, Small Interfering; Signal Transduction; T Cell Transcription Factor 1; TCF Transcription Factors; Trans-Activators; Transcription, Genetic; Tumor Cells, Cultured; Tumor Stem Cell Assay; Wnt Proteins

  To study the clinicopathological and prognostic value of cyclin D1 overexpression in patients with breast carcinoma..   Immunohistochemistry was performed on paraffin-embedded tissue specimens from 290 invasive breast carcinomas to detect the proteins cyclin D1, oestrogen receptor (ER), progesterone receptor (PR), p53, c-erbB2, and topoisomerase IIα (topoIIα). Cyclin D1 staining was quantified using a computerized image analysis method. Cyclin D1 overexpression characterized smaller, ER-positive and PR-positive tumours (P = 0.017, P < 0.0001, and P < 0.0001, respectively), of a lower histological and nuclear grade (P = 0.011 and P < 0.0001, respectively), and with reduced expression of topoIIα (P = 0.001) and p53 (P < 0.001). Cyclin D1 was found to have an independent favourable impact on the overall survival of both the unselected cohort of patients (P = 0.011) and of patients with ER-negative and lymph node-positive tumours (P = 0.034 and P = 0.015, respectively). In triple-negative tumours, cyclin D1 overexpression was found to have independent favourable impacts on both overall and relapse-free survival (P = 0.002 for both)..   This is the first immunohistochemical study to dissociate the advantageous prognostic effect of cyclin D1 overexpression from its association with ER expression, and to provide evidence that cyclin D1 overexpression may be a marker of prolonged survival in patient subgroups with aggressive phenotypes.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Ductal, Breast; Cyclin D1; Disease-Free Survival; Female; Humans; Image Interpretation, Computer-Assisted; Immunohistochemistry; Middle Aged; Neoplasm Grading; Neoplasm Staging; Phenotype; Prognosis; Proportional Hazards Models; Receptors, Estrogen

Breast cancer constitutes a major health problem for women worldwide. However, its incidence varies between populations and geographical locations. These variations could be diet-related, since there are several carcinogenic compounds in the modern diet, while natural products contain various anti-cancer elements. Several lines of evidence indicate that, in addition to their clear preventive effect, these compounds could also be used as therapeutic agents. In the present report we have shown that oleuropein, a pharmacologically safe natural product of olive leaf, has potent anti-breast cancer properties. Indeed, oleuropein exhibits specific cytotoxicity against breast cancer cells, with higher effect on the basal-like MDA-MB-231 cells than on the luminal MCF-7 cells. This effect is mediated through the induction of apoptosis via the mitochondrial pathway. Moreover, oleuropein inhibits cell proliferation by delaying the cell cycle at S phase and up-regulated the cyclin-dependent inhibitor p21. Furthermore, oleuropein inhibited the anti-apoptosis and pro-proliferation protein NF-κB and its main oncogenic target cyclin D1. This inhibition could explain the great effect of oleuropein on cell proliferation and cell death of breast cancer cells. Therefore, oleuropein warrants further investigations to prove its utility in preventing/treating breast cancer, especially the less-responsive basal-like type.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Cycle; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Electrophoresis, Polyacrylamide Gel; Female; Flow Cytometry; Humans; Iridoid Glucosides; Iridoids; MCF-7 Cells; Mitochondria; NF-kappa B; Olive Oil; Plant Oils; Pyrans; Receptors, Estrogen; Up-Regulation

Transferrin (Tf) conjugates of monomeric artemisinin (ART) and artemisinin dimer were synthesized. The two conjugates, ART-Tf and dimer-Tf, retained the original protein structure, and formed stable aggregates in aqueous buffer. ART-Tf induced declines in proteins involved in apoptosis (survivin), cell cycling (cyclin D1), oncogenesis (c-myelocytomatosis oncogene product (c-MYC)), and dysregulated WNT signaling (beta-catenin) in both the human prostate (DU145) and breast (MCF7) cancer cell lines. Both ART-Tf and dimer-Tf induced down-regulation of survivin, c-MYC and mutated human epidermal growth factor receptor-2 (ERBB2 or HER2) in the BT474 breast cancer cell line. To our knowledge, this is the first demonstration that an ART derivative can cause a decline of ERBB2 in a human cancer cell line. Potential mechanisms for the observed effects are presented. Both transferrin conjugates strongly inhibited the growth of BT474 cells in the same concentration range that the conjugates caused declines in the levels of ERBB2, survivin, and c-MYC, while showing essentially no toxicity towards MCF10A normal breast cells.

Topics: Apoptosis; Artemisinins; beta Catenin; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Humans; Inhibitor of Apoptosis Proteins; Male; Prostatic Neoplasms; Protein Multimerization; Proto-Oncogene Proteins c-myc; Receptor, ErbB-2; Survivin; Transferrin; Wnt Signaling Pathway

Hepatitis B X-interacting protein (HBXIP) is an important oncoprotein that plays critical role in the development of cancer. In this study, we report that HBXIP activates LIM-only protein 4 (LMO4), a transcriptional coregulatory protein, in promotion of cell proliferation. We observed that the messenger RNA (mRNA) expression levels of HBXIP were positively associated with those of LMO4 in clinical breast cancer tissues. We further identified that HBXIP upregulated LMO4 at the levels of promoter, mRNA and protein in MCF-7 and LM-MCF-7 breast cancer cell lines. The expression of cyclin D1 and cyclin E, downstream effectors of LMO4, could be upregulated by HBXIP through LMO4. Then, chromatin immunoprecipitation (ChIP) assay revealed that HBXIP was able to interact with the promoter region of LMO4. Electrophoretic mobility shift assay showed that HBXIP occupied the -237/-206 region of LMO4 promoter containing Sp1 binding element. The mutant of Sp1 binding site in the LMO4 promoter impeded the interaction of HBXIP with the promoter. Co-immunoprecipitation, ChIP and luciferase reporter gene assays showed that HBXIP activated LMO4 promoter through binding to Sp1. In function, flow cytometry, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 5-ethynyl-2'-deoxyuridine (EdU) incorporation assays and animal transplantation assays demonstrated that HBXIP-enhanced cell proliferation of breast cancer through upregulating LMO4 in vitro and in vivo. Thus, we concluded that oncoprotein HBXIP is able to activate the transcriptional coregulatory protein LMO4 through transcription factor Sp1 in promotion of proliferation of breast cancer cells. HBXIP may serve as a driver gene to activate transcription in the development of cancer.

Topics: Adaptor Proteins, Signal Transducing; Animals; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Chromatin Immunoprecipitation; Cyclin D1; Cyclin E; Disease Progression; Electrophoretic Mobility Shift Assay; Female; Gene Expression Regulation, Neoplastic; Humans; LIM Domain Proteins; MCF-7 Cells; Mice; Mice, Inbred BALB C; Promoter Regions, Genetic; RNA Interference; RNA, Messenger; RNA, Small Interfering; Sp1 Transcription Factor; Transcription, Genetic

Curcumin, a principal component of turmeric (Curcuma longa), has potential therapeutic activities against breast cancer through multiple signaling pathways. Increasing evidence indicates that curcumin reverses chemo-resistance and sensitizes cancer cells to chemotherapy and targeted therapy in breast cancer. To date, few studies have explored its potential antiproliferation effects and resistance reversal in antiestrogen-resistant breast cancer. In this study, we therefore investigated the efficacy of curcumin alone and in combination with tamoxifen in the established antiestrogen-resistant breast cancer cell lines MCF-7/LCC2 and MCF-7/LCC9. We discovered that curcumin treatment displayed anti-proliferative and pro-apoptotic activities and induced cell cycle arrest at G2/M phase. Of note, the combination of curcumin and tamoxifen resulted in a synergistic survival inhibition in MCF-7/LCC2 and MCF-7/LCC9 cells. Moreover, we found that curcumin targeted multiple signals involved in growth maintenance and resistance acquisition in endocrine resistant cells. In our cell models, curcumin could suppress expression of pro-growth and anti-apoptosis molecules, induce inactivation of NF-κB, Src and Akt/mTOR pathways and downregulate the key epigenetic modifier EZH2. The above findings suggested that curcumin alone and combinations of curcumin with endocrine therapy may be of therapeutic benefit for endocrine-resistant breast cancer.

Topics: Antineoplastic Agents, Hormonal; Apoptosis; Apoptosis Regulatory Proteins; Breast Neoplasms; Cell Proliferation; Cell Survival; Curcumin; Cyclin D1; Down-Regulation; Drug Resistance, Neoplasm; Drug Synergism; Extracellular Signal-Regulated MAP Kinases; Female; G2 Phase Cell Cycle Checkpoints; Gene Expression; Humans; MCF-7 Cells; NF-kappa B; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-myc; Signal Transduction; Tamoxifen; TOR Serine-Threonine Kinases

To evaluate the expression levels of cyclin D1 in breast papillomas and papillary carcinomas, and to analyze the types of cells that co-express cyclin D1 with cytokeratin 5/6 (CK 5/6) or with cytokeratin 8/18(CK 8/18).. Fifty-nine cases of papillary lesions including 36 papillomas and 23 intracystic papillary carcinomas were examined. Cyclin D1, CK 5/6 and CK 8/18 expression levels were evaluated by double immunostaining.. Cyclin D1 is highly expressed in papillary carcinomas (27.54% ± 15.43%) compared with papillomas (8.81% ± 8.41%, p < 0.01). Cyclin D1 is predominantly expressed in cytokeratin 8/18- expressing cells, rather than in cytokeratin 5/6-expressing cells, regardless of the type of lesion. In papillomas, cyclin D1 exhibited a mean 11.42% (11.42% ± 10.17%) co-expression rate with cytokeratin 8/18 compared with a mean 2.50% (2.50% ± 3.24%) co-expression rate with cytokeratin 5/6 (p < 0.01). In papillary carcinomas, cyclin D1 exhibited a mean 34.74% (34.74% ± 16.32%) co-expression rate with cytokeratin 8/18 compared with a co-expression rate of 0.70% (0.70% ± 0.93%) with cytokeratin 5/6 (p < 0.01).. The increase in cyclin D1 suggests an association of cyclin D1 staining with papillary carcinomas. Although cyclin D1 is an effective marker for the differential diagnosis of other papillary lesions, it cannot be used to distinguish between papilloma and papillary carcinoma lesions because its expression occurs in both lesions. Our results show that cyclin D1 and CK 5/6 staining could be used in concert to distinguish between the diagnosis of papilloma (cyclin D1 < 4.20%, CK 5/6 positive) or papillary carcinoma (cyclin D1 > 37.00%, CK 5/6 negative). In addition, our data suggest that cyclin D1 is expressed only in the cancer stem or progenitor cells that co-immunostained with CK 8/18 in papillary carcinomas, and predominantly with CK 8/18 in the papillomas.. The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/7299340558756848.

Topics: Adolescent; Adult; Aged; Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Papillary; Cyclin D1; Female; Humans; Immunohistochemistry; Keratin-18; Keratin-5; Keratin-6; Keratin-8; Middle Aged; Papilloma; Young Adult

Stat3 is a signaling node for multiple oncogenic pathways and is therefore frequently active in breast cancer. As experimental and clinical evidence reveals that progestins are key players in controlling mammary gland tumorigenesis, we studied Stat3 participation in this event. We have previously shown that progestins induce Stat3Tyr705 phosphorylation and its transcriptional activation in breast cancer cells. In this study, we demonstrate that progestins also induce Stat3 phosphorylation at Ser727 residue, which occurs via activation of c-Src/p42/p44 MAPK pathways in murine progestin-dependent C4HD cells and in T-47D cells. Expression of a Stat3S727A vector, which carries a serine-to-alanine substitution at codon 727, shows that Stat3Ser727 phosphorylation is required for full transcriptional activation of cyclin D1 gene expression by progestins and for in vivo Stat3 recruitment on cyclin D1 promoter. Transfection of Stat3S727A in murine and human breast cancer cells abolished progestin-induced in vitro and in vivo growth. Moreover, we found a positive correlation between progesterone receptor expression and nuclear localization of Stat3Ser727 phosphorylation in breast cancer biopsies. These data highlight Stat3 phosphorylation in Ser727 residue as a nongenomic action by progestins, necessary to promote breast cancer growth.

Topics: Animals; Breast Neoplasms; Cell Proliferation; Cyclin D1; Female; Humans; Medroxyprogesterone Acetate; Mice; Mice, Inbred BALB C; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phosphorylation; STAT3 Transcription Factor

Loss of the retinoblastoma protein tumor suppressor gene (RB) coding for a nuclear phosphoprotein that regulates the cell cycle is found in many human cancers and probably leads to disruption of the p16-cyclin D1-CDK4/6-RB pathway. Cyclin D1 is known to activate CDK4, which then phosphorylates the RB protein, leading to cell cycle progression. p16 inhibits CDK4, keeping RB hypophosphorylated and preventing cell cycle progression. The significance of these three markers, cyclin D1, CDK4 and p16, for breast cancer and carcinogenesis is nevertheless still controversial.. The material consisted of 102 formalin-fixed human breast cancer samples, in which cyclin D1, CDK4 and p16 expression was evaluated immunohistochemically. The amounts of cyclin D1 mRNA present were analyzed by quantitative real time PCR.. High cyclin D1 expression statistically significantly correlated with lower tumor grade, estrogen and progesterone receptor positivity and lower proliferation activity in breast tumors and increased breast cancer-specific survival and overall survival. Tumors with high cyclin D1 protein had 1.8 times higher expression of cyclin D1 mRNA. CDK4 expression did not correlate with cyclin D1 expression or the survival data. p16 expression was associated with Human Epidermal Growth Factor Receptor 2 (HER2) negativity and increased breast cancer-specific survival and disease-free survival. No statistical correlations between cyclin D1, CDK4 and p16 were found.. Cyclin D1 was associated with a good breast cancer prognosis but functioned independently of CDK4. High cyclin D1 expression may be partially due to increased CCND1 transcription. p16 correlated with a better prognosis and may function without CDK4. In conclusion, it appears that cyclin D1, CDK4 and p16 function independently in human breast cancer.

Topics: Adult; Aged; Aged, 80 and over; Breast Neoplasms; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p16; Female; Gene Expression Regulation, Neoplastic; Humans; Middle Aged; Prognosis; Signal Transduction; Tumor Suppressor Proteins

Fangchinoline, an alkaloid derived from the dry roots of Stephaniae tetrandrine S. Moore (Menispermaceae), has been shown to possess cytotoxic, anti-inflammatory, and antioxidant properties. In this study, we used Fangchinoline to inhibit breast cancer cell proliferation and to investigate its underlying molecular mechanisms. Human breast cancer cell lines, MCF-7 and MDA-MB-231, were both used in this study. We found that Fangchinoline significantly decreased cell proliferation in a dose-dependent manner and induced G1-phase arrest in both cell lines. In addition, upon analysis of expression of cell cycle-related proteins, we found that Fangchinoline reduced expression of cyclin D1, cyclin D3, and cyclin E, and increased expression of the cyclin-dependent kinase (CDK) inhibitors, p21/WAF1, and p27/KIP1. Moreover, Fangchinoline also inhibited the kinase activities of CDK2, CDK4, and CDK6. These results suggest that Fangchinoline can inhibit human breast cancer cell proliferation and thus may have potential applications in cancer therapy.

Topics: Antineoplastic Agents, Phytogenic; Benzylisoquinolines; Breast Neoplasms; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin D3; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Female; G1 Phase; Humans; Oncogene Proteins

Strong evidence implicates cyclin D1 in human breast cancer. Nevertheless, the prognostic value of cyclin D1 overexpression in breast cancer is still controversial. This work aims to assess the predictive value of cyclin D1 immunohistochemical expression in invasive breast carcinomas and evaluate its association with clinicopathological parameters, in addition to hormone receptor status and Her2/neu immunohistochemical expression.. This study was conducted on 71 cases of invasive breast carcinoma selected according to the availability of clinical data and paraffin-embedded tissue specimens. Immunohistochemistry was performed for estrogen receptors (ER); progesterone receptors (PR); Her2/neu and cyclin D1. Cyclin D1 expression was assessed and compared to the patients' age, tumor histology, grade, nodal status, tumor size and ER; PR; Her2/neu immunostaining results.. Cyclin D1 nuclear expression was detected in 35% of invasive breast carcinomas. There were statistically significant associations between the cyclin D1 and younger age, small tumor size, negative nodal status, well differentiated and lobular types of breast cancer and estrogen receptor positivity. Cyclin D1 had no association with progesterone receptor or Her2/neu.. Cyclin D1 immunohistochemical expression associates strongly with the approved favourable prognostic factors in primary breast carcinoma, suggesting a favourable predictive value of cyclin D1.

Topics: Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Lobular; Cyclin D1; Female; Humans; Lymphatic Metastasis; Middle Aged; Neoplasm Grading; Prognosis; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone

Ursolic acid (UA), a naturally occurring pentacyclic triterpene, is a potent in vitro anticancer agent, acting through control of growth, apoptosis, and differentiation. As the anticancer effect and the mechanism of action of ursolic acid on human breast cancer cells has not been extensively studied, we performed an evaluation of the effects of UA on apoptosis in MCF-7 cells. UA was found to inhibit the proliferation of MCF-7 cells in a concentration and time-dependent manner. After treatment, UA-induced apoptosis was accompanied by a significant decrease in CyclinD1/CDK4 expression, which can be regulated by FoxM1. Previous studies demonstrated that FoxM1 orchestrates the transcription of genes that are essential for cell cycle progression and cell proliferation. The result of Western blot suggested that ursolic acid inhibited the expression of FoxM1. Taken together, the data suggest that the proapoptotic effect of UA on MCF-7 cells is mediated by inhibition of FoxM1 and is highly correlated with inactivation of CyclinD1/CDK4.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Female; Forkhead Box Protein M1; Forkhead Transcription Factors; Gene Expression; Humans; Reverse Transcriptase Polymerase Chain Reaction; Triterpenes; Ursolic Acid

Docetaxel is a chemotherapy drug to treat breast cancer, however as with many chemotherapeutic drugs resistance to docetaxel occurs in 50% of patients, and the underlying molecular mechanisms of drug resistance are not fully understood. Gene regulation through microRNAs (miRNA) has been shown to play an important role in cancer drug resistance. By directly targeting mRNA, miRNAs are able to inhibit genes that are necessary for signalling pathways or drug induced apoptosis rendering cells drug resistant. This study investigated the role of differential miRNA expression in two in vitro breast cancer cell line models (MCF-7, MDA-MB-231) of acquired docetaxel resistance. MiRNA microarray analysis identified 299 and 226 miRNAs altered in MCF-7 and MDA-MB-231 docetaxel-resistant cells, respectively. Docetaxel resistance was associated with increased expression of miR-34a and miR-141 and decreased expression of miR-7, miR-16, miR-30a, miR-125a-5p, miR-126. Computational target prediction revealed eight candidate genes targeted by these miRNAs. Quantitative PCR and western analysis confirmed decreased expression of two genes, BCL-2 and CCND1, in docetaxel-resistant cells, which are both targeted by miR-34a. Modulation of miR-34a expression was correlated with BCL-2 and cyclin D1 protein expression changes and a direct interaction of miR-34a with BCL-2 was shown by luciferase assay. Inhibition of miR-34a enhanced response to docetaxel in MCF-7 docetaxel-resistant cells, whereas overexpression of miR-34a conferred resistance in MCF-7 docetaxel-sensitive cells. This study is the first to show differences in miRNA expression, in particular, increased expression of miR-34a in an acquired model of docetaxel resistance in breast cancer. This serves as a mechanism of acquired docetaxel resistance in these cells, possibly through direct interactions with BCL-2 and CCND1, therefore presenting a potential therapeutic target for the treatment of docetaxel-resistant breast cancer.

Topics: Antineoplastic Agents; Base Sequence; Breast Neoplasms; Cell Line, Tumor; Cyclin D1; Docetaxel; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Proto-Oncogene Proteins c-bcl-2; Reproducibility of Results; Taxoids

In this study we investigated the effect of glucose on GSK3β and β-catenin expression and the involvement of the N-linked glycosylation and hexosamine pathways in the Wnt canonical pathway in response to in vitro conditions resembling normoglycemia (5  mmol) and hyperglycemia (20  mmol) in the metastatic breast cancer-derived cell line MDA-MB-231. We also investigated the relationship between this circuitry and the thioredoxin-interacting protein (TXNIP) regulation that seems to be related. MDA-MB-231 cells were grown either in 5 or 20  mM glucose chronically prior to plating. For glucose shift (5/20), cells were plated in 5  mM glucose and shifted to 20  mM at time 0. Both protein and mRNA levels for GSK3β but only the protein expression for β-catenin, were increased in response to high glucose. Furthermore, we assessed the response of GSK3β, β-catenin, and TXNIP to inhibition of the N-linked glycosylation, hexosamine, and Wnt pathways. Wnt signaling pathway activation was validated by specific reporter assay. We show that high levels of glucose regulate mRNA and protein expression of GSK3β, and consequently higher levels of activated β-catenin protein, which locates to the nucleus and is associated with increased levels of cyclin D1 expression. This event coincides with increased level of N-terminal Ser 9 phosphorylation of GSK3β protein. The inhibition of both the hexosamine pathway and N-linked glycosylation along with Wnt signaling pathway by sFRP1 and DKK1 is associated with significant decrease of the protein levels of GSK3β, β-catenin, and TXNIP RNA. Our work illuminates a novel and never described before function of this signaling pathway that relates glucose metabolism with redox regulation mechanism.

Topics: beta Catenin; Breast Neoplasms; Carrier Proteins; Cell Line, Tumor; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Glucose; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Signal Transduction; Wnt Proteins

The human POK family members are transcription factors with a POZ domain and zinc-fingers that act primarily as transcriptional repressors. Several members of this family are involved in oncogenesis and this prompted us to assess whether expression levels of individual POK family members are associated with clinical outcomes in cancer. We have observed that ZBTB4 (zinc-finger and BTB domain containing 4) is downregulated in breast cancer patients, and that its expression is significantly correlated with relapse-free survival. Further integrative analysis of mRNA and microRNA (miR) expression data from the NCI-60 cell lines revealed an inverse correlation between ZBTB4 and oncogenic miRs derived from the miR-17-92 cluster and its paralogs. The experimental results using MDA-MB-231 and MCF-7 human breast cancer cells confirm that miRNAs derived from these clusters, containing miR-17-5p, miR-20a, miR-106a, miR-106b and miR-93, negatively regulate ZBTB4 expression. Overexpression of ZBTB4 or restoration of ZBTB4 by using an antagomir inhibit growth and invasion of breast cancer cells, and this effect is due, in part, to ZBTB4-dependent repression of the specificity protein 1 (Sp1), Sp3 and Sp4 genes, and subsequent downregulation of several Sp-dependent oncogenes, in part, through competition between ZBTB4 and Sp transcription factors for GC-rich promoter sequences. These results confirm that ZBTB4 functions as a novel tumor-suppressor gene with prognostic significance for breast cancer survival, and the oncogenic miR-17-92/ZBTB4/Sp axis may be a potential therapeutic target.

Topics: Binding, Competitive; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Disease-Free Survival; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; Kaplan-Meier Estimate; Luciferases; MicroRNAs; Oncogenes; Prognosis; Promoter Regions, Genetic; Protein Binding; Repressor Proteins; RNA Interference; RNA, Long Noncoding; Sp Transcription Factors; Tumor Suppressor Proteins

ATM protein kinase plays a critical role in maintaining genome integrity by activating a biochemical chain reaction that in turn leads to cell cycle checkpoint activation and repair of DNA damage. Cyclin D1 acts in regulating the G1 phase of the cell cycle. Experimental and clinical studies suggest them to be involved in transformation and tumour progression. To elucidate the role of ATM and cyclin D1 expression in sporadic breast cancer, we investigated the possible link between their RNA expression levels in ductal carcinoma and normal adjacent versus normal breast tissues measured by Taqman real-time PCR in 119 breast tissues. Results showed that cyclin D1 over-expressed in 51.4% of breast tumours, whereas ATM expression was down regulated in 55% of breast tumours compared to both normal adjacent and normal controls (P ≤ 0.01). Cyclin D1 expression in adjacent normal and normal tissues was not significantly differed, whereas ATM expression in normal adjacent was lower than normal control (P ≤ 0.01). Over-expression of cyclin D1 correlated with ER(+) and/or PR(+) (oestrogen/progesterone receptor) status, whereas it mostly under-expressed in HER2(+) (human epidermal growth factor 2) tumours. ATM under-expression was more observed in triple-negative tumours (ER(-), PR(-) and HER2(-)). Our results indicated that reduced expression of the ATM and aberrant cyclin D1 expressions may contribute to the development and/or malignant progression of breast carcinomas also the latter could be involved in the regulation of hormone sensitivity associated with ER and PR.

Topics: Adult; Aged; Aged, 80 and over; Ataxia Telangiectasia Mutated Proteins; Biomarkers, Tumor; Breast; Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Cycle Proteins; Cyclin D1; DNA-Binding Proteins; Female; Humans; Iran; Middle Aged; Neoplasm Staging; Protein Serine-Threonine Kinases; Real-Time Polymerase Chain Reaction; Tumor Suppressor Proteins

The cucurbitacins are tetracyclic triterpenes found in plants of the family Cucurbitaceae. Cucurbitacins have been shown to have anti-cancer and anti-inflamatory activities. We investigated the anti-cancer activity of cucurbitacin B extracted from Thai medicinal plant Trichosanthes cucumerina Linn. Cell viability was assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Results indicated that cucurbitacin B from T. cucumerina Linn. has a cytotoxic effect on breast cancer cell lines SKBR-3 and MCF-7 with an IC50 of 4.60 and 88.75 µg/ml, respectively. Growth inhibition was attributed to G2/M phase arrest and apoptosis. Cyclin D1, c-Myc, and β-catenin expression levels were reduced. Western blot analysis showed increased PARP cleavage and decreased Wnt-associated signaling molecules β-catenin, galectin-3, cyclin D1 and c-Myc, and corresponding changes in phosphorylated GSK-3β levels. Cucurbitacin B treatment inhibited translocation to the nucleus of β-catenin and galectin-3. The depletion of β-catenin and galectin-3 in the nucleus was confirmed by cellular protein fractionation. T-cell factor (TCF)/lymphoid enhancer factor (LEF)-dependent transcriptional activity was disrupted in cucurbitacin B treated cells as tested by a TCF reporter assay. The relative luciferase activity was reduced when we treated cells with cucurbitacin B compound for 24 h. Our data suggest that cucurbitacin B may in part induce apoptosis and exert growth inhibitory effect via interruption the Wnt signaling.

Topics: Apoptosis; beta Catenin; Breast Neoplasms; Cell Cycle; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Cell Survival; Cyclin D1; Female; Galectin 3; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Plant Extracts; Poly(ADP-ribose) Polymerases; Protein Transport; Proto-Oncogene Proteins c-myc; TCF Transcription Factors; Trichosanthes; Triterpenes; Wnt Proteins; Wnt Signaling Pathway

Lapatinib plus capecitabine emerged as an efficacious therapy in metastatic breast cancer (mBC). We aimed to identify germline single-nucleotide polymorphisms (SNPs) in genes involved in capecitabine catabolism and human epidermal receptor signaling that were associated with clinical outcome to assist in selecting patients likely to benefit from this combination.. DNA was extracted from 240 of 399 patients enrolled in EGF100151 clinical trial (NCT00078572; clinicaltrials.gov) and SNPs were successfully evaluated in 234 patients. The associations between SNPs and clinical outcome were analyzed using Fisher's exact test, Kaplan-Meier curves, log-rank tests, likelihood ratio test within logistic or Cox regression model, as appropriate.. There were significant interactions between CCND1 A870G and clinical outcome. Patients carrying the A-allele were more likely to benefit from lapatinib plus capecitabine versus capecitabine when compared with patients harboring G/G (P = 0.022, 0.024 and 0.04, respectively). In patients with the A-allele, the response rate (RR) was significantly higher with lapatinib plus capecitabine (35%) compared with capecitabine (11%; P = 0.001) but not between treatments in patients with G/G (RR = 24% and 32%, respectively; P = 0.85). Time to tumor progression (TTP) was longer in patients with the A-allele treated with lapatinib plus capecitabine compared with capecitabine (median TTP = 7.9 and 3.4 months; P < 0.001), but not in patients with G/G (median TTP = 6.1 and 6.6 months; P = 0.92).. Our findings suggest that CCND1A870G may be useful in predicting clinical outcome in HER2-positive mBC patients treated with lapatinib plus capecitabine.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Capecitabine; Cyclin D1; Deoxycytidine; Disease-Free Survival; Female; Fluorouracil; Genetic Association Studies; Humans; Kaplan-Meier Estimate; Lapatinib; Middle Aged; Multivariate Analysis; Neoplasm Metastasis; Polymorphism, Single Nucleotide; Proportional Hazards Models; Quinazolines; Randomized Controlled Trials as Topic; Receptor, ErbB-2; Treatment Outcome

Amplification of chromosomal region 11q13, containing the cell cycle regulatory gene CCND1, is frequently found in breast cancer and other malignancies. It is associated with the favourable oestrogen receptor (ER)-positive breast tumour phenotype, but also with poor prognosis and treatment failure. 11q13 spans almost 14 Mb and contains more than 200 genes and is affected by various patterns of copy number gains, suggesting complex mechanisms and selective pressure during tumour progression. In this study, we used 32 k tiling BAC array CGH to analyse 94 CCND1-amplified breast tumours from sporadic, hereditary, and familial breast cancers to fine map chromosome 11q13. A set containing 281 CCND1-non-amplified breast tumours was used for comparisons. We used gene expression data to further validate the functional effect of gene amplification. We identified six core regions covering 11q13.1-q14.1 that were amplified in different combinations. The major core contained CCND1, whereas two cores were found proximal of CCND1 and three distal. The majority of the CCND1-amplified tumours were ER-positive and classified as luminal B. Furthermore, we found that CCND1 amplification is associated with a more aggressive phenotype within histological grade 2 tumours and luminal A subtype tumours. Amplification was equally prevalent in familial and sporadic tumours, but strikingly rare in BRCA1- and BRCA2-mutated tumours. We conclude that 11q13 includes many potential target genes in addition to CCND1.

Topics: Breast Neoplasms; Chromosome Mapping; Chromosomes, Human, Pair 11; Cluster Analysis; Comparative Genomic Hybridization; Cyclin D1; DNA Copy Number Variations; Family; Female; Gene Amplification; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Proteins; Nuclear Proteins; Repressor Proteins; Survival Analysis

In the female population in Asia, systematic investigation concerning alterations in cancer-related genes in breast carcinoma is rare, and the correlation among oncogene or suppressor gene expression with tumor cell apoptosis, cell cycle regulation and tumor cell autophagy remains to be clarified. In this study, a tissue microarray consisting of 360 individual samples from three different breast tissues was generated. By comparing the expression of the tumor-suppressor genes (BRCA1, BECN1, CCND1, PTEN and UVRAG) in ductal breast cancer and normal breast tissues, respectively, we were able to assign changes in the expression of these mRNAs to specific stages and allocate them to define the roles in the multi‑step process of breast carcinogenesis. Tumor-suppressor genes, such as BRCA1 and BECN1, usually had lower signals in the carcinomatous tissues (10.2 and 6.6%) compared to the normal tissues (31 and 32.6%), while stronger positive dots (positive cells >30%) usually existed in the normal tissues. The patients in the oldest age group had the lowest expression rate. Only BECN1 and CCND1 expression showed a significant association with patient age (p=0.030 and p=0.003). A significant association was observed between BRCA1 and BECN1 expression and tumor size (p=0.028 and p=0.021). BECN1 gene expression was positively correlated with UVRAG and PTEN expression (p=0.006 and p=0.000). CCND1 was negatively correlated with PTEN, BECN1 and BRCA1 expression (p=0.011, p=0.000 and p=0.000). Abnormal expression of BRCA1, BECN1, CCND1, PTEN and UVRAG may play a role in human breast carcinogenesis through dysregulated mRNA expression. Overexpressed CCND1 may shorten the G1 phase of the cell cycle, suppress cell apoptosis and contribute to the formation of invasive ductal carcinoma (IDC).

Topics: Adult; Aged; Apoptosis Regulatory Proteins; Beclin-1; BRCA1 Protein; Breast Neoplasms; Carcinoma, Ductal, Breast; Cyclin D1; Female; G1 Phase; Humans; Membrane Proteins; Middle Aged; PTEN Phosphohydrolase; RNA, Messenger; Tumor Suppressor Proteins

The transcription factor CCAAT/enhancer-binding protein delta (C/EBPδ, CEBPD) is a tumor suppressor that is downregulated during breast cancer progression but may also promote metastasis. Here, we have investigated the mechanism(s) regulating C/EBPδ expression and its role in human breast cancer cells. We describe a novel pathway by which the tyrosine kinase Src downregulates C/EBPδ through the SIAH2 E3 ubiquitin ligase. Src phosphorylates SIAH2 in vitro and leads to tyrosine phosphorylation and activation of SIAH2 in breast tumor cell lines. SIAH2 interacts with C/EBPδ, but not C/EBPβ, and promotes its polyubiquitination and proteasomal degradation. Src/SIAH2-mediated inhibition of C/EBPδ expression supports elevated cyclin D1 levels, phosphorylation of retinoblastoma protein (Rb), motility, invasive properties, and survival of transformed cells. Pharmacological inhibition of Src family kinases by SKI-606 (bosutinib) induces C/EBPδ expression in an SIAH2-dependent manner, which is necessary for "therapeutic" responses to SKI-606 in vitro. Ectopic expression of degradation-resistant mutants of C/EBPδ, which do not interact with SIAH2 and/or cannot be polyubiquitinated, prevents full transformation of MCF-10A cells by activated Src (Src truncated at amino acid 531 [Src-531]) in vitro. These data reveal that C/EBPδ expression can be regulated at the protein level by oncogenic Src kinase signals through SIAH2, thus contributing to breast epithelial cell transformation.

Topics: Breast; Breast Neoplasms; CCAAT-Enhancer-Binding Protein-delta; Cell Line, Tumor; Cell Transformation, Neoplastic; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Nuclear Proteins; RNA, Messenger; Signal Transduction; src-Family Kinases; Ubiquitin-Protein Ligases; Ubiquitination

Breast cancer detected by screening has an unexplained prognostic advantage beyond stage shift compared with cancers detected clinically. The aim was to investigate biological factors in invasive breast cancer, with reference to mode of detection and rate of death from breast cancer.. Histology, oestrogen receptor α and β, progesterone receptor, human epidermal growth factor receptor (HER) 2, cyclin D1, p27, Ki-67 and perinodal growth were analysed in 466 tumours from a prospective cohort, the Malmö Diet and Cancer Study. Using logistic regression, odds ratios were calculated to investigate the relationship between tumour characteristics and mode of detection. The same tumour factors were analysed in relation to standard prognostic features. Death from breast cancer was analysed using Cox regression with adjustments for standard tumour factors; differences following adjustment were analysed by means of Freedman statistics.. None of the biological tumour characteristics varied with mode of detection of breast cancer. After adjustment for age, tumour size, axillary lymph node involvement (ALNI) and grade, women with cancer detected clinically had an increased risk of death from breast cancer (hazard ratio 2·48, 95 per cent confidence interval 1·34 to 4·59), corresponding to a 37·2 per cent difference compared with the unadjusted model. Additional adjustment for biological tumour factors studied caused only minor changes.. None of the biological tumour markers investigated explained the improved prognosis in breast cancer detected by screening. None of the factors was related to ALNI, suggesting that other mechanisms may be responsible for tumour spread.

Topics: Aged; Biomarkers, Tumor; Breast Neoplasms; Cyclin D1; ErbB Receptors; Female; Humans; Ki-67 Antigen; Logistic Models; Lymphatic Metastasis; Mass Screening; Middle Aged; Odds Ratio; Predictive Value of Tests; Prognosis; Proliferating Cell Nuclear Antigen; Proportional Hazards Models; Prospective Studies; Receptors, Estrogen; Receptors, Progesterone; Risk Assessment; Risk Factors; Sweden

Early growth response-1 (Egr-1) is an immediate early gene involved in relevant biological events including the proliferation of diverse types of cell tumors. In a microarray analysis performed in breast cancer cells, 17β-estradiol (E2) and the estrogen receptor antagonist 4-hydroxitamoxifen (OHT) up-regulated Egr-1 through the G protein-coupled receptor named GPR30/GPER. Hence, in this study, we aimed to provide evidence regarding the ability of E2, OHT and the selective GPER ligand G-1 to regulate Egr-1 expression and function through the GPER/EGFR/ERK transduction pathway in both Ishikawa (endometrial) and SkBr3 (breast) cancer cells. Interestingly, we demonstrate that Egr-1 is involved in the transcription of genes regulating cell proliferation like CTGF and cyclin D1 and required for the proliferative effects induced by E2, OHT, and G-1 in both Ishikawa and SkBr3 cells. In addition, we show that GPER mediates the expression of Egr-1 also in carcinoma-associated fibroblasts (CAFs). Our data suggest that Egr-1 may represent an important mediator of the biological effects induced by E2 and OHT through GPER/EGFR/ERK signaling in breast and endometrial cancer cells. The results obtained in CAFs provide further evidence regarding the potential role exerted by the GPER-dependent Egr-1 up-regulation in tumor development and progression. Therefore, Egr-1 may be included among the bio-markers of estrogen and antiestrogen actions and may be considered as a further therapeutic target in both breast and endometrial tumors.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Connective Tissue Growth Factor; Cyclin D1; Early Growth Response Protein 1; Endometrial Neoplasms; ErbB Receptors; Estradiol; Estrogen Antagonists; Extracellular Signal-Regulated MAP Kinases; Female; Fibroblasts; Gene Expression Regulation, Neoplastic; Humans; Promoter Regions, Genetic; Receptors, Estrogen; Receptors, G-Protein-Coupled; Signal Transduction; Tamoxifen

Despite the improvement of strategies against cancer therapy, the multidrug resistance (MDR)is the critical problem for successful cancer therapy. Recurrent cancers after initial treatment with chemotherapy are generally refractory to second treatments with these anticancer therapies. Therefore, it is necessary to elucidate the therapy-resistant mechanism for development of effective therapeutic modalities against tumors. Here we demonstrate a phase-specific chemotherapy resistance due to epidermal growth factor receptor (EGFR) in human breast cancer cells. Thymidine-induced G1-arrested cultures showed upregulated chemosensitivity, whereas S-phase arrested cells were more resistant to chemotherapeutic agents. Overexpression of EGFR promoted the MDR phenotypes in breast cancer cells via accelerating the G1/S phase transition, whereas depletion of EGFR exerted the opposite effects. Furthermore, CyclinD1, a protein related to cell cycle, was demonstrated to be involved in above EGFR-mediated effects since EGFR increased the expression of CyclinD1, and the specific RNA interference against CyclinD1 could primarily abolish the EGFR-induced MDR phenotypes. These data provide new insights into the mode by which MDR breast cancers evade cytoxic attacks from chemotherapeutic agents and also suggest a role for EGFR-CyclinD1 axis in this process.

Topics: Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Breast Neoplasms; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase 4; Drug Resistance, Multiple; Drug Resistance, Neoplasm; ErbB Receptors; Female; G1 Phase; Gene Expression Regulation, Neoplastic; Humans; Inhibitory Concentration 50; Neoplasm Proteins; Phenotype; RNA, Messenger; S Phase; Up-Regulation

Insulin like growth factor-1 (IGF-1) stimulates increased proliferation and survival of mammary epithelial cells and also promotes mammary tumorigenesis. To study the effects of IGF-1 on the mammary gland in vivo, we used BK5.IGF-1 transgenic (Tg) mice. In these mice, IGF-1 overexpression is controlled by the bovine keratin 5 promoter and recapitulates the paracrine exposure of breast epithelium to stromal IGF-1 that is seen in women. Studies have shown that BK5.IGF-1 Tg mice are more susceptible to mammary tumorigenesis than wild-type littermates. Investigation of the mechanisms underlying increased mammary cancer risk, reported here, revealed that IGF-1 preferentially activated the PI3K/Akt pathway in glands from prepubertal Tg mice, resulting in increased cyclin D1 expression and hyperplasia. However, in glands from postpubertal Tg mice, a pathway switch occurred and activation of the Ras/Raf/MAPK pathway predominated, without increased cyclin D1 expression or proliferation. We further showed that in prepubertal Tg glands, signaling was mediated by formation of an ERα/IRS-1 complex, which activated IRS-1 and directed signaling via the PI3K/Akt pathway. Conversely, in postpubertal Tg glands, reduced ERα expression failed to stimulate formation of the ERα/IRS-1 complex, allowing signaling to proceed via the alternate Ras/Raf/MAPK pathway. These in vivo data demonstrate that changes in ERα expression at different stages of development direct IGF-1 signaling and the resulting tissue responses. As ERα levels are elevated during the prepubertal and postmenopausal stages, these may represent windows of susceptibility during which increased IGF-1 exposure maximally enhances breast cancer risk.

Topics: Animals; Breast Neoplasms; Cattle; Cell Proliferation; Cyclin D1; Estradiol; Estrogen Receptor alpha; Female; Gene Expression Regulation, Developmental; Humans; Insulin Receptor Substrate Proteins; Insulin-Like Growth Factor I; Mammary Glands, Animal; MAP Kinase Signaling System; Mice; Mice, Knockout; Mice, Transgenic; Models, Biological; Paracrine Communication; Proto-Oncogene Proteins c-raf; Sexual Maturation; Signal Transduction

Artemisinin, a sesquiterpene phytolactone derived from Artemisia annua, is a potent antimalarial compound with promising anticancer properties, although the mechanism of its anticancer signaling is not well understood. Artemisinin inhibited proliferation and induced a strong G1 cell cycle arrest of cultured MCF7 cells, an estrogen-responsive human breast cancer cell line that represents an early-stage cancer phenotype, and effectively inhibited the in-vivo growth of MCF7 cell-derived tumors from xenografts in athymic nude mice. Artemisinin also induced a growth arrest of tumorigenic human breast cancer cell lines with preneoplastic and late stage cancer phenotypes, but failed to arrest the growth of a nontumorigenic human mammary cell line. Concurrent with the cell cycle arrest of MCF7 cells, artemisinin selectively downregulated the transcript and protein levels of the CDK2 and CDK4 cyclin-dependent kinases, cyclin E, cyclin D1, and the E2F1 transcription factor. Analysis of CDK2 promoter-luciferase reporter constructs showed that the artemisinin ablation of CDK2 gene expression was accounted for by the loss of CDK2 promoter activity. Chromatin immunoprecipitation revealed that artemisinin inhibited E2F1 interactions with the endogenous MCF7 cell CDK2 and cyclin E promoters. Moreover, constitutive expression of exogenous E2F1 prevented the artemisinin-induced cell cycle arrest and downregulation of CDK2 and cyclin E gene expression. Taken together, our results demonstrate that the artemisinin disruption of E2F1 transcription factor expression mediates the cell cycle arrest of human breast cancer cells and represents a critical transcriptional pathway by which artemisinin controls human reproductive cancer cell growth.

Topics: Animals; Antineoplastic Agents; Artemisinins; Breast Neoplasms; Cell Line, Tumor; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; E2F1 Transcription Factor; Female; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Nude; Xenograft Model Antitumor Assays

Aromatase is responsible for the rate-determining reaction in estrogen synthesis and is a prime target for treating estrogen-receptor-positive breast cancer. Previous in vitro study has demonstrated that apigenin (APG), naringenin (NGN) and hesperetin (HSP) are three of the most potent natural aromatase inhibitors. Because the enzyme inhibition could potentially block breast cancer development, we employed an established postmenopausal breast cancer model to examine the chemopreventive effect of these flavonoids in vivo. Athymic mice were ovariectomized and transplanted with aromatase-overexpressing MCF-7 cells. Dietary administration of HSP at 1000 ppm and 5000 ppm significantly deterred the xenograft growth, while a null effect was observed in mice treated with APG or NGN. Further study illustrated that plasma estrogen in HSP-treated mice was reduced. Messenger RNA expression of the estrogen-responsive gene pS2 was also decreased in the tumors of mice treated with 1000 and 5000 ppm HSP. On the other hand, western analysis indicated that cyclin D1, CDK4 and Bcl-x(L) were reduced in the tumors. This study suggested that HSP could be a potential chemopreventive agent against breast carcinogenesis through aromatase inhibition.

Topics: Animals; Aromatase; Aromatase Inhibitors; bcl-X Protein; Breast Neoplasms; Cell Proliferation; Citrus; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p57; Estradiol; Estrogens; Female; Hesperidin; Humans; MCF-7 Cells; Mice; Mice, Nude; Ovariectomy; RNA, Messenger; Xenograft Model Antitumor Assays

Cyclin D1 gene (CCND1) plays pivotal roles in the development of several human cancers, including breast cancer, functioning as an oncogene. The aim of this study was to better understand the molecular dynamics of ductal carcinomas with regard to proliferation and the ageing process.. 130 cases of ductal breast cancer in postmenopausal women, aged 52-96 in 3 age classes were selected. Tumoral tissues preserved in formaldehyde solution and subsequently embedded in paraffin were subjected to analysis Fluorescence in situ Hybridization (FISH), Reverse Transcription-Polymerase Chain Reaction (RT- PCR) and immuno-histochemical tests. The molecular variables studied were estimated in relation to the patients' age.. The results obtained suggest that the increment of the levels of cyclin D1 in intra-ductal breast tumors in older woman that we have examined is significantly associated with a lower proliferation rate.. Cyclin D1, which characterizes tumor in young women as molecular director involved in strengthening tumoral proliferation mechanisms, may be seen as a potential blocking molecular switch in corresponding tumours in old women.

Topics: Aged; Aged, 80 and over; Aging; Biomarkers, Tumor; Breast Neoplasms; Cell Cycle; Cell Cycle Proteins; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin D1; Female; Genes, Tumor Suppressor; Humans; Middle Aged; Up-Regulation

We report that in breast cancer cells, tyrosine phosphorylation of the estradiol receptor alpha (ERalpha) by Src regulates cytoplasmic localization of the receptor and DNA synthesis. Inhibition of Src or use of a peptide mimicking the ERalpha p-Tyr537 sequence abolishes ERalpha tyrosine phosphorylation and traps the receptor in nuclei of estradiol-treated MCF-7 cells. An ERalpha mutant carrying a mutation of Tyr537 to phenylalanine (ER537F) persistently localizes in nuclei of various cell types. In contrast with ERalpha wt, ER537F does not associate with Ran and its interaction with Crm1 is insensitive to estradiol. Thus, independently of estradiol, ER537F is retained in nuclei, where it entangles FKHR-driving cell cycle arrest. Chromatin immunoprecipitation analysis reveals that overexpression of ER537F in breast cancer cells enhances FKHR interaction with cyclin D1 promoter. This mutant also counteracts cell transformation by the activated forms of Src or PI3-K. In conclusion, in addition to regulating receptor localization, ERalpha phosphorylation by Src is required for hormone responsiveness of DNA synthesis in breast cancer cells.

Topics: Active Transport, Cell Nucleus; Animals; Breast Neoplasms; Cell Cycle Checkpoints; Cell Growth Processes; Cell Line; Cell Line, Tumor; Cell Nucleus; Chlorocebus aethiops; COS Cells; Cyclin D1; Cytoplasm; Estradiol; Estrogen Receptor alpha; Exportin 1 Protein; Female; Forkhead Transcription Factors; Humans; Karyopherins; MCF-7 Cells; Mice; Mutation; NIH 3T3 Cells; Phenylalanine; Phosphatidylinositol 3-Kinases; Phosphorylation; Promoter Regions, Genetic; ran GTP-Binding Protein; Receptors, Cytoplasmic and Nuclear; S Phase; src-Family Kinases; Transcription, Genetic; Tyrosine

Various endocrine disrupting chemicals (EDCs) are exogenous compounds found in the environment and have the potential to interfere with the endocrine system and hormonal regulation. Among EDCs, bisphenol A (BPA) and 1,1,1-trichloro-2,2-bis(4-methoxyphenol)-ethane [methoxychlor (MXC)] have estrogenic activity resulting in a variety of dysfunctions in the E2-mediated response by binding to estrogen receptors (ERs), causing human health problems such as abnormal reproduction and carcinogenesis. In this study, we investigated the effects of BPA and MXC on cell proliferation facilitated by ER signaling in human breast cancer cells. MCF-7 cells are known to be ERα-positive and to be a highly E2-responsive cancer cell line; these cells are, therefore, a useful in vitro model for detecting estrogenic activity in response to EDCs. We evaluated cancer cell proliferation following BPA and MXC treatment using an MTT assay. We analyzed alterations in the expression of genes associated with the cell cycle in MCF-7 cells by semi-quantitative reverse-transcription PCR following treatment with BPA or MXC compared to EtOH. To determine whether BPA and MXC stimulate cancer cell growth though ER signaling, we co-treated the cells with agonists (propyl pyrazoletriol, PPT; and diarylpropionitrile, DPN) or an antagonist (ICI 182,780) of ER signaling and reduced ERα gene expression via siRNA in MCF-7 cells before treatment with EDCs. These studies confirmed the carcinogenicity of EDCs in vitro. As a result, BPA and MXC induced the cancer cell proliferation by the upregulation of genes that promote the cell cycle and the downregulation of anti-proliferative genes, especially ones affecting the G1/S transition via ERα signaling. These collective results confirm the carcinogenicity of these EDCs in vitro. Further studies are required to determine whether EDCs promote carcinogenesis in vivo.

Topics: Benzhydryl Compounds; Breast Neoplasms; Carcinogens; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Estrogen Receptor alpha; Estrogens, Non-Steroidal; Female; Gene Expression Regulation, Neoplastic; Genes, cdc; Humans; Insecticides; Methoxychlor; Phenols; Signal Transduction

Chromosomal instability (CIN) in tumors is characterized by chromosomal abnormalities and an altered gene expression signature; however, the mechanism of CIN is poorly understood. CCND1 (which encodes cyclin D1) is overexpressed in human malignancies and has been shown to play a direct role in transcriptional regulation. Here, we used genome-wide ChIP sequencing and found that the DNA-bound form of cyclin D1 occupied the regulatory region of genes governing chromosomal integrity and mitochondrial biogenesis. Adding cyclin D1 back to Ccnd1(-/-) mouse embryonic fibroblasts resulted in CIN gene regulatory region occupancy by the DNA-bound form of cyclin D1 and induction of CIN gene expression. Furthermore, increased chromosomal aberrations, aneuploidy, and centrosome abnormalities were observed in the cyclin D1-rescued cells by spectral karyotyping and immunofluorescence. To assess cyclin D1 effects in vivo, we generated transgenic mice with acute and continuous mammary gland-targeted cyclin D1 expression. These transgenic mice presented with increased tumor prevalence and signature CIN gene profiles. Additionally, interrogation of gene expression from 2,254 human breast tumors revealed that cyclin D1 expression correlated with CIN in luminal B breast cancer. These data suggest that cyclin D1 contributes to CIN and tumorigenesis by directly regulating a transcriptional program that governs chromosomal stability.

Topics: Animals; Binding Sites; Breast Neoplasms; Cell Line, Tumor; Chromatin Immunoprecipitation; Chromosomal Instability; Chromosome Aberrations; Cyclin D1; Female; Fibroblasts; Gene Expression Regulation, Neoplastic; Genome-Wide Association Study; Humans; Karyotyping; Mice; Mice, Transgenic; Transcription, Genetic

MUC1 is a heterodimeric glycoprotein that is overexpressed in breast cancers. The present studies demonstrate that the oncogenic MUC1 C-terminal subunit (MUC1-C) associates with the TCF7L2 transcription factor. The MUC1-C cytoplasmic domain (MUC1-CD) binds directly to the TCF7L2 C-terminal region. MUC1-C blocks the interaction between TCF7L2 and the C-terminal-binding protein (CtBP), a suppressor of TCF7L2-mediated transcription. TCF7L2 and MUC1-C form a complex on the cyclin D1 gene promoter and MUC1-C promotes TCF7L2-mediated transcription by the recruitment of β-catenin and p300. Silencing MUC1-C in human breast cancer cells down-regulated activation of the cyclin D1 promoter and decreased cyclin D1 expression. In addition, a MUC1-C inhibitor blocked the interaction with TCF7L2 and suppressed cyclin D1 levels. These findings indicate that the MUC1-C oncoprotein contributes to TCF7L2 activation and thereby promotes cyclin D1 expression in breast cancer cells.

Topics: Alcohol Oxidoreductases; beta Catenin; Breast Neoplasms; Cell Line, Tumor; Cyclin D1; DNA-Binding Proteins; Female; Gene Expression Regulation, Neoplastic; Humans; Mucin-1; p300-CBP Transcription Factors; Protein Binding; Protein Structure, Tertiary; Transcription Factor 7-Like 2 Protein; Transcription, Genetic

Although a high frequency of androgen receptor (AR) expression in human breast cancers has been described, exploiting this knowledge for therapy has been challenging. This is in part because androgens can either inhibit or stimulate cell proliferation in pre-clinical models of breast cancer. In addition, many breast cancers co-express other steroid hormone receptors that can affect AR signaling, further obfuscating the effects of androgens on breast cancer cells.. To create better-defined models of AR signaling in human breast epithelial cells, we took estrogen receptor (ER)-α-negative and progesterone receptor (PR)-negative human breast epithelial cell lines, both cancerous and non-cancerous, and engineered them to express AR, thus allowing the unambiguous study of AR signaling. We cloned a full-length cDNA of human AR, and expressed this transgene in MCF-10A non-tumorigenic human breast epithelial cells and MDA-MB-231 human breast-cancer cells. We characterized the responses to AR ligand binding using various assays, and used isogenic MCF-10A p21 knock-out cell lines expressing AR to demonstrate the requirement for p21 in mediating the proliferative responses to AR signaling in human breast epithelial cells.. We found that hyperactivation of the mitogen-activated protein kinase (MAPK) pathway from both AR and epidermal growth factor receptor (EGFR) signaling resulted in a growth-inhibitory response, whereas MAPK signaling from either AR or EGFR activation resulted in cellular proliferation. Additionally, p21 gene knock-out studies confirmed that AR signaling/activation of the MAPK pathway is dependent on p21.. These studies present a new model for the analysis of AR signaling in human breast epithelial cells lacking ERα/PR expression, providing an experimental system without the potential confounding effects of ERα/PR crosstalk. Using this system, we provide a mechanistic explanation for previous observations ascribing a dual role for AR signaling in human breast cancer cells. As previous reports have shown that approximately 40% of breast cancers can lack p21 expression, our data also identify potential new caveats for exploiting AR as a target for breast cancer therapy.

Topics: Androgen Antagonists; Androgens; Anilides; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Enzyme Activation; Epithelial Cells; Estrogen Receptor alpha; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression; Humans; MAP Kinase Signaling System; Metribolone; Nitriles; Receptors, Androgen; Recombinant Proteins; Tosyl Compounds; Up-Regulation

The GLI-Krüppel zinc finger factor yin yang-1 (YY1) is a complex protein that regulates a variety of processes including transcription, proliferation, development and differentiation. YY1 inhibits cell growth in a cell type-specific manner. The role played by YY1 in its control of tumor cell growth is unclear and controversial. We show here that YY1 can suppress the growth of different tumor cell types in vitro, including human breast carcinoma cells and glioblastoma cells. YY1 also blocked the growth of 13762 MAT mammary adenocarcinoma isografts in rats. YY1 inhibited 13762 MAT tumor growth by approximately 80% compared with the GFP alone group 21 days after injection. YY1 inhibited proliferating cell nuclear antigen (PCNA) expression and pRbSer249/Thr252 phosphorylation without influencing tumor microvascular density. Moreover, YY1 inhibited p21WAF1/Cip1 complex formation with cdk4 and cyclin D1. These findings demonstrate that YY1 can negatively regulate the growth of multiple malignant cell types.

Topics: Adenocarcinoma; Animals; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p21; Female; Green Fluorescent Proteins; Humans; Microvessels; Phosphorylation; Proliferating Cell Nuclear Antigen; Rats; Rats, Inbred F344; Retinoblastoma Protein; Signal Transduction; Transfection; Tumor Burden; YY1 Transcription Factor

The Mediator subunit MED1 is essential for mammary gland development and lactation, whose contribution through direct interaction with estrogen receptors (ERs) is restricted to involvement in pubertal mammary gland development and luminal cell differentiation. Here, we provide evidence that the MED24-containing submodule of Mediator functionally communicates specifically with MED1 in pubertal mammary gland development. Mammary glands from MED1/MED24 double heterozygous knockout mice showed profound retardation in ductal branching during puberty, while single haploinsufficient glands developed normally. DNA synthesis of both luminal and basal cells were impaired in double mutant mice, and the expression of ER-targeted genes encoding E2F1 and cyclin D1, which promote progression through the G(1)/S phase of the cell cycle, was attenuated. Luciferase reporter assays employing double mutant mouse embryonic fibroblasts showed selective impairment in ER functions. Various breast carcinoma cell lines expressed abundant amounts of MED1, MED24, and MED30, and attenuated expression of MED1 and MED24 in breast carcinoma cells led to attenuated DNA synthesis and growth. These results indicate functional communications between the MED1 subunit and the MED24-containing submodule that mediate estrogen receptor functions and growth of both normal mammary epithelial cells and breast carcinoma cells.

Topics: Aging; Animals; Breast Neoplasms; Cell Line, Tumor; Cyclin D1; E2F1 Transcription Factor; Female; G1 Phase; Humans; Mammary Glands, Animal; Mammary Neoplasms, Animal; Mediator Complex; Mediator Complex Subunit 1; Mice; S Phase

Estrogens are involved in the complex regulation of cell proliferation and apoptosis of hormone sensitive tumors including breast and endometrial cancers. Sulfation is the main pathway for estrogen metabolism, which is believed to be involved in the inactivation of estrogens in target tissues. SULT1E1 and PAPSS (PAPSS1 and PAPSS2) are responsible for the estrogen sulfation by providing catalyzing enzyme and universal sulfate donor. The present study showed the expression patterns of SULT1E1 and PAPSS in the breast and endometrial tissues by tissue array analysis and the assessment of clinical samples. The estrogen sulfation enzymes were comparatively higher in the tumorous tissues than their adjacent normal tissues. SULT1E1 overexpression inhibited the tumorigenesis in subcutaneous xenograft model. By CCK-8 assay and flow cytometry assay, overexpression of SULT1E1 and PAPSS1 by adenovirus blocked the estrogen pro-proliferating effect and promoted cell apoptosis induced by H(2)O(2) in MCF-7 cells. By real-time reverse transcription-polymerase chain reaction and western-blot assays, overexpression of SULT1E1 and PAPSS1 suppressed cell growth and triggered apoptosis by downregulating the levels of c-myc, cyclin D1 and bcl-2, meanwhile, upregulating bax expression. In conclusion, the discrepancies in expressions of SULT1E1 and PAPSS between breast and endometrial tumorous tissues and their adjacent normal tissues were prominent. Overexpression of SULT1E1 and PAPSS1 retarded MCF-7 cells growth in vivo and in vitro by arresting cell cycles and inducing apoptosis. Thus, targeting SULT1E1 and PAPSS expressions might be an important approach for estrogen-dependent cancers.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Breast; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin D1; Endometrial Neoplasms; Endometrium; Estrogens; Female; Humans; Hydrogen Peroxide; Mice; Mice, Inbred BALB C; Mice, Nude; Multienzyme Complexes; Neoplasm Transplantation; Neoplasms, Hormone-Dependent; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; Sulfate Adenylyltransferase; Sulfotransferases; Tissue Array Analysis; Transplantation, Heterologous

Synthetic progesterone used in contraception drugs (progestins) can promote breast cancer growth, but the mechanisms involved are unknown. Moreover, it remains unclear whether cytoplasmic interactions between the progesterone receptor (PR) and estrogen receptor alpha (ERα) are required for PR activation. In this study, we used a murine progestin-dependent tumor to investigate the role of ERα in progestin-induced tumor cell proliferation. We found that treatment with the progestin medroxyprogesterone acetate (MPA) induced the expression and activation of ERα, as well as rapid nuclear colocalization of activated ERα with PR. Treatment with the pure antiestrogen fulvestrant to block ERα disrupted the interaction of ERα and PR in vitro and induced the regression of MPA-dependent tumor growth in vivo. ERα blockade also prevented an MPA-induced increase in CYCLIN D1 (CCND1) and MYC expression. Chromatin immunoprecipitation studies showed that MPA triggered binding of ERα and PR to the CCND1 and MYC promoters. Interestingly, blockade or RNAi-mediated silencing of ERα inhibited ERα, but not PR binding to both regulatory sequences, indicating that an interaction between ERα and PR at these sites is necessary for MPA-induced gene expression and cell proliferation. We confirmed that nuclear colocalization of both receptors also occurred in human breast cancer samples. Together, our findings argued that ERα-PR association on target gene promoters is essential for progestin-induced cell proliferation.

Topics: Animals; Breast Neoplasms; Cell Growth Processes; Cell Nucleus; Cyclin D1; Estradiol; Estrogen Receptor alpha; Female; Fulvestrant; Gene Expression Regulation, Neoplastic; Genes, myc; Genes, Reporter; Humans; Mammary Neoplasms, Experimental; Medroxyprogesterone Acetate; Mice; Mice, Inbred BALB C; Neoplasms, Hormone-Dependent; Promoter Regions, Genetic; Receptors, Progesterone

Chronic stress and a high-fat diet are well-documented risk factors associated with the renin-angiotensin system in the development of breast cancer. The angiotensin II type 1 receptor (AT1R) is a novel component of the renin-angiotensin system. Several recent studies have focused on the function of AT1R in cell proliferation during cancer development. Thus, we hypothesized that angiotensin II (Ang Ⅱ) can promote proliferation of breast cancer via activated AT1R; the activation of AT1R may play an important role in promoting breast cancer growth, and AT1R blocker (ARB) may suppress the promotional effect on proliferation by antagonizing AT1R. The expression level of AT1R was found to be significantly upregulated in breast cancer cells by immunohistochemistry, but no correlation between AT1R expression and ER/PR/Her-2 expression was observed. The AT1R(+)-MCF-7 cell line exhibited high expression of AT1R protein, and we generated the AT1R(-)-MCF-7 cell line using RNA interference. ARBs, and in particular irbesartan, effectively inhibited the effects of Ang II on cell proliferation, cell cycle development and downstream AT1R signaling events, including the activation of the Ras-Raf-MAPK pathway and the transcription factors NF-κB and CREB. Irbesartan also significantly altered p53, PCNA and cyclin D1 expression, which was also influenced by activated AT1R in AT1R(+)-MCF-7 cells. These results suggest that ARBs may be useful as a novel preventive and therapeutic strategy for treating breast cancer.

Topics: Angiotensin II; Angiotensin II Type 1 Receptor Blockers; Apoptosis; Biphenyl Compounds; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; CREB-Binding Protein; Cyclin D1; Female; Humans; Irbesartan; Mitogen-Activated Protein Kinases; NF-kappa B; Proliferating Cell Nuclear Antigen; raf Kinases; ras Proteins; Receptor, Angiotensin, Type 1; RNA Interference; RNA, Small Interfering; Signal Transduction; Tetrazoles; Tumor Suppressor Protein p53

It is well established that estrogen is a potent mitogen in cells expressing estrogen receptors (ER). However, a large body of evidence has demonstrated that the effects of mitogenic estrogen signaling exhibit a non-monotonic or biphasic, dose-response curve; estrogen at low concentrations, elicits a mitogenic signaling pathway to stimulate cell proliferation, while at high concentrations, estrogen inhibits cell growth. The molecular mechanism underlying this paradoxical effect of estrogen on cell proliferation remains largely unknown. Recently, we reported that ER-α36, a variant of ER-α, mediates mitogenic estrogen signaling in ER-negative breast cancer cells. Here, we investigated the molecular mechanisms underlying the biphasic estrogen signaling in MDA-MB-231 and MDA-MB-436 ER-negative breast cancer cells. We found that 17β-estradiol (E2β) at l nM induced the phosphorylation of Src-Y416, an event that activates Src, while at 5 µM failed to induce Src-Y416 phosphorylation but induced Src-Y527 phosphorylation an event that inactivates Src. E2β at 1 nM, but not at 5 µM, also induced phosphorylation of MAPK/ERK and activated Cyclin D1 promoter activity through the Src/EGFR/STAT5 pathway. Knockdown of ER‑α36 abrogated the biphasic estrogen signaling in these cells. Our results thus indicate that in ER-negative breast cancer cells Src functions as a switch in ER‑α36-mediated biphasic estrogen signaling through the EGFR/STAT5 pathway.

Topics: Breast Neoplasms; Cell Line, Tumor; Cyclin D1; ErbB Receptors; Estradiol; Estrogen Receptor alpha; Extracellular Signal-Regulated MAP Kinases; Female; Gene Knockdown Techniques; Humans; Phosphorylation; Promoter Regions, Genetic; Signal Transduction; src-Family Kinases; STAT5 Transcription Factor

The metastatic ability of breast cancer cells with chemoresistant properties is higher when compared to that of their parental wild-type cells. Expression of AnnexinA2 (Anxa2), a 36-kDa calcium-dependent phospholipid binding protein, is increased in metastatic tumours and has been found to be associated with the phenotype of drug resistance and metastasis.. In the present study, we found that up-regulation of Anxa2 correlates with enhanced migration and invasion ability of MCF-7 breast cancer cells both in vitro and in vivo. Western blot analysis revealed that exposure to chemotherapeutic drugs may induce elevated expression of Anxa2. In addition, our data have shown that Anxa2 might influence proliferation, migration and invasion of MCF-7 cells by increasing expression of c-myc and cyclin D1 via activation of Erk1/2 signalling pathways.. Our findings suggest that up-regulation of Anxa2 may play an important role in modulating proliferation and invasion of breast cancer MCF-7 cells through regulation of many relevant downstream target genes.

Topics: Animals; Annexin A2; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Drug Resistance, Neoplasm; Female; Humans; Mice; Mice, Nude; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neoplasm Invasiveness; Proto-Oncogene Proteins c-myc; RNA Interference; RNA, Small Interfering; S Phase; Signal Transduction; STAT3 Transcription Factor; Transplantation, Heterologous; Up-Regulation

Fibroblast growth factor-8 (FGF-8) is implicated in the development and progression of breast cancer and its levels are frequently elevated in breast tumors. The mechanisms driving FGF-8-mediated tumorigenesis are not well understood. Herein we aimed to identify target genes associated with FGF-8b-mediated breast cancer cell proliferation by carrying out a cDNA microarray analysis of genes expressed in estrogen receptor negative S115 breast cancer cells treated with FGF-8b for various time periods in comparison with those expressed in non-treated cells. Gene and protein expression was validated for selected genes by qPCR and western blotting respectively. Furthermore, using TRANSBIG data, the expression of human orthologs of FGF-8-regulated genes was correlated to the Nottingham prognostic index and estrogen receptor status. The analysis revealed a number of significantly up- and down-regulated genes in response to FGF-8b at all treatment times. The most differentially expressed genes were genes related to cell cycle regulation, mitosis, cancer, and cell death. Several key regulators of early cell cycle progression such as Btg2 and cyclin D1, as well as regulators of mitosis, including cyclin B, Plk1, survivin, and aurora kinase A, were identified as novel targets for FGF-8b, some of which were additionally shown to correlate with prognosis and ER status in human breast cancer. The results suggest that in stimulation of proliferation FGF-8b not only promotes cell cycle progression through the G1 restriction point but also regulates key proteins involved in chromosomal segregation during mitosis and cytokinesis of breast cancer cells.

Topics: Animals; Apoptosis; Aurora Kinase A; Aurora Kinases; Breast Neoplasms; Cell Cycle; Cell Cycle Proteins; Cell Line, Transformed; Cell Line, Tumor; Cell Proliferation; Cyclin B; Cyclin D1; Female; Fibroblast Growth Factor 8; Gene Expression Regulation, Neoplastic; Humans; Immediate-Early Proteins; Inhibitor of Apoptosis Proteins; Mammary Neoplasms, Animal; Mice; Mitosis; Oligonucleotide Array Sequence Analysis; Polo-Like Kinase 1; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Receptors, Estrogen; RNA Interference; RNA, Small Interfering; Signal Transduction; Survivin; Tumor Suppressor Proteins

The ubiquitin-proteasome system (UPS) is usurped by many if not all cancers to regulate their survival, proliferation, invasion, angiogenesis and metastasis. Bioflavonoids curcumin and chalcones exhibit anti-neoplastic selectivity through inhibition of the 26S proteasome-activity within the UPS. Here, we provide evidence for a novel mechanism of action of chalcone-based derivatives AM146, RA-9 and RA-14, which exert anticancer activity by targeting deubiquitinating enzymes (DUB) without affecting 20S proteasome catalytic-core activity. The presence of the α,β-unsaturated carbonyl group susceptible to nucleophilic attack from the sulfhydryl of cysteines in the active sites of DUB determines the capacity of novel small-molecules to act as cell-permeable, partly selective DUB inhibitors and induce rapid accumulation of polyubiquitinated proteins and deplete the pool of free ubiquitin. These chalcone-derivatives directly suppress activity of DUB UCH-L1, UCH-L3, USP2, USP5 and USP8, which are known to regulate the turnover and stability of key regulators of cell survival and proliferation. Inhibition of DUB-activity mediated by these compounds downregulates cell-cycle promoters, e.g., cyclin D1 and upregulates tumor suppressors p53, p27(Kip1) and p16(Ink4A). These changes are associated with arrest in S-G 2/M, abrogated anchorage-dependent growth and onset of apoptosis in breast, ovarian and cervical cancer cells without noticeable alterations in primary human cells. Altogether, this work provides evidence of antitumor activity of novel chalcone-based derivatives mediated by their DUB-targeting capacity; supports the development of pharmaceuticals to directly target DUB as a most efficient strategy compared with proteasome inhibition and also provides a clear rationale for the clinical evaluation of these novel small-molecule DUB inhibitors.

Topics: Antineoplastic Agents; Apoptosis; Benzylidene Compounds; Boronic Acids; Bortezomib; Breast Neoplasms; Catalytic Domain; Cell Cycle Checkpoints; Cell Proliferation; Cell Survival; Chalcone; Cyclin D1; Cysteine; Dose-Response Relationship, Drug; Endopeptidases; Female; HeLa Cells; Humans; Ovarian Neoplasms; Phenotype; Piperidones; Protease Inhibitors; Proteasome Endopeptidase Complex; Pyrazines; Tumor Suppressor Protein p53; Ubiquitin Thiolesterase; Ubiquitins

The purposes of this study were to investigate the role of Aplysia Ras Homolog I (ARHI) on cell growth, proliferation, apoptosis, and other biological characteristics of HER2-positive breast cancer cells. Our goal was to provide experimental evidence for the development of future effective treatments of HER2-positive breast cancer. A pcDNA3.1-ARHI eukaryotic expression vector was constructed and transfected into the human HER2-positive breast cancer cell lines SK-BR-3 and JIMT-1. Then, various experimental methods were utilized to analyze the biological characteristics of ARHI-expressing breast cancer cells and to examine the impact of expression of the ARHI gene on cyclin D1, p27(Kip1), and calpain1 expression. We further analyzed the cells in each group after treatment with trastuzumab to examine the effects of this drug on various cellular characteristics. When we compared pcDNA3.1-ARHI-expressing SK-BR-3 and JIMT-1 cells to their respective empty vector and control groups, we found that cell viability was significantly lower (p < 0.05) in the ARHI-expressing cells, and the proportions of G1 phase cells and apoptotic cells were significantly higher in the ARHI-expressing cells (p < 0.05). In all groups of SK-BR-3 cells, trastuzumab treatment significantly decreased cell growth (p < 0.05). The proportion of cells in G1 phase and the number of apoptotic cells in the pcDNA3.1-ARHI-expressing group were significantly higher than that in the empty vector group and the control group (p < 0.05). The growth of pcDNA3.1-ARHI-transfected JIMT-1 cells was significantly decreased (p < 0.05), while the proportion of apoptotic cells was significantly increased (p < 0.05). Cell growth, viability, and the percentage of apoptotic cells were similar between the JIMT-1 empty vector and control groups. ARHI expression inhibited cyclin D1 expression in SK-BR-3 cells and JIMT-1 cells, while it promoted p27(Kip1) and calpain1 expression in these cells. ARHI expression inhibits the growth and proliferation of HER2-positive breast cancer cells, while it also promotes apoptosis in these cells. ARHI expression also improves the sensitivity of JIMT-1 cells to trastuzumab by inducing apoptosis.

Topics: Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Calpain; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Female; Gene Expression; Humans; PTEN Phosphohydrolase; rho GTP-Binding Proteins; Trastuzumab

The use of aromatase inhibitors (AI) in the treatment of estrogen receptor (ER)-positive, postmenopausal breast cancer has proven efficacy. However, inappropriate activation of ER target genes has been implicated in the development of resistant tumors. The ER coactivator protein AIB1 has previously been associated with initiation of breast cancer and resistance to endocrine therapy.. Here, we investigated the role of AIB1 in the deregulation of ER target genes occurring as a consequence of AI resistance using tissue microarrays of patients with breast cancer and cell line models of resistance to the AI letrozole.. Expression of AIB1 associated with disease recurrence (P = 0.025) and reduced disease-free survival time (P = 0.0471) in patients treated with an AI as first-line therapy. In a cell line model of resistance to letrozole (LetR), we found ERα/AIB1 promoter recruitment and subsequent expression of the classic ER target genes pS2 and Myc to be constitutively upregulated in the presence of both androstenedione and letrozole. In contrast, the recruitment of the ERα/AIB1 transcriptional complex to the nonclassic ER target cyclin D1 and its subsequent expression remained sensitive to steroid treatment and could be inhibited by treatment with letrozole. Molecular studies revealed that this may be due in part to direct steroid regulation of c-jun-NH(2)-kinase (JNK), signaling to Jun and Fos at the cyclin D1 promoter.. This study establishes a role for AIB1 in AI-resistant breast cancer and describes a new mechanism of ERα/AIB1 gene regulation which could contribute to the development of an aggressive tumor phenotype.

Topics: Androstenedione; Aromatase Inhibitors; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Disease-Free Survival; Drug Resistance, Neoplasm; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Humans; JNK Mitogen-Activated Protein Kinases; Letrozole; MAP Kinase Signaling System; Neoplasm Recurrence, Local; Nitriles; Nuclear Receptor Coactivator 3; Promoter Regions, Genetic; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Proto-Oncogene Proteins c-myc; Transcription, Genetic; Trefoil Factor-1; Triazoles; Tumor Suppressor Proteins; Up-Regulation

Nuclear protein peptidyl-prolyl isomerase Pin1-mediated prolyl isomerization is an essential and novel regulatory mechanism for protein phosphorylation. Therefore, tight regulation of Pin1 localization and catalytic activity is crucial for its normal nuclear functions. Pin1 is commonly dysregulated during oncogenesis and likely contributes to these pathologies; however, the mechanism(s) by which Pin1 catalytic activity and nuclear localization are increased is unknown. Here we demonstrate that mixed-lineage kinase 3 (MLK3), a MAP3K family member, phosphorylates Pin1 on a Ser138 site to increase its catalytic activity and nuclear translocation. This phosphorylation event drives the cell cycle and promotes cyclin D1 stability and centrosome amplification. Notably, Pin1 pSer138 is significantly up-regulated in breast tumors and is localized in the nucleus. These findings collectively suggest that the MLK3-Pin1 signaling cascade plays a critical role in regulating the cell cycle, centrosome numbers, and oncogenesis.

Topics: Active Transport, Cell Nucleus; Breast Neoplasms; Catalysis; Cell Cycle; Cell Nucleus; Cell Transformation, Neoplastic; Centrosome; Cyclin D1; Female; Green Fluorescent Proteins; HEK293 Cells; HeLa Cells; Humans; MAP Kinase Kinase Kinases; Mitogen-Activated Protein Kinase Kinase Kinase 11; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; Phosphorylation; Serine; Signal Transduction

The emergence of automated image analysis algorithms has aided the enumeration, quantification, and immunohistochemical analyses of tumor cells in both whole section and tissue microarray samples. To date, the focus of such algorithms in the breast cancer setting has been on traditional markers in the common invasive ductal carcinoma subtype. Here, we aimed to optimize and validate an automated analysis of the cell cycle regulator cyclin D1 in a large collection of invasive lobular carcinoma and relate its expression to clinicopathologic data. The image analysis algorithm was trained to optimally match manual scoring of cyclin D1 protein expression in a subset of invasive lobular carcinoma tissue microarray cores. The algorithm was capable of distinguishing cyclin D1-positive cells and illustrated high correlation with traditional manual scoring (κ=0.63). It was then applied to our entire cohort of 483 patients, with subsequent statistical comparisons to clinical data. We found no correlation between cyclin D1 expression and tumor size, grade, and lymph node status. However, overexpression of the protein was associated with reduced recurrence-free survival (P=.029), as was positive nodal status (P<.001) in patients with invasive lobular carcinoma. Finally, high cyclin D1 expression was associated with increased hazard ratio in multivariate analysis (hazard ratio, 1.75; 95% confidence interval, 1.05-2.89). In conclusion, we describe an image analysis algorithm capable of reliably analyzing cyclin D1 staining in invasive lobular carcinoma and have linked overexpression of the protein to increased recurrence risk. Our findings support the use of cyclin D1 as a clinically informative biomarker for invasive lobular breast cancer.

Topics: Adult; Aged; Aged, 80 and over; Algorithms; Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Lobular; Cell Nucleus; Cyclin D1; Disease-Free Survival; Female; Gene Amplification; Humans; Image Processing, Computer-Assisted; Mastectomy; Middle Aged; Neoplasm Invasiveness; Neoplasm Staging; Prognosis; Survival Rate; Sweden

Ligand-dependent activation of the estrogen receptor (ER) as well as of the insulin-like growth factor type 1 (IGF1R) induces the proliferation of luminal breast cancer cells. These two pathways cooperate and are interdependent. We addressed the question of the mechanisms of crosstalk between the ER and IGF1R.. We evaluated the mitogenic effects of estradiol (E2; agonist ligand of ER) and of insulin (a ligand of IGF1R) in the MCF-7 cells by flow cytometry and by analyzing the cell levels of cell cycle-related proteins (immunoblotting) and mRNA (RT-QPCR). To verify the requirement for the kinase activity of Akt (a downstream target of IGF1R) in the mitogenic action of estradiol, we used shRNA strategy and shRNA-resistant expression vectors.. The activation of the ER by E2 is unable to induce the cell cycle progression when the phosphatidyl inositol-3 kinase (PI3K)/Akt signaling is blocked by a chemical inhibitor (LY 294002) or by shRNA targeting Akt1 and Akt2. shRNA-resistant Akt wild-type constructs efficiently complemented the mitogenic signaling activity of E2 whereas constructs with inactivated kinase function did not. In growth factor-starved cells, the residual PI3K/Akt activity is sufficient to complement the mitogenic action of E2. Conversely, when ER function is blocked by the antiestrogen ICI 182780, IGF1R signaling is intact but does not lead to efficient reinitiation of the cell cycle in quiescent, growth factor-starved MCF-7 cells. The basal transcription-promoting activity of ligand-free ER in growth factor-starved cells is sufficient to complement the mitogenic action of the IGF1R-dependent signaling.. The basal ER activity in the absence of ligand is sufficient to allow efficient mitogenic action of IGF1R agonists and needs to be blocked to prevent the cell cycle progression.

Topics: Autocrine Communication; Breast Neoplasms; Cell Cycle; Cell Proliferation; Cyclin A; Cyclin D1; Estradiol; Female; Gene Expression Regulation, Neoplastic; Humans; Insulin; MCF-7 Cells; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-myc; Receptor, IGF Type 1; Receptors, Estrogen; Signal Transduction

Sox2 (sex-determining region Y-box protein 2) is a transcription factor regulating pluripotency in embryonic stem cells. Sox2 is aberrantly expressed in breast and other cancers, though its biological significance remains widely unexplored. To understand the significance of this aberrancy, we assessed the transcription activity of Sox2 in two Sox2-expressing breast cancer cell lines, MCF7 and ZR751, using a lentiviral Sox2 GFP reporter vector. Surprisingly, Sox2 transcription activity, as measured by GFP expression encoded in a Sox2 reporter construct, was detectable only in a small subset of cells in both cell lines. Purification of GFP+ cells (cells with Sox2 activity) and GFP- cells (cells without Sox2 activity) was enriched for two phenotypically distinct cell populations in both MCF7 and ZR751 cell lines. Specifically, GFP+ cells formed significantly more colonies in methylcellulose and more mammospheres in vitro compared to GFP- cells. These phenotypic differences are directly linked to Sox2 as siRNA knockdown of Sox2 in GFP+ cells abolished these abilities. To provide a mechanistic explanation to our observations, we performed gel shift and chromatin immunoprecipitation studies; Sox2 was found to bind to its DNA binding consensus sequence and the promoters of Cyclin D1 and Nanog (two known Sox2 downstream targets) only in GFP+ cells. GFP+ cells also up-regulated CD49f, phospho-GSK3β, and β-catenin. In summary, we have identified two novel phenotypically distinct cell subsets in two breast cancer cell lines based on their differential Sox2 transcription activity. We demonstrate that Sox2 transcription activity, and not its protein expression alone, underlies the tumorigenicity and cancer stem cell-like phenotypes in breast cancers.

Topics: beta Catenin; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; DNA; Female; Genes, Reporter; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Homeodomain Proteins; Humans; Integrin alpha6; MCF-7 Cells; Nanog Homeobox Protein; Phenotype; RNA Interference; RNA, Small Interfering; SOXB1 Transcription Factors; Transcription, Genetic

As part of a cell's inherent protection against carcinogenesis, p14ARF is upregulated in response to hyperproliferative signalling to induce cell cycle arrest. This property makes p14ARF a leading candidate for cancer therapy. This study explores the consequences of reactivating p14ARF in breast cancer and the potential of targeting p14ARF in breast cancer treatment. Our results show that activation of the p14ARF-p53-p21-Rb pathway in the estrogen sensitive MCF-7 breast cancer cells induces many hallmarks of senescence including a large flat cell morphology, multinucleation, senescence-associated-β-gal staining, and rapid G1 and G2/M phase cell cycle arrest. P14ARF also induces the expression of the proto-oncogene cyclin D1, which is most often associated with a transition from G1-S phase and is highly expressed in breast cancers with poor clinical prognosis. In this study, siRNA knockdown of cyclin D1, p21 and p53 show p21 plays a pivotal role in the maintenance of high cyclin D1 expression, cell cycle and growth arrest post-p14ARF induction. High p53 and p14ARF expression and low p21/cyclin D1 did not cause cell-cycle arrest. Knockdown of cyclin D1 stops proliferation but does not reverse senescence-associated cell growth. Furthermore, cyclin D1 accumulation in the nucleus post-p14ARF activation correlated with a rapid loss of nucleolar Ki-67 protein and inhibition of DNA synthesis. Latent effects of the p14ARF-induced cellular processes resulting from high nuclear cyclin D1 accumulation included a redistribution of Ki-67 into the nucleoli, aberrant nuclear growth (multinucleation), and cell proliferation. Lastly, downregulation of cyclin D1 through inhibition of ER abrogated latent recurrence. The mediation of these latent effects by continuous expression of p14ARF further suggests a novel mechanism whereby dysregulation of cyclin D1 could have a double-edged effect. Our results suggest that p14ARF induced-senescence is related to late-onset breast cancer in estrogen responsive breast cancers and/or the recurrence of more aggressive breast cancer post-therapy.

Topics: Breast Neoplasms; Cell Cycle; Cell Nucleus; Cyclin D1; Female; Gene Knockdown Techniques; Humans; MCF-7 Cells; Prognosis; Proto-Oncogene Mas; RNA Processing, Post-Transcriptional; RNA, Small Interfering; Tumor Suppressor Protein p14ARF

To investigate markers for predicting breast cancer progression, we performed a candidate gene-based study that assessed expression change of three genes, cyclin D1, β-catenin, and metastasis-associated protein-1 (MTA1), involving in aggressive phenotypes of cancerous cells, namely hyperproliferation, epithelial-mesenchymal transition, and global transcriptional regulation.. Specimens were from 150 enrolled female patients, with invasive ductal carcinoma, followed up for more than 10 years. mRNA expression of cyclin D1, β-catenin, and MTA1 in cancerous and noncancerous cells microdissected from the primary tumor site was determined by quantitative real-time PCR. The relationship between mRNA expression levels of the genes and clinicopathologic features was assessed by statistical analysis. Disease-free and overall survival (DFS and OS) were analyzed by Kaplan-Meier analysis with log-rank test and a multivariate Cox regression model.. Cyclin D1 was shown to be overexpressed in late-stage breast cancer (stage III/IV). Breast cancer with lymph node metastasis (LNM) showed significantly higher frequency of overexpressed cyclin D1, β-catenin, and MTA1 (P < 0.05). Patients carrying greater numbers of overexpressed genes had joint effects on increased risk in tumors of advanced stages (P ( trend ) = 0.03) and LNM (P ( trend ) < 0.01). In the LNM-negative group, patients whose tumors with greater number of cyclin D1, β-catenin, and MTA1 overexpressions were associated with poorer clinical outcomes, with hazard ratio of 14.79 for OS (P = 0.015) and 7.54 for DFS (P = 0.015) using multivariate Cox regression analysis during the 10-year follow-up.. Higher expression of cyclin D1, β-catenin, and MTA1 mRNAs in breast cancers may prove effective in predicting unfavorable outcomes of breast cancer.

Topics: Adult; Aged; Aged, 80 and over; beta Catenin; Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Ductal, Breast; Cyclin D1; Female; Follow-Up Studies; Histone Deacetylases; Humans; Immunoenzyme Techniques; Laser Capture Microdissection; Middle Aged; Neoplasm Grading; Neoplasm Staging; Prognosis; Real-Time Polymerase Chain Reaction; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Survival Rate; Trans-Activators

Recent molecular data pointed towards the possibility of a stepwise dedifferentiation in a subgroup of invasive breast cancer (BC) cases. It was hypothesized that oestrogen receptor positive (ER+) grade 3 (G3) ductal invasive BCs are the end stage of a dedifferentiation process of luminal BC. A progression of luminal A towards luminal B BCs associated with a 'progression through grade' and an increased cell proliferation seemed the obvious explanation. In order to verify this hypothesis on a morphological and immunohistochemical level, we investigated 865 invasive BC cases. All cases were reviewed for the presence of intratumoural heterogeneity in grade of the invasive cancer and the presence of associated ductal carcinoma in situ (DCIS). With the use of tissue microarrays, the molecular subtype was determined and correlated with clinico-pathological features. In addition, all cases were stained for p21, p27, Ki-67, Cyclin D1, bcl-2, p53, and p16 and the results subjected to a biomathematical dependency analysis. The frequency of ER-positivity decreased with tumour size. The frequency of luminal A BC decreased as well, whereas the number of luminal B BCs remained constant. A gradual increase of the frequency of basal-like, HER2-driven and non-expressor BCs with tumour size was seen. In only 1 out of 865 BC cases, both a G1 and a G3 invasive cancer component was seen within the same BC. In two cases, a ductal invasive G1 carcinoma was associated with a poorly-differentiated DCIS. The frequency of columnar cell lesions was evenly distributed over ER+ and ER- ductal invasive G3 carcinomas. The biomathematical analysis gave striking hints against an obligate progression of BC trough grade. In conclusion, our results show that a morphological recognizable striking 'progression through grade' at least in its extreme form from G1 towards G3 is a very rare event in the natural course of invasive BC, including luminal BC.

Topics: Adult; Aged; Aged, 80 and over; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Intraductal, Noninfiltrating; Cyclin D1; Female; Humans; Ki-67 Antigen; Middle Aged; Neoplasm Grading; Proto-Oncogene Proteins c-bcl-2; Receptor, ErbB-2; Receptors, Estrogen; Tumor Suppressor Protein p53

In C4-HD murine mammary carcinomas and in human breast cancer T47D cells, we showed that medroxyprogesterone acetate (MPA) induces a nuclear physical association between estrogen receptor alpha (ERa) and progesterone receptors (PR). The blockade of ERa inhibits cell proliferation mediated by progestins. We hypothesized that this nuclear association between ERa/PR is necessary to trigger progestin-induced cell proliferation and tumor growth. We demonstrated that fulvestrant (FUL, ICI182.780) induced complete regression of C4-HD tumors growing with progestins. MPA treatment induced an early increase in both CCND1 and MYC expression in T47D cells. The blockade of ERa prevented the MPA-dependent transcription of both genes. Specific binding of PR/ERa was observed at the same MPA-sensitive regions at the CCND1 and MYC gene promoters after chromatin immunoprecipitation (ChIP) analysis. ICI inhibited binding of ERa to both gene regulatory sequences while PR binding was unaffected. The nuclear colocalization between both receptors in T47D cells was confirmed by: confocal microscopy, Duolink assays and co-immunoprecipitation assays. In breast cancer samples we also observed a nuclear interaction between both steroid receptors. Our results indicate that the presence of ERa interacting with activated PR at the CCND1 and MYC promoters is required to trigger progestin-induced gene transcription and cell proliferation in breast cancer cells.

Topics: Animals; Antineoplastic Agents, Hormonal; Breast Neoplasms; Carcinoma; Cell Proliferation; Chromatin Immunoprecipitation; Cyclin D1; Estradiol; Estrogen Receptor alpha; Female; Fulvestrant; Genes, myc; Humans; Mammary Neoplasms, Experimental; Medroxyprogesterone Acetate; Murinae; Progestins; Receptors, Progesterone; Transcription, Genetic

Eukaryotic elongation factor 2 kinase (eEF-2K), through its phosphorylation of elongation factor 2 (eEF2), provides a mechanism by which cells can control the rate of the elongation phase of protein synthesis. The activity of eEF-2K is increased in rapidly proliferating malignant cells, is inhibited during mitosis, and may contribute to the promotion of autophagy in response to anti-cancer therapies. The purpose of this study was to examine the therapeutic potential of targeting eEF-2K in breast cancer tumors. Through the systemic administration of liposomal eEF-2K siRNA (twice a week, i.v. 150 µg/kg), the expression of eEF-2K was down-regulated in vivo in an orthotopic xenograft mouse model of a highly aggressive triple negative MDA-MB-231 tumor. This targeting resulted in a substantial decrease in eEF2 phosphorylation in the tumors, and led to the inhibition of tumor growth, the induction of apoptosis and the sensitization of tumors to the chemotherapy agent doxorubicin. eEF-2K down-modulation in vitro resulted in a decrease in the expression of c-Myc and cyclin D1 with a concomitant increase in the expression of p27(Kip1). A decrease in the basal activity of c-Src (phospho-Tyr-416), focal adhesion kinase (phospho-Tyr-397), and Akt (phospho-Ser-473) was also detected following eEF-2K down-regulation in MDA-MB-231 cells, as determined by Western blotting. Where tested, similar results were seen in ER-positive MCF-7 cells. These effects were also accompanied by a decrease in the observed invasive phenotype of the MDA-MB-231 cells. These data support the notion that the disruption of eEF-2K expression in breast cancer cells results in the down-regulation of signaling pathways affecting growth, survival and resistance and has potential as a therapeutic approach for the treatment of breast cancer.

Topics: Animals; Antibiotics, Antineoplastic; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Disease Models, Animal; Doxorubicin; Drug Resistance, Neoplasm; Elongation Factor 2 Kinase; Female; Focal Adhesion Protein-Tyrosine Kinases; Gene Expression Regulation, Neoplastic; Gene Silencing; Genes, src; Humans; Mice; Neoplasm Invasiveness; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-myc; RNA Interference

It is well established that epidermal growth factor (EGF) is a potent mitogen in cells expressing EGF receptor (EGFR). However, a body of evidence indicated that the effects of mitogenic EGF signaling exhibit a non-monotonic, or biphasic dose response curve; EGF at low concentrations elicits a mitogenic signaling pathway to stimulate cell proliferation while at high concentrations, EGF inhibits cell growth. However, the molecular mechanism underlying this paradoxical effect of EGF on cell proliferation remains largely unknown. Here, we investigated the molecular mechanisms underlying the biphasic EGF signaling in ER-negative breast cancer MDA-MB-231 and MDA-MB-436 cells, both of which express endogenous EGFR. We found that EGF at low concentrations induced the phosphorylation of the Src-Y416 residue, an event to activate Src, while at high concentrations allowed Src-Y527 phosphorylation that inactivates Src. EGF at 10 ng/ml also induced phosphorylation of the MAPK/ERK and activated cyclin D1 promoter activity through the Src/EGFR/STAT5 pathways but not at a higher concentration (500 ng/ml). Our results thus demonstrated that Src functions as a switch of EGF signaling depending on concentrations of EGF.

Topics: Breast Neoplasms; Cell Line, Tumor; Cyclin D1; Dose-Response Relationship, Drug; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Neoplastic; Humans; Mitogen-Activated Protein Kinases; Promoter Regions, Genetic; Proto-Oncogene Proteins pp60(c-src); Receptors, Estrogen; Signal Transduction; STAT5 Transcription Factor

The aim of this work is to study the validity of cyclin D1 expression, a cell cycle regulatory protein, on (fine needle aspiration cytology) FNAC samples in patients with breast carcinoma using immunostaining technique.. This is a study done on 70 patients with primary breast carcinoma, presented to Cytology Unit, Pathology Department, National Cancer Institute, Cairo University. They underwent preoperative FNAC and diagnosed as breast carcinoma. The cytologic and tissue section slides were subjected to cyclin D1 immunocytochemical staining. Only the nuclear immunoreactivity for cyclin D1 was considered specific. The rate of concordance, and discordance, and kappa value were calculated. Relation between cytologic expression of cyclin D1 and different clinicopathologic parameters was evaluated.. Cyclin D1 immunocytochemical expression was observed in 53/70 cases (75.7%) in cytologic smears. In histologic sections of the corresponding cases, cyclin D1was detected in 48/70 cases (68.6%). The concordance rate of cyclin D1 expression in the FNA and histologic sections was 87.1% while the discordance rate was 12.9%. Kappa showed a value of 0.65. A statistically significant relation was found between cyclin D1 immunocytochemical expression and hormonal status as well as nuclear grade.. Cyclin D1 immunocytochemical expression can be performed successfully on cytologic samples with a high concordance rate and agreement with histologic results. This can help in determining tumor biology, and plan for patients' treatment. The marker showed a significant relation with hormone receptor status and nuclear grade.

Topics: Biomarkers, Tumor; Biopsy, Fine-Needle; Breast; Breast Neoplasms; Carcinoma, Ductal, Breast; Cyclin D1; Female; Humans; Immunohistochemistry; Lymphatic Metastasis; Neoplasm Grading

Glycogen synthase kinase 3β (GSK3β) is phosphorylated and inactivated by the phosphoinositide 3 kinase PI3K/Akt pathway. Activation of Akt phosphorylates GSK3β preventing phosphorylation of cyclin D1 which leads to accumulation and nuclear localisation of cyclin D1, activation of CDK4/6 and cell cycle progression. The CCND1 gene found at chromosome 11q13 has been shown to be amplified in approximately 15 % of breast cancers. Cyclin D1, the product of the CCND1 gene, is one of the most commonly overexpressed proteins in breast cancer. Protein expression for GSK3β, phosphorylated-GSK3β (p-GSK3β), cyclin D1 and gene expression of CCND1 were examined in tissue microarrays of 1,686 patients from the Edinburgh Breast Conservation Series. High GSK3β expression was associated with reduced distant relapse-free survival (DRFS), while no association between p-GSK3β and breast cancer-specific survival was seen. CCND1 amplification is also associated with poor DRFS. On the contrary, cyclin D1 overexpression is associated with an increase in DRFS. Multivariate analysis was performed. We suggest that analysis of both GSK3β and cyclin D1 expressions can be considered as a marker of good prognosis in early breast cancer.

Topics: Aged; Breast Neoplasms; Cyclin D1; Disease-Free Survival; Female; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Middle Aged; Phosphatidylinositol 3-Kinases; Prognosis; Proto-Oncogene Proteins c-akt; Signal Transduction; Tamoxifen; Tissue Array Analysis; Treatment Outcome

The phosphatidylinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway is a key potential target in breast cancer therapy. Because some cancer cell lines are resistant to mTOR inhibition, we combined the mTOR inhibitor with the PI3K inhibitor and assayed the inhibitory effect of this combination versus that of a single inhibitor.. The proliferation of MCF7, SK-BR-3, T-47D, and MDA-MB-231 cells was measured by MTT assay in the presence of GDC-0941 and/or rapamycin. Afterwards, we determined the visible changes in signaling in the PI3K/AKT/mTOR pathway by Western blotting.. GDC-0941 exhibited excellent inhibition on MCF7, T-47D and SK-BR-3 cells with different characteristics. In addition, GDC-0941 blocked the feedback of PI3K/Akt through S6K1, resulting in decreased Akt activity by rapamycin activation. The combination of GDC-0941 and rapamycin downregulated the key components of the cell cycle machinery, such as cyclin D1 and upregulated the apoptotic markers.. Our findings suggest that GDC-0941, either alone or in combination with rapamycin, may serve as a new, promising approach for breast cancer treatment.

Topics: Antibiotics, Antineoplastic; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; Female; Humans; Indazoles; MCF-7 Cells; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; Sulfonamides; TOR Serine-Threonine Kinases

One-third of breast cancers display amplifications of the ERBB2 gene encoding the HER2 kinase receptor. Trastuzumab, a humanized antibody directed against an epitope on subdomain IV of the extracellular domain of HER2 is used for therapy of HER2-overexpressing mammary tumors. However, many tumors are either natively resistant or acquire resistance against Trastuzumab. Antibodies directed to different epitopes on the extracellular domain of HER2 are promising candidates for replacement or combinatorial therapy. For example, Pertuzumab that binds to subdomain II of HER2 extracellular domain and inhibits receptor dimerization is under clinical trial. Alternative antibodies directed to novel HER2 epitopes may serve as additional tools for breast cancer therapy. Our aim was to generate novel anti-HER2 monoclonal antibodies inhibiting the growth of breast cancer cells, either alone or in combination with tumor necrosis factor-α (TNF-α).. Mice were immunized against SK-BR-3 cells and recombinant HER2 extracellular domain protein to produce monoclonal antibodies. Anti-HER2 antibodies were characterized with breast cancer cell lines using immunofluorescence, flow cytometry, immunoprecipitation, western blot techniques. Antibody epitopes were localized using plasmids encoding recombinant HER2 protein variants. Antibodies, either alone or in combination with TNF-α, were tested for their effects on breast cancer cell proliferation.. We produced five new anti-HER2 monoclonal antibodies, all directed against conformational epitope or epitopes restricted to the native form of the extracellular domain. When tested alone, some antibodies inhibited modestly but significantly the growth of SK-BR-3, BT-474 and MDA-MB-361 cells displaying ERBB2 amplification. They had no detectable effect on MCF-7 and T47D cells lacking ERBB2 amplification. When tested in combination with TNF-α, antibodies acted synergistically on SK-BR-3 cells, but antagonistically on BT-474 cells. A representative anti-HER2 antibody inhibited Akt and ERK1/2 phosphorylation leading to cyclin D1 accumulation and growth arrest in SK-BR-3 cells, independently from TNF-α.. Novel antibodies against extracellular domain of HER2 may serve as potent anti-cancer bioactive molecules. Cell-dependent synergy and antagonism between anti-HER2 antibodies and TNF-α provide evidence for a complex interplay between HER2 and TNF-α signaling pathways. Such complexity may drastically affect the outcome of HER2-directed therapeutic interventions.

Topics: Animals; Antibodies, Monoclonal; Antibody Affinity; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Dose-Response Relationship, Drug; Drug Antagonism; Drug Synergism; Epitopes; Female; Fluorescent Antibody Technique, Indirect; Humans; In Situ Hybridization, Fluorescence; MCF-7 Cells; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phosphorylation; Protein Binding; Proto-Oncogene Proteins c-akt; Receptor, ErbB-2; Tumor Necrosis Factor-alpha

Constitutive activation of Signal Transducers and Activators of Transcription 3 (STAT3) signaling is frequently detected in breast and pancreatic cancer. Inhibiting constitutive STAT3 signaling represents a promising molecular target for therapeutic approach. Using structure-based design, we developed a non-peptide cell-permeable, small molecule, termed as XZH-5, which targeted STAT3 phosphorylation. XZH-5 was found to inhibit STAT3 phosphorylation (Tyr705) and induce apoptosis in human breast and pancreatic cancer cell lines expressing elevated levels of phosphorylated STAT3. XZH-5 could also inhibit interleukin-6-induced STAT3 phosphorylation in cancer cell lines expressing low phosphorylated STAT3. Inhibition of STAT3 signaling by XZH-5 was confirmed by the down-regulation of downstream targets of STAT3, such as Cyclin D1, Bcl-2, and Survivin at mRNA level. In addition, XZH-5 inhibited colony formation, cell migration, and enhanced the cytotoxicity of chemotherapeutic drugs when combined with Doxorubicin or Gemcitabine. Our results indicate that XZH-5 may be a potential therapeutic agent for breast and pancreatic cancers with constitutive STAT3 signaling.

Topics: Animals; Apoptosis; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cyclin D1; Female; HeLa Cells; Histidine; Humans; Inhibitor of Apoptosis Proteins; Interleukin-6; Mice; Mice, SCID; Pancreatic Neoplasms; Phenylurea Compounds; Phosphorylation; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; STAT1 Transcription Factor; STAT3 Transcription Factor; Survivin

Protein denitrosylation by thioredoxin reductase (TrxR) is key for maintaining S-nitrosothiol (SNO) homeostasis, although its role in tumor progression is unknown. Therefore, the present study aimed to assess the role of altered SNO homeostasis in breast cancer cells.. The impairment of SNO homeostasis in breast cancer cells was achieved with the highly specific TrxR inhibitor auranofin and/or exposure to S-nitroso-L-cysteine. S-nitrosylated proteins were detected using the biotin switch assay. Estrogen receptor (ER) alpha knockdown was achieved using RNA silencing technologies and subcellular localization of ERα was analyzed by confocal microscopy. The Oncomine database was explored for TrxR1 (TXNRD1) expression in breast tumors and TrxR1, ER and p53 expression was analyzed by immunohistochemistry in a panel of breast tumors.. The impairment of SNO homeostasis enhanced cell proliferation and survival of ER+ MCF-7 cells, but not of MDA-MB-231 (ER-, mut p53) or BT-474 (ER+, mut p53) cells. This enhanced cell growth and survival was associated with Akt, Erk1/2 phosphorylation, and augmented cyclin D1 expression and was abolished by the ER antagonist fulvestrant or the p53 specific inhibitor pifithrin-α. The specific silencing of ERα expression in MCF-7 cells also abrogated the growth effect of TrxR inhibition. Estrogenic deprivation in MCF-7 cells potentiated the pro-proliferative effect of impaired SNO homeostasis. Moreover, the subcellular distribution of ERα was altered, with a predominant nuclear localization associated with phosphorylation at Thr311 in those cells with impaired SNO homeostasis. The impairment of SNO homeostasis also expanded a cancer stem cell-like subpopulation in MCF-7 cells, as indicated by the increase of percentage of CD44+ cells and the augmented capability to form mammospheres in vitro. Notably, ER+ status in breast tumors was significantly associated with lower TXNDR1 mRNA expression and immunohistochemical studies confirmed this association, particularly when p53 abnormalities were absent.. The ER status in breast cancer may dictate tumor response to different nitrosative environments. Impairment of SNO homeostasis confers survival advantages to ER+ breast tumors, and these molecular mechanisms may also participate in the development of resistance against hormonal therapies that arise in this type of mammary tumors.

Topics: Antineoplastic Agents, Hormonal; Antirheumatic Agents; Auranofin; Benzothiazoles; Breast Neoplasms; CD24 Antigen; Cell Proliferation; Cell Survival; Cyclin D1; Cysteine; Estradiol; Estrogen Receptor alpha; Extracellular Signal-Regulated MAP Kinases; Female; Fulvestrant; Homeostasis; Humans; Hyaluronan Receptors; MCF-7 Cells; Phosphorylation; Proto-Oncogene Proteins c-akt; RNA Interference; RNA, Messenger; RNA, Small Interfering; S-Nitrosothiols; Spheroids, Cellular; Thioredoxin-Disulfide Reductase; Toluene; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Curcumin exhibits growth-suppressive activity against a variety of cancer cells, but low bioavailability prevents its use in chemotherapeutic applications. One strategy for circumventing this problem has been the creation of synthetic analogs. In this study we synthesized an analog of curcumin bis-1,7-2(hydroxyphenil)-hepta-1,6diene-3,5diore (BDMC-A) and investigated its anti-breast cancer property.. We compared the impact of bis-1,7-2(hydroxyphenil)-hepta-1,6diene-3,5diore (BDMC-A) with that of curcumin in human breast cancer cell line MCF-7. MTT [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide] cell viability assay was used to examine the cell viability/proliferation. LDH assay and cell counts were performed to assess the cytotoxicity and anti-proliferative effects of the compound respectively. Flow cytometry followed by Western blot were performed to investigate the cell cycle distribution.. BDMC-A has an inhibitory effect on MCF-7 cells comparably equivalent to that of curcumin as determined by MTT assay. Cytotoxicity of the cells by both curcumin and BDMC-A were confirmed by LDH release assay and cell count assay. Flow cytometric studies showed accumulation of cells in the G2/M phase which confirms the cell cycle arrest. This was further confirmed by immunoblotting of the protein Cyclin D1, whose expression were found to be decreased in both curcumin and BDMC-A treatment.. The results indicate that the curcumin analog exhibit potent inhibitory activity which is comparable to that of curcumin in human breast cancer cells. Since the solubility of BDMC-A was higher in aqueous medium, it is expected to be more bioavailable, and hence more active in vivo. Further evaluation might reveal its role on various molecular targets.

Topics: Antineoplastic Agents, Phytogenic; Biomarkers, Tumor; Blotting, Western; Breast Neoplasms; Cell Proliferation; Cell Survival; Curcumin; Cyclin D1; Dose-Response Relationship, Drug; Female; Flow Cytometry; G2 Phase Cell Cycle Checkpoints; Humans; L-Lactate Dehydrogenase; MCF-7 Cells; Time Factors

The Cyclin D1(CCND1) G870A polymorphism may be associated with breast cancer, but the evidence from individual studies is inconclusive. The aim of this study was to investigate the correlation between the CCND1 G870A polymorphism and breast cancer risk in a meta-analysis.. We searched Pubmed and analysed 11 articles on 5,528 cases and 5,353 controls before February 1, 2012.. we found there are significant association for AA versus GG and AA versus GA/GG. No significant associations were found for GA versus GG, GA/AA versus GG. There are significant association for AA versus GG ,and AA versus GA/GG in Caucasians. We didn't find any significant main effects for G870A polymorphism on breast cancer risk either in recessive or dominant models in Asians.. This meta-analysis suggests that AA of the CCND1 G870A polymorphism is associated with breast cancer susceptibility.

Topics: Breast Neoplasms; Case-Control Studies; Cyclin D1; Female; Genetic Predisposition to Disease; Humans; Polymorphism, Genetic; Prognosis; Risk Factors

The behavior of breast epithelial cells is influenced by their microenvironment which includes stromal cells and extracellular matrix (ECM). During cancer progression, the tissue microenvironment fails to control proliferation and differentiation, resulting in uncontrolled growth and invasion. Upon invasion, the ECM encountered by breast cancer cells changes from primarily laminin and collagen IV to primarily collagen I. We show here that culturing invasive breast cancer cells in 3-dimensional (3D) collagen I inhibits proliferation through direct regulation of cyclin E1, a G(1)/S regulator that is overexpressed in breast cancer. When the breast cancer cell line MDA-MB-231 was cultured within 3D collagen I gels, the G(1)/S transition was inhibited as compared to cells cultured on conventional 2D collagen or plastic dishes. Cells in 3D collagen downregulated cyclin E1 protein and mRNA, with no change in cyclin D1 level. Cyclin D1 was primarily cytoplasmic in 3D cultures, and this was accompanied by decreased phosphorylation of Rb, a nuclear target for both cyclin E1- and cyclin D1-associated kinases. Positive regulators of cyclin E1 expression, the transcription factor c-Myc and cold-inducible RNA binding protein (CIRP), were decreased in 3D collagen cultures, while the collagen I receptor β1 integrin was greatly increased. Inhibition of β1 integrin function rescued proliferation and cyclin E1 expression as well as c-Myc expression and Rb phosphorylation, but cyclin D1 remained cytoplasmic. We conclude that cyclin E1 is repressed independent of effects on cyclin D1 in a 3D collagen environment and dependent on β1 integrin interaction with collagen I, reducing proliferation of invasive breast cancer cells.

Topics: Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Collagen; Cyclin D1; Cyclin E; Cytoplasm; Down-Regulation; Female; Humans; Integrin beta1; Oncogene Proteins; Phosphorylation; Protein Transport; Proto-Oncogene Proteins c-myc; Retinoblastoma Protein

The purpose of this study was to evaluate the importance of biological markers to predict pathologic complete response (pCR) to neoadjuvant docetaxel plus epirubicin combination chemotherapy in patients with locally advanced breast cancer (LABC). Two hundred and twenty consecutive patients with LABC who had received neoadjuvant chemotherapy (NCT) with docetaxel and epirubicin from March 2006 to March 2009 were included in this retrospective study. The pre- and post-neoadjuvant chemotherapy (NCT) treatment expression levels and changes of Ki-67 proliferation index, estrogen receptor (ER), progesterone receptor (PgR), epidermal growth factor receptor 2 (HER-2), cyclin D1, and nm23-H1 were detected by immunohistochemistry (IHC). The pCR rate was 9.1% (95% CI, 5.3-12.9%). In univariate analysis, poor tumor differentiation, OR after 2 cycles of NCT, both negative of ER/PgR, negative HER-2, positive cyclin D1, and positive nm23-H1 were found to be significantly predictive of a pCR. Histological grade and ER/PgR status were significant for pCR on multivariate analysis (P = 0.023 and 0.003, respectively). The expression levels of cyclin D1 (median, 8% vs. 9%; P = 0.016) after NCT treatment increased significantly, while the median Ki-67 proliferation index was dramatically decreased after NCT treatment from 35 to 15% (P = 0.036). However, after a Bonferroni adjustment, only the difference of Ki-67 proliferation index was still significant (P = 0.026). Histological grade and ER/PgR status are independent predictive factors of pCR to neoadjuvant docetaxel plus epirubicin combination chemotherapy in locally advanced breast cancer. Expression of HER-2, Ki-67, cyclin D1, and nm23-H1 were not predictive for pCR.

Topics: Adult; Aged; Biomarkers, Tumor; Breast Neoplasms; Cyclin D1; Docetaxel; Epirubicin; Female; Humans; Ki-67 Antigen; Middle Aged; Neoadjuvant Therapy; NM23 Nucleoside Diphosphate Kinases; Predictive Value of Tests; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone; Retrospective Studies; Taxoids; Young Adult

Somatic and germline mutations in PTEN (phosphatase and tensin homolog deleted on chromosome 10) are found in sporadic cancers and Cowden syndrome patients, respectively. Recent identification of naturally occurring cancer and germline mutations within the ATP-binding motifs of PTEN (heretofore referred to as PTEN ATP-binding mutations) has revealed that these mutations disrupted the subcellular localization and tumor-suppressor activity of PTEN. However, very little is known about the underlying mechanisms of PTEN ATP-binding mutations in tumorigenesis. Here we show that these mutations impair PTEN's function both qualitatively and quantitatively. On the one hand, PTEN ATP-binding mutants lose their phosphatase activity and the effect of downregulation of cyclin D1. On the other, the mislocalized mutant PTEN results in a significantly decreased nuclear p53 protein level and transcriptional activity, enhanced production of reactive oxygen species, induction of Cu/Zn superoxide dismutase as well as dramatically increased DNA double-strand breaks (DSBs). When compared with wild-type PTEN, the ATP-binding mutant PTEN has reduced half-life in vitro and decreased protein expression levels in vivo. Our data, thus, reveal a novel mechanism of tumorigenesis in patients with germline or somatic mutations affecting PTEN ATP-binding motifs, i.e. qualitative and quantitative impairment of PTEN due to the loss of its phosphatase activity, and nuclear mislocalization, resulting in rapid PTEN protein degradation, suppression of p53-mediated transcriptional activity, loss of protection against oxidative stress as well as accumulation of spontaneous DNA DSBs.

Topics: Adenosine Triphosphate; Binding Sites; Breast Neoplasms; Cell Line, Tumor; Cell Nucleus; Cyclin D1; DNA Breaks, Double-Stranded; DNA Damage; Female; Gene Expression Regulation, Neoplastic; Germ-Line Mutation; Hamartoma Syndrome, Multiple; Humans; Mutation; Oxidative Stress; Protein Transport; PTEN Phosphohydrolase; Superoxide Dismutase; Tumor Suppressor Protein p53

Human estrogen receptor α (ERα) is a nuclear transcription factor that displays a major therapeutic target for breast cancer. The transcriptional activity of ERα can be regulated by particular estrogen receptor modulators. Celastrol, a quinine methide triterpene extracted from a Chinese medicine (Trypterygium wilfordii Hook F.), has been reported to have therapeutic efficacy against various cancer cells, including prostate cancer, leukemia, and melanoma cells. However, ERα regulation by Celastrol has not been reported. In this study, we investigated the effects of Celastrol on the growth of breast cancer cells. We observed that Celastrol decreased expression of ERα at both the mRNA and the protein levels in MCF7 and T47D human breast cancer cells. Results from a luciferase assay showed that Celastrol decreased the transcriptional activity of ERα. Also, Celastrol treatment inhibited ERα target gene expression, including expressions of cyclin D(1), progesterone receptor (PR), and c-Myb leading to cell cycle arrest and growth inhibition of breast cancer cells. We propose that Celastrol, an anti-cancer drug extracted from natural sources, induces inhibition of cell growth through modulation of ERα in estrogen positive breast cancer cells and is a candidate for use in cancer chemotherapy for human breast cancer.

Topics: Antineoplastic Agents, Phytogenic; Breast Neoplasms; Cell Line, Tumor; Cell Nucleus; Cyclin D1; Cytoplasm; Estrogen Receptor alpha; Female; Humans; Pentacyclic Triterpenes; Proto-Oncogene Proteins c-myb; Receptors, Estrogen; RNA, Messenger; Triterpenes

UbcH5c, a member of the UbcH5 family of protein ubiquitin conjugase E2 enzymes, is a critical component of biological processes in human cells, being the initial ubiquitinating enzyme of substrates like IκB, TP53, and cyclin D1. We report here that the metastasis regulator protein SLUG inhibits the expression of UbcH5c directly through chromatin remodeling and thus, among other downstream effects, elevates the level of cyclin D1, thus enhancing the growth rates of breast cancer cells. Overexpression of SLUG in the SLUG-deficient breast cancer cells significantly decreased the levels of mRNA and protein of UbcH5c but only elevated the protein levels of cyclin D1. On the contrary, knockdown of SLUG in SLUG-high breast cancer cells elevated the levels of UbcH5c while decreasing the level of cyclin D1 protein. SLUG is recruited at the E2-box sequence at the UbcH5c gene promoter along with the corepressor CtBP1 and the effector HDAC1 to silence the expression of this gene. Knockdown of UbcH5c in the SLUG-deficient human breast cells elevated the level of cyclin D1 as well as the rates of proliferation and invasiveness of these cells. Whereas the growth rates of the cells are enhanced due to overexpression of SLUG or knockdown of UbcH5c in the breast cancer cells tested, ER(+) cells also acquire resistance to the anti-estrogen 4-hydroxytamoxifen due to the rise of cyclin D1 levels in these cells. This study thus implicates high levels of SLUG and low levels of UbcH5c as a determinant in the progression of metastatic breast cancer.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Chromatin Assembly and Disassembly; Cyclin D1; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Neoplasm Invasiveness; Promoter Regions, Genetic; Proteasome Endopeptidase Complex; Snail Family Transcription Factors; Tamoxifen; Transcription Factors; Ubiquitin-Conjugating Enzymes; Ubiquitination

The purpose of this study was to evaluate the importance of biological markers to predict pathologic complete response (pCR) to neoadjuvant chemotherapy (NCT) in patients with locally advanced triple-negative breast cancers (TNBCs). Forty-one patients (18.6%) among 220 breast cancer patients were identified as TNBCs from March 2006 to 2009 were included in this prospective study. The pre-NCT treatment expression levels of Ki-67 proliferation index, estrogen receptor (ER), progesterone receptor (PgR), epidermal growth factor receptor 2 (HER-2), CK5/6, epidermal growth factor receptor (EGFR), cyclin D1, and nm23-H1 were detected by immunohistochemistry (IHC). A total of 180 cycles were administered with the median number of four cycles per patient (range, 4-6). The pCR rate was 34.1% (95% CI, 19.6-48.6%). In univariate analysis, early T stage, clinical response after 2 cycles, negative basal-like, negative EGFR, high Ki-67 proliferation index, and positive nm23-H1 were found to be significantly predictive of a pCR (P = 0.010, 0.040, 0.007, 0.001, 0.019, and 0.010, respectively). Basal-like status and nm23-H1 status were significant for pCR on multivariate analysis (P = 0.004 and 0.031, respectively). Basal-like status and nm23-H1 are independent predictive factors of pCR to neoadjuvant docetaxel plus epirubicin combination chemotherapy in patients with TNBCs.

Topics: Adult; Aged; Biomarkers, Tumor; Breast Neoplasms; Cyclin D1; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Keratin-5; Keratin-6; Ki-67 Antigen; Middle Aged; Neoadjuvant Therapy; NM23 Nucleoside Diphosphate Kinases; Predictive Value of Tests; Prospective Studies; Treatment Outcome

Obesity is a risk factor for breast cancer, and low blood concentrations of adiponectin are associated with high incidence and poor prognosis of breast cancer. However, the molecular mechanisms underlying such inhibitory effects of adiponectin remain to be defined. By using MCF7 cells, we investigated the mechanisms underlying the inhibitory effects of adiponectin on breast cancer cells, particularly in the context of opposing IGF-1-induced proliferation. We found that adiponectin, at 20 and 40 μg/ml, reduced MCF7 cell growth in the absence and presence of IGF-1. These inhibitory effects were primarily the result of the significant increase of cells at G(1)/G(0) phase and concomitant decrease of cells at S phase. In addition, the percentage of apoptotic cells increased more than two-fold. Within 30-min of adiponectin addition, the phosphorylation of AMPKα was sharply elevated, and the level of IGF-1-activated Akt was decreased. Prolonged exposure to adiponectin resulted in reduction of cyclin D1 and cyclin E2 expression. Adiponectin also increased intracellular levels of cAMP and the activity of protein kinase-A (PKA). The inhibitors of PKA completely abolished the adiponectin's effects on cell growth. In conclusion, our studies revealed an important cellular mechanism underlying the relationship between reduced adiponectin concentrations and breast cancer development.

Topics: Adiponectin; Apoptosis; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclin D1; Cyclins; Female; Humans; Receptors, Adiponectin; Signal Transduction

Molecular effects of obesity, a well-established risk factor for breast cancer progression, are mediated by adipocytokine leptin. Given the important role of leptin in breast cancer growth and metastasis, novel strategies to antagonize biological effects of this adipocytokine are much desired. We showed previously that benzyl isothiocyanate (BITC), a constituent of edible cruciferous vegetables (e.g. garden cress), confers significant protection against mammary carcinogenesis in a transgenic mouse model. The present study provides first evidence for the efficacy of BITC against oncogenic effects of leptin. The BITC treatment circumvented leptin-induced clonogenicity and anchorage-independent growth of MDA-MB-231 and MCF-7 human breast cancer cells. Leptin-stimulated migration and invasion of these cells was also inhibited in the presence of BITC. Analysis of the underlying molecular mechanisms revealed that BITC treatment suppressed leptin-induced Stat3 phosphorylation and cyclin D1 transactivation. The BITC-mediated inhibition of MDA-MB-231 xenograft growth correlated with a modest yet significant decrease in levels of Tyr705 phosphorylated Stat3. The BITC treatment efficiently inhibited Stat3 and SRC1 recruitment to cyclin D1 promoter in a chromatin immunoprecipitation analysis. Furthermore, overexpression of constitutively active Stat3 imparted significant protection against BITC-mediated inhibition of cyclin D1 transactivation, whereas RNA interference of Stat3 resulted in a significant increase in BITC-mediated inhibition of cyclin D1 transactivation in the presence of leptin. These results indicate that Stat3 plays an important role in BITC-mediated inhibition of leptin-induced cyclin D1 transactivation. In conclusion, BITC could potentially be a rational therapeutic strategy for breast carcinoma in obese patients with high leptin levels.

Topics: Apoptosis; Blotting, Western; Breast Neoplasms; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Transformation, Neoplastic; Chromatin Immunoprecipitation; Cyclin D1; Humans; Immunoenzyme Techniques; Isothiocyanates; Leptin; Luciferases; Oncogenes; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; STAT3 Transcription Factor; Wound Healing

We previously reported that CG0006, a novel hydroxamate-based pan-histone deacetylase inhibitor (HDACI), suppresses the growth of human cancer cells. Here, we tested the ability of CG0006 to inhibit breast cancer cell proliferation in relation to estrogen receptor (ER) status, and examined changes in the expression of cell-cycle regulatory proteins. CG0006 effects on the proliferation of multiple human cancer cell lines were tested using MTT and MTS assays. Changes in estrogen-signaling proteins and cell-cycle regulatory proteins were examined by western blotting and quantitative RT-PCR, and cell-cycle effects were tested using flow cytometry. CG0006 increased histone H3 and H4 acetylation, up-regulated p21 protein, and promoted cell-cycle arrest, inducing G(2)/M-phase accumulation in ER-positive MCF7 cells, and G(1)- and G(2)/M-phase accumulation in ER-negative MDA-MB-231 cells. In both cell types, CG0006 treatment (1 μM) reduced the levels of the estrogen-signaling proteins ERα and cyclin D1, and promoted massive degradation of cell-cycle regulatory proteins. CG0006 down-regulated the histone deacetylase HDAC6 at the protein level in association with a subsequent increase in Hsp90 and α-tubulin acetylation. HDAC6 depletion using small interfering RNA produced a protein-degradation phenotype similar to that of CG0006 treatment. These findings suggest that CG0006 inhibits breast cancer cell growth by two different pathways: a histone acetylation-dependent pathway, and a non-epigenetic pathway that disrupts chaperone function.

Topics: Acetylation; Antineoplastic Agents, Hormonal; Apoptosis; Breast Neoplasms; Caspase 9; Cell Cycle Proteins; Cell Line, Tumor; Cell Survival; Cyclin D1; Enzyme Activation; Estrogen Receptor alpha; Female; Gene Expression; Gene Knockdown Techniques; Histone Deacetylase 6; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; HSP90 Heat-Shock Proteins; Humans; Hydroxamic Acids; Mitogen-Activated Protein Kinases; Phosphorylation; Piperidines; Protein Biosynthesis; Proteolysis; Receptor, ErbB-2; RNA Interference; Signal Transduction

ICI182,780 is used in adjuvant therapies of breast cancer. As a complete estrogen receptor (ER) blocker, ICI182,780 may antagonize the effects of estrogen on the cardiovascular system. Estrogen inhibits the proliferation of vascular smooth muscle cells (VSMCs), which is one of the mechanisms that estrogen can exert cardioprotective effects. In the present study, to assess the effects of ICI182,780 on the proliferation of VSMCs, we cultured VSMCs isolated from rat aorta with or without the ER antagonist ICI182,780. The results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, bromodeoxyuridine incorporation assay, viable cell count, immunochemical staining for proliferating cell nuclear antigen (PCNA), and S-phase ratio determined by flow cytometry revealed a remarkable proliferation of VSMCs after ICI182,780 treatment. ICI182,780 significantly enhanced cell growth in a dose-dependent manner (10(-8)-10(-5) M). Furthermore, the number of PCNA-positive cells and the S-phase progression of VSMCs increased after treatment with ICI182,780. Reverse transcriptase-polymerase chain reaction (RT-PCR) and western blot analysis showed that the mRNA and protein level of cyclin D1 in VSMCs increased under the treatment of ICI182,780. These data suggested that ICI182,780 can promote the growth of VSMCs, which might produce some adverse effects on the cardiovascular system.

Topics: Animals; Breast Neoplasms; Cell Division; Cell Line; Cyclin D1; Dose-Response Relationship, Drug; Estradiol; Estrogen Antagonists; Female; Fulvestrant; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Proliferating Cell Nuclear Antigen; Rats; Rats, Wistar; Receptors, Estrogen; S Phase

EMSY is a putative oncogene amplified in a minority of breast carcinomas, its protein product interacts with and transcriptionally silences BRCA2. We hypothesized that breast tumors from BRCA2 mutation carriers would be less likely than other familial breast cancers to exhibit EMSY amplification. As EMSY is located on 11q13 in proximity to CCND1, an established breast cancer oncogene, we also examined the amplification of CCND1 in the same tumor cohort. Amplification of EMSY and CCND1 were examined in 58 BRCA1-associated, 64 BRCA2-associated, and 242 familial non-BRCA1/BRCA2 breast cancers using fluorescent in situ hybridization (FISH). All tumors had a centralized pathology review and underwent molecular phenotyping by immunohistochemical profiling on tissue microarrays (TMAs). Tumors with amplification of EMSY and/or CCND1 were compared with non-amplified tumors for morphological appearance, molecular subtype, and overall survival. EMSY amplification was detected in 8% of BRCA1-associated, 0% of BRCA2-associated, and 9% of familial non-BRCA1/BRCA2 breast tumors (P = 0.036). CCND1 was amplified in 4% of BRCA1-associated, 13% of BRCA2-associated and 21% of non-BRCA1/BRCA2 breast tumors (P = 0.054). EMSY was amplified independently of CCND1 in 38% of cases. EMSY amplification was associated with increased tumor stage only; whereas CCND1 amplification was associated with high tumor grade, ER positivity, and inversely associated with the basal-like phenotype. There was a trend toward worse overall survival in ER-positive non-BRCA1/BRCA2 familial breast cancer patients whose tumors exhibited EMSY and CCND1 co-amplification. BRCA2-associated breast tumors are less likely than non-BRCA1/BRCA2 familial breast cancers to exhibit EMSY amplification. BRCA1-associated breast cancers are less likely than non-BRCA1/BRCA2 familial breast cancers to exhibit CCND1 amplification. EMSY amplification does occur independently of CCND1 amplification in a minority of familial breast cancers, supporting its role as a possible breast cancer oncogene.

Topics: Biomarkers, Tumor; BRCA1 Protein; BRCA2 Protein; Breast Neoplasms; Canada; Cyclin D1; Female; Gene Amplification; Gene Silencing; Genetic Predisposition to Disease; Humans; Neoplasm Proteins; Nuclear Proteins; Receptors, Estrogen; Repressor Proteins; Tissue Array Analysis

To investigate expression, regulation, potential role and targets of miR-195 and miR-497 in breast cancer.. The expression patterns of miR-195 and miR-497 were initially examined in breast cancer tissues and cell lines by Northern blotting and quantitative real-time PCR. Combined bisulfite restriction analysis and bisulfite sequencing were carried out to study the DNA methylation status of miR-195 and miR-497 genes. Breast cancer cells stably expressing miR-195 and miR-497 were established to study their role and targets. Finally, normal, fibroadenoma and breast cancer tissues were employed to analyze the correlation between miR-195/497 levels and malignant stages of breast tumor tissues.. MiR-195 and miR-497 were significantly downregulated in breast cancer. The methylation state of CpG islands upstream of the miR-195/497 gene was found to be responsible for the downregulation of both miRNAs. Forced expression of miR-195 or miR-497 suppressed breast cancer cell proliferation and invasion. Raf-1 and Ccnd1 were identified as novel direct targets of miR-195 and miR-497. miR-195/497 expression levels in clinical specimens were found to be correlated inversely with malignancy of breast cancer.. Our data imply that both miR-195 and miR-497 play important inhibitory roles in breast cancer malignancy and may be the potential therapeutic and diagnostic targets.

Topics: 3' Untranslated Regions; Base Sequence; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; CpG Islands; Cyclin D1; DNA Methylation; Down-Regulation; Female; Gene Silencing; Genes, Reporter; Humans; Luciferases, Renilla; MicroRNAs; Neoplasm Invasiveness; Proto-Oncogene Proteins c-raf; Restriction Mapping

The majority of estrogen receptor (ER)-positive breast cancers are treated with endocrine therapy. While this is effective, acquired resistance to therapies targeted against ER is a major clinical challenge. Here, model systems of ER-positive breast cancers with differential susceptibility to endocrine therapy were employed to define common nodes for new therapeutic interventions. These analyses revealed that cell cycle progression is effectively uncoupled from the activity and functional state of ER in these models. In this context, cyclin D1 expression and retinoblastoma tumor suppressor protein (RB) phosphorylation are maintained even with efficient ablation of ER with pure antagonists. These therapy-resistant models recapitulate a key feature of deregulated RB/E2F transcriptional control. Correspondingly, a gene expression signature of RB-dysfunction is associated with luminal B breast cancer, which exhibits a relatively poor response to endocrine therapy. These collective findings suggest that suppression of cyclin D-supported kinase activity and restoration of RB-mediated transcriptional repression could represent a viable therapeutic option in tumors that fail to respond to hormone-based therapies. Consistent with this hypothesis, a highly selective CDK4/6 inhibitor, PD-0332991, was effective at suppressing the proliferation of all hormone refractory models analyzed. Importantly, PD-0332991 led to a stable cell cycle arrest that was fundamentally distinct from those elicited by ER antagonists, and was capable of inducing aspects of cellular senescence in hormone therapy refractory cell populations. These findings underscore the clinical utility of downstream cytostatic therapies in treating tumors that have experienced failure of endocrine therapy.

Topics: Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cyclin D1; Female; Gene Expression Profiling; Humans; Molecular Targeted Therapy; Neoplasms, Hormone-Dependent; Piperazines; Protein Kinase Inhibitors; Pyridines; Receptors, Estrogen; Retinoblastoma Protein; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Zeranol (Z) is a non-steroidal anabolic growth promoter with potent estrogenic activity that is widely used as a growth promoter in the US beef industry. Consumption of beef derived from zeranol-implanted cattle may be a risk factor for breast cancer.. The effect of serum on the proliferation of human breast cancer MCF-7 cell line and primary cultured human breast epithelial cells (PCHBECs) was investigated. ACI rats were implanted with 12 mg zeranol pellet and the serum was harvested at day 110 after implantation. The effect of zeranol-serum on mRNA expression of cell cycle regulating gene (cyclin D1) and tumor suppressor genes (p53, and p21) was evaluated using real-time RT-PCR.. The serum derived from ACI rats 110 days post-zeranol implantation significantly promoted the proliferation of MCF-7 cells and primary cultured human breast epithelial cells compared to control serum. Zeranol-serum up-regulated cyclin D1 and down-regulated p53 and p21 expression in PCHBECs compared with control serum.. Bio-active zeranol metabolites contained in meat produced from cattle after zeranol implantation may be a risk factor for breast cancer.

Topics: Animals; Breast Neoplasms; Cell Growth Processes; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Estrogens, Non-Steroidal; Female; Gene Expression Regulation, Neoplastic; Humans; Rats; Rats, Inbred ACI; RNA, Messenger; Serum; Tumor Suppressor Protein p53; Zeranol

Melatonin suppresses breast cancer cell proliferation by inhibiting the upregulation of estrogen-induced cyclin D1 via its G-protein-coupled receptor MT1. Additionally, melatonin stimulates the expression of the estrogen sulfotransferase, SULT1E1. However, metabolism of melatonin via 6-hydroxylation by CYP1A1/1A2 and subsequent sulfonation by SULT1A1/1A3 decreases its intracellular concentration. This could have a negative impact on its oncostatic action in breast cancer.. In this pilot study, we performed immunohistochemical (IHC) analysis of MT1 and cyclin D1 in breast cancer specimens from 33 patients. Also, we investigated the expression of CYP1A1/1A2, SULT1A1/1A3/1E1,and cyclin D1 in cancer (CANC) and adjacent non-cancer (NCANC) specimens from 10 representative breast cancer patients using quantitative real-time reverse transcription polymerase chain reaction.. CYP1A1-mRNA-expression was found only in three CANC and in one NCANC. CYP1A2 mRNA was below the detection limit in all patients. SULT1A1 was observed only in two of the 10 CANC and one of the 10 NCANC specimens. But, all 10 CANC and NCANC samples showed high SULT1A3 levels. Cyclin D1 mRNA levels were found in all 10 CANC and NCANC specimens. Furthermore, IHC-staining of cyclin D1 was observed in 27 of 33 CANC and correlated positively with estrogen receptor positivity (p = 0.015).. The low or even absent expression of CYP1A1 or CYP1A2 in breast cancer specimens suggested that melatonin might be involved in cell cycle arrest.

Topics: Biotransformation; Breast Neoplasms; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Melatonin; Neoplasm Invasiveness; Neoplasm Proteins; Receptor, Melatonin, MT1; RNA, Messenger

We have previously reported that induction of epidermal growth factor receptor and ErbB2 in response to antihormonal agents may provide an early mechanism to allow breast cancer cells to evade the growth-inhibitory action of such therapies and ultimately drive resistant cell growth. More recently, the other two members of the ErbB receptor family, ErbB3 and ErbB4, have been implicated in antihormone resistance in breast cancer. In the present study, we have investigated whether induction of ErbB3 and/or ErbB4 may provide an alternative resistance mechanism to antihormonal action in a panel of four oestrogen receptor (ER)-positive breast cancer cell lines.. MCF-7, T47D, BT474 and MDAMB361 cell lines were exposed to fulvestrant (100 nM) for seven days, and effects on ErbB3/4 expression and signalling, as well as on cell growth, were assessed. Effects of heregulin β1 (HRGβ1) were also examined in the absence and presence of fulvestrant to determine the impact of ER blockade on the capacity of this ErbB3/4 ligand to promote signalling and cell proliferation.. Fulvestrant potently reduced ER expression and transcriptional activity and significantly inhibited growth in MCF-7, T47D, BT474 and MDAMB361 cells. However, alongside this inhibitory activity, fulvestrant also consistently induced protein expression and activity of ErbB3 in MCF-7 and T47D cells and ErbB4 in BT474 and MDAMB361 cell lines. Consequently, fulvestrant treatment sensitised all cell lines to the actions of the ErbB3/4 ligand HRGβ1 with enhanced ErbB3/4-driven signalling activity, reexpression of cyclin D1 and significant increases in cell proliferation being observed when compared to untreated cells. Indeed, in T47D and MDAMB361 HRGβ1 was converted from a ligand having negligible or suppressive growth activity into one that potently promoted cell proliferation. Consequently, fulvestrant-mediated growth inhibition was completely overridden by HRGβ1 in all four cell lines.. These findings suggest that although antihormones such as fulvestrant may have potent acute growth-inhibitory activity in ER-positive breast cancer cells, their ability to induce and sensitise cells to growth factors may serve to reduce and ultimately limit their inhibitory activity.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Drug Resistance, Neoplasm; ErbB Receptors; Estradiol; Estrogen Receptor Modulators; Female; Fulvestrant; Humans; Intracellular Signaling Peptides and Proteins; Neuregulin-1; Receptor, ErbB-3; Receptor, ErbB-4; Receptors, Estrogen; Signal Transduction

To investigate the correlation between CCND1 amplification and CHK1 deletion in breast cancer, and to explore their role in tumorigenesis and progression, a comparative study of the gene copy number changes of CCND1 and CHK1 was performed with dual-colour fluorescence in-situ hybridization (FISH).. Sixty-one infiltrating ductal breast carcinomas with foci of ductal carcinoma in situ (DCIS) components were selected for dual-colour FISH. A strong correlation was found between CCND1 amplification and CHK1 deletion (P<0.0001). Fourteen cases were detected that demonstrated both CCND1 amplification and CHK1 deletion. Interestingly, when comparing the infiltrating and non-invasive areas for the same tumour, we found three cases with CCND1 amplification in the infiltrating areas but not in the DCIS areas. We did not find a CHK1 gene profile difference between infiltrating and DCIS areas in the same lesions.. Our findings suggest that CCND1 amplification and CHK1 deletion are common events in breast cancer, and that the two genetic alterations often coexist. Our data also suggest that CHK1 deletion is an early genetic event in the development of breast cancer and can be detected at the DCIS stage, whereas CCND1 amplification is more likely to be associated with tumour progression.

Topics: Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Intraductal, Noninfiltrating; Cell Transformation, Neoplastic; Checkpoint Kinase 1; Cyclin D1; Disease Progression; Female; Gene Amplification; Gene Deletion; Gene Dosage; Humans; Immunohistochemistry; In Situ Hybridization, Fluorescence; Protein Kinases

To examine the histopathological features of 24 surgically resected carcinoma in situ (CIS) involving sclerosing adenosis (SA), with special reference to the topographical relationship between CIS and SA.. In 13 (54%) lesions, CIS was entirely surrounded by SA (type A) and in 11 (46%), CIS involved SA at least focally but was not confined to the SA area (type B). The mean size of CIS in type B (30.45 mm) was significantly larger than in type A (18.00 mm). The mean size of SA in type A (39.46 mm) was significantly larger than in type B (19.54 mm). Most type A CIS were non-high-grade, and the oestrogen receptor (ER)(+)/progesterone receptor (PgR)(+)/HER2(-) immunophenotype predominated. Most type B CIS were high-grade and six (54%) were ER(-)/PgR(-). Most type A were bcl-2(+)/p53(-) in both SA and CIS areas, but two (18%) apocrine ductal CIS of type B were bcl-2(-)/p53(+) in both SA and CIS areas. Expression of ER and cyclin D1 in SA was not different from that of SA unassociated with cancer.. Most CIS involving SA arises within SA and high-grade DCIS tends to grow beyond SA. Occasional CIS may arise outside SA and secondarily involve SA.

Topics: Adult; Aged; Breast Neoplasms; Calcium-Binding Proteins; Calponins; Carcinoma in Situ; Carcinoma, Intraductal, Noninfiltrating; Comorbidity; Cyclin D1; Female; Fibrocystic Breast Disease; Humans; Membrane Proteins; Microfilament Proteins; Middle Aged; Proto-Oncogene Proteins c-bcl-2; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone; Retrospective Studies; Sclerosis; Tumor Suppressor Protein p53

The G-protein coupled receptor associated protein β-arrestin-1 is crucial for the regulation of numerous biological processes involved in cancer progression, such as intracellular signaling and cell motility. The encoding gene ARRB1 is harbored in the same chromosomal region as the CCND1 gene (11q13). Amplification of CCND1, frequently encountered in breast cancer, often involves coamplification of additional oncogenes, as well as deletion of distal 11q genes. We investigated the clinical relevance of β-arrestin-1 in breast cancer and elucidated a potential link between β-arrestin-1 expression and CCND1 amplification. β-Arrestin-1 protein expression was evaluated in two breast cancer patient cohorts, comprising 179 patients (cohort I) and 500 patients randomized to either tamoxifen or no adjuvant treatment (cohort II). Additionally, migration after β-arrestin-1 overexpression or silencing was monitored in two breast cancer cell lines. Overexpression of β-arrestin-1 reduced the migratory propensity of both cell lines, whereas silencing increased migration. In cohort I, high expression of stromal β-arrestin-1 was linked to reduced patient survival, whereas in cohort II both high and absent stromal expression predicted a poor clinical outcome. Patients exhibiting low or moderate levels of stromal β-arrestin-1 did not benefit from tamoxifen, in contrast to patients exhibiting absent or high expression. Furthermore, CCND1 amplification was inversely correlated with tumor cell expression of β-arrestin-1, indicating ARRB1 gene deletion in CCND1-amplified breast cancers.

Topics: Adult; Aged; Aged, 80 and over; Arrestins; beta-Arrestin 1; beta-Arrestins; Biomarkers, Tumor; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Checkpoint Kinase 1; Cyclin D1; Female; Gene Amplification; Gene Expression Regulation, Neoplastic; Humans; Middle Aged; Prognosis; Protein Kinases; Stromal Cells; Survival Analysis; Tamoxifen; Treatment Outcome

HuR, a ubiquitously expressed member of the Hu family, selectively binds and stabilizes ARE-containing mRNAs encoding proto-oncogenes, cell cycle regulators, cytokines and growth factors. The role of HuR and its cellular function in breast cancer remains unclear. This study aimed to provide new insights into the implication of HuR in breast cancer. We show that MCF7 and MDA-MB-231 breast cancer cells stably transfected with a hammerhead ribozyme transgene specifically targeted to HuR (MCF7HuRKO and MDA-MB-231HuRKO) have reduced HuR expression both at mRNA and protein levels. This study reveals that HuR knockdown dramatically reduced cell growth in MCF7 cells (P<0.001) and invasive properties in MDA-MB-231 cells (P<0.001). Furthermore, we report that the decreased cell growth rate in MCF7 cells is seen together with a reduction in cyclin D1 transcript and protein levels and that the change in invasiveness in MDA-MB-231 cells seems to be linked with decreased MMP-9 levels. Our study shows that targeting HuR can influence breast cancer cell growth and invasion and suggests a role for HuR in vitro in enhancing breast cancer cell growth and invasion. These changes may be facilitated through changes in the levels of cyclin D1 and MMP-9.

Topics: Antigens, Surface; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; ELAV Proteins; ELAV-Like Protein 1; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Matrix Metalloproteinase 9; Neoplasm Invasiveness; RNA-Binding Proteins; RNA, Catalytic; Transgenes; Wound Healing

MTA1(metastasis associated-1) is a tumor metastasis associated candidate gene and overexpression in many human tumors, including breast cancer. In this study, we investigated depressive effect on MTA1 by MTA1-specific short hairpin RNA(shRNA) expression plasmids in human breast cancer cell lines MDA-MB-231 and MCF-7, and effect on protein levels of ER alpha, MMP-9, cyclinD1, and tumor cell invasion, proliferation.. ShRNA expression vectors targeting MTA1 was constructed and transfected into human breast cancer cell lines MDA-MB-231 and MCF-7. The transfection efficiency was evaluated by fluorescence microscopy, mRNA levels of MTA1 were detected by reverse transcription-polymerase chain reaction (RT-PCR), protein levels of ER alpha, MMP-9 and cyclinD1 were detected by Western blotting, respectively. Tumor cells invasive ability were evaluated by Boyden chamber assay, the cells proliferation were evaluated using cell growth curve and MTT analysis, the cell cycle analysis was performed using flow cytometry.. Down-regulation of MTA1 by RNAi approach led to re-expression of ER alpha in ER-negative breast cancer cell lines MDA-MB-231, and reduced protein levels of MMP-9 and CyclinD1, as well as decreased tumor cell invasion and proliferation, more cells were blocked in G0/G1 stage(P < 0.05). However, after inhibiting mRNA levels of MTA1, protein expression of ER alpha, MMP-9, cyclinD1 and the changes of cancer cells invasiveness, proliferation, cells cycle were no statistical difference in ER-positive human breast cancer cell lines MCF-7 (P > 0.05).. ShRNA targeted against MTA1 could specifically mediate the MTA1 gene silencing and consequentially recover the protein expression of ER alpha, resulting in increase sensitivity of antiestrogens, as well as suppress the protein levels of MMP-9 and cyclinD1 in ER-negative human breast cancer cell lines MDA-MB-231. Silencing effect of MTA1 could efficiently inhibit the invasion and proliferation in MDA-MB-231 cells. The shRNA interference targeted against MTA1 may have potential therapeutic utility in human breast cancer.

Topics: Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; Histone Deacetylases; Humans; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Repressor Proteins; RNA, Messenger; RNA, Small Interfering; Trans-Activators; Transfection

Triple negative breast cancer (TNBC) has increased recurrence and poor survival, despite a high response rate to neoadjuvant chemotherapy. The aim of this study was to determine whether current drug treatment(s) eliminates bulk of tumor cells, but it has a minimal effect on cancer stem cells (CSCs) leading to tumor recurrence. We studied the effects of PARP inhibitors (AZD2281 and BSI-201), paclitaxel, docetaxel, cisplatin and cisplatin plus TRAIL on CSCs derived from CRL-2335 and MDA-MB-468 TNBC cells in vitro. The in vitro data indicate that cisplatin plus TRIAL treatment was most effective in eliminating CSCs compared to PARP inhibitors, cisplatin, paclitaxel and docetaxel. Treatment with cisplatin plus TRAIL also inhibits Wnt-1 signaling and its downstream target, β-catenin, phospho β-catenin, cyclin D1, increased apoptosis, reduced proliferation and mammosphere formation. Inhibition of Wnt-1 by siRNA significantly reduced the ability of CSCs to form mammospheres compared to control. However, maximum effect was seen in cisplatin plus TRAIL-treated cells. Taken together the data suggest that cisplatin plus TRAIL treatment has the potential of providing a new strategy for improving the therapeutic outcome in TNBC patients.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; beta Catenin; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cisplatin; Cyclin D1; Drug Synergism; Female; Humans; Neoplasm Recurrence, Local; Neoplastic Stem Cells; RNA, Small Interfering; Signal Transduction; TNF-Related Apoptosis-Inducing Ligand; Wnt1 Protein

Bazedoxifene (BZA) is a third-generation selective estrogen receptor modulator (SERM) that has been approved for the prevention and treatment of postmenopausal osteoporosis. It has antitumor activity; however, its mechanism of action remains unclear. In the present study, we characterized the effects of BZA and several other SERMs on the proliferation of hormone-dependent MCF-7 and T47D breast cancer cells and hormone-independent MCF-7:5C and MCF-7:2A cells and examined its mechanism of action in these cells. We found that all of the SERMs inhibited the growth of MCF-7, T47D, and MCF-7:2A cells; however, only BZA and fulvestrant (FUL) inhibited the growth of hormone-independent MCF-7:5C cells. Cell cycle analysis revealed that BZA and FUL induced G(1) blockade in MCF-7:5C cells; however, BZA down-regulated cyclin D1, which was constitutively overexpressed in these cells, whereas FUL suppressed cyclin A. Further analysis revealed that small interfering RNA knockdown of cyclin D1 reduced the basal growth of MCF-7:5C cells, and it blocked the ability of BZA to induce G(1) arrest in these cells. BZA also down-regulated estrogen receptor-α (ERα) protein by increasing its degradation and suppressing cyclin D1 promoter activity in MCF-7:5C cells. Finally, molecular modeling studies demonstrated that BZA bound to ERα in an orientation similar to raloxifene; however, a number of residues adopted different conformations in the induced-fit docking poses compared with the experimental structure of ERα-raloxifene. Together, these findings indicate that BZA is distinct from other SERMs in its ability to inhibit hormone-independent breast cancer cell growth and to regulate ERα and cyclin D1 expression in resistant cells.

Topics: Binding Sites; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Crystallography, X-Ray; Cyclin D1; Down-Regulation; Estrogen Receptor alpha; Female; Gene Knockdown Techniques; Humans; Indoles; Luciferases, Renilla; Selective Estrogen Receptor Modulators

Understanding the molecular etiology of cancer and increasing the number of drugs and their targets are critical to cancer management. In our attempt to unravel novel breast-cancer associated proteins, we previously conducted protein expression profiling of the MCF10AT model, which comprises a series of isogenic cell lines that mimic different stages of breast cancer progression. NRD1 expression was found to increase during breast cancer progression. Here, we attempted to confirm the relevance of NRD1 in clinical breast cancer and understand the functional role and mechanism of NRD1 in breast cancer cells. Immunohistochemistry data show that NRD1 expression was elevated in ductal carcinoma in situ and invasive ductal carcinomas compared with normal tissues in 30% of the 26 matched cases studied. Examination of NRD1 expression in tissue microarray comprising >100 carcinomas and subsequent correlation with clinical data revealed that NRD1 expression was significantly associated with tumor size, grade, and nodal status (P < 0.05). Silencing of NRD1 reduced MCF10CA1h and MDA-MD-231 breast-cancer-cell proliferation and growth. Probing the oncogenic EGF signaling pathways revealed that NRD1 knock down did not affect overall downstream tyrosine phosphorylation cascades including AKT and MAPK activation. Instead, silencing of NRD1 resulted in a reduction of overall cyclin D1 expression, a reduction of EGF-induced increase in cyclin D1 expression and an increase in apoptotic cell population compared with control cells.

Topics: Apoptosis; Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Disease Progression; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Immunohistochemistry; Metalloproteases; Mitogen-Activated Protein Kinase Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Real-Time Polymerase Chain Reaction; RNA, Small Interfering; Signal Transduction; Tyrosine; Up-Regulation

Gene amplification and protein overexpression of erbB2 (Her2/neu) has been observed in approximately 20-30% of breast cancers. ErbB2-positive breast cancer is tend to be more aggressive than other types of breast cancer and therefore further investigation on the signaling pathways of erbB2 is needed for the therapeutic target for breast cancer treatment. Here we report that microRNA-205 (miR-205), a molecule also reported to be associated with breast cancer, is negatively regulated by erbB2 overexpression. Breast epithelial cells exogenously overexpressed with erbB2 decreased the expression of miR-205, whereas increased the expression of cyclin D1, cyclin E, cyclin-dependent kinase 2 (CDK2), cyclin-dependent kinase 4 (CDK4), and cyclin-dependent kinase 6 (CDK6). The decreased expression of miR-205 slightly increased by the transfection of erbB2 siRNA into the erbB2-overexpressing breast cancer epithelial cells. Overexpression of erbB2 enabled breast epithelial cells to grow anchorage-independently in soft agar, and the transfection of the precursor of miR-205 into the cells leaded to the decrease in the ability to grow in soft agar. These results suggest that down-regulation of miR-205 in erbB2-overexpressing breast epithelial cells is essential for erbB2-induced tumorigenesis, and miR-205 may have the potential to be a novel important alternative therapeutic target for erbB2-positive breast cancer.

Topics: Breast Neoplasms; Cell Cycle Proteins; Cell Line; Cell Transformation, Neoplastic; Cyclin D1; Female; Humans; MicroRNAs; Receptor, ErbB-2

The use of celecoxib is associated with a significant decrease in breast cancer risk. However, the long-term use of high-dose celecoxib might be limited owing to cardiovascular side effects. In this study, we found that acetylbritannilactone (ABL), extract from a Chinese medicinal herb, could reduce celecoxib dose and potentiate the growth-inhibitory effect in breast cancer cells. ABL enhanced the apoptotic effect of celecoxib in COX-2-expressing cells, but had little effect in COX-2-negative cells. The apoptosis induced by the combination treatment disappeared when COX-2 was knocked down, whereas the lack of apoptotic effects in COX-2-negative cells was reversed after COX-2 transfection. However, the combination treatment induced a G(0)/G(1) phase arrest independent of whether or not the cells expressed COX-2. The G(0)/G(1) arrest was attributed to a decreased expression of cyclinD1, cyclinE, CDK2 and CDK6, especially the upregulation of p21. In addition, inhibition of Akt and p38 signaling pathways was required by the synergism, as the constitutively active Akt and p38 protected cells against apoptosis and cell cycle arrest induced by the combination treatment. In vivo, administration of celecoxib and ABL were more effective than the individual agents against xenograft tumor growth. Thus, our data suggested that the combinatorial approach of celecoxib and ABL might be helpful for breast cancer treatment.

Topics: Animals; Apoptosis; Breast Neoplasms; Celecoxib; Cell Line, Tumor; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinase Inhibitor p21; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Drugs, Chinese Herbal; Female; G1 Phase; Humans; Lactones; Mice; Mice, Nude; p38 Mitogen-Activated Protein Kinases; Proto-Oncogene Proteins c-akt; Pyrazoles; Resting Phase, Cell Cycle; Signal Transduction; Sulfonamides; Transplantation, Heterologous

Multidrug resistance is the most predominant phenomenon leading to chemotherapy treatment failure in breast cancer patients. Despite many studies having suggested that overexpression of epidermal growth factor receptor (EGFR) is a potent predictor of malignancy in cancers, systematic research of EGFR in multidrug resistant (MDR) breast cancer cells is lacking. In order to clarify the role of EGFR in MDR breast cancer cells, MCF7/Adr expressing relatively higher EGFR, and its parental cell line MCF7 expressing relatively lower EGFR, were chosen for this study. Knockdown of EGFR by siRNA in MCF7/Adr cells showed that EGFR siRNA inhibits cell migration, invasion and proliferation in vitro; converse effects were observed in MCF7 cells transfected with pcDNA3.0-EGFR plasmid. Moreover, we found that EGFR upregulated migration and invasion via EMMPRIN, MMP2 and MMP9 in addition to promoting cell cycle passage via elevation of cyclin D1 and CDK4 in MDR breast cancer cells. Interestingly, MCF7/Adr cells not expressing EGFR showed significant decrease of P-glycoprotein (P-gp) and ABCG2 expression levels, and became more sensitive to treatment of adriamycin (ADR) and paclitaxel (Taxol); the above results indicated that MDR of cancer cells is related to S-phase arrest. In conclusion, EGFR is an important factor enhancing the malignancy of MDR breast cancer cells, partially, inducing MDR. Anti-EGFR therapy may improve outcome in chemorefractory breast cancer patients.

Topics: Basigin; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Drug Resistance, Multiple; Drug Resistance, Neoplasm; ErbB Receptors; Female; Humans; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Phenotype; RNA, Small Interfering

Current clinical strategies for treating hormonal breast cancer involve the use of anti-estrogens that block estrogen receptor (ER)α functions and aromatase inhibitors that decrease local and systemic estrogen production. Both of these strategies improve outcomes for ERα-positive breast cancer patients, however, development of therapy resistance remains a major clinical problem. Divergent molecular pathways have been described for this resistant phenotype and interestingly, the majority of downstream events in these resistance pathways converge upon the modulation of cell cycle regulatory proteins including aberrant activation of cyclin dependent kinase 2 (CDK2). In this study, we examined whether the CDK inhibitor roscovitine confers a tumor suppressive effect on therapy-resistant breast epithelial cells.. Using various in vitro and in vivo assays, we tested the effect of roscovitine on three hormonal therapy-resistant model cells: (a) MCF-7-TamR (acquired tamoxifen resistance model); (b) MCF-7-LTLTca (acquired letrozole resistance model); and (c) MCF-7-HER2 that exhibit tamoxifen resistance (ER-growth factor signaling cross talk model).. Hormonal therapy-resistant cells exhibited aberrant activation of the CDK2 pathway. Roscovitine at a dose of 20 μM significantly inhibited the cell proliferation rate and foci formation potential of all three therapy-resistant cells. The drug treatment substantially increased the proportion of cells in G2/M cell cycle phase with decreased CDK2 activity and promoted low cyclin D1 levels. Interestingly, roscovitine also preferentially down regulated the ERα isoform and ER-coregulators including AIB1 and PELP1. Results from xenograft studies further showed that roscovitine can attenuate growth of therapy-resistant tumors in vivo.. Roscovitine can reduce cell proliferation and survival of hormone therapy-resistant breast cancer cells. Our results support the emerging concept that inhibition of CDK2 activity has the potential to abrogate growth of hormonal therapy-resistant cells.

Topics: Animals; Antineoplastic Agents, Hormonal; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Co-Repressor Proteins; Cyclin D1; Cyclin-Dependent Kinase 2; Drug Resistance, Neoplasm; Estrogen Receptor alpha; Female; Humans; Mice; Mice, Nude; Nuclear Receptor Coactivator 3; Protein Kinase Inhibitors; Purines; Roscovitine; Transcription Factors; Xenograft Model Antitumor Assays

Tamoxifen resistance is common in estrogen receptor-α (ERα)-positive breast cancers. Pawpaw and soursop are anticancer annonaceous plants in complementary medicine. Thus, we studied the effects of annonacin, an annonaceous acetogenin, in breast cancer cells.. Cell growth and ERα-related pathways were studied. The effects of annonacin were tested in MCF-7 xenografts in nude mice.. In ERα-positive MCF-7 cells, annonacin (half-effective dose ED(50) = 0.31 μM) and 4-hydroxytamoxifen (ED(50) = 1.13 μM) decreased cell survival whereas annonacin (0.5-1 μM) increased cell death at 48 h. Annonacin and 4-hydroxytamoxifen were additive in inhibiting cell survival. Annonacin (0.1 μM) induced G(0)/G(1) growth arrest while increasing p21(WAF1) and p27(kip1) and decreasing cyclin D1 protein expression. Annonacin (0.1μM) decreased cyclin D1 protein expression more than 4-hydroxytamoxifen (1 μM). Annonacin (0.1 μM) increased apoptosis while decreasing Bcl-2 protein expression. The combination of annonacin (0.1 μM) and 4-hydroxytamoxifen (1 μM) decreased Bcl-2 protein expression and ERα transcriptional activity more than annonacin (0.1 μM) did alone. Annonacin, but not 4-hydroxytamoxifen, decreased ERα protein expression. Moreover, annonacin decreased phosphorylation of ERK1/2, JNK and STAT3. In nude mice, annonacin decreased MCF-7 xenograft tumor size at 7-22 days. Moreover, annonacin decreased ERα, cyclin D1 and Bcl-2 protein expression in the xenograft at 22 days.. Annonacin induced growth arrest and apoptosis in ERα-related pathways in MCF-7 cells. Annonacin and 4-hydroxytamoxifen were additive in inhibiting cell survival and ERα transcriptional activity. Moreover, annonacin attenuated MCF-7 xenograft tumor growth while inhibiting ERα, cyclin D1 and Bcl-2 protein expressions in nude mice.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Breast Neoplasms; Cell Cycle Checkpoints; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Dose-Response Relationship, Drug; Estrogen Antagonists; Estrogen Receptor alpha; Extracellular Signal-Regulated MAP Kinases; Female; Furans; Humans; JNK Mitogen-Activated Protein Kinases; Lactones; Mice; Mice, Inbred BALB C; Mice, Nude; Phosphorylation; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; STAT3 Transcription Factor; Tamoxifen; Time Factors; Transfection; Tumor Burden; Xenograft Model Antitumor Assays

Clioquinol, a metal-binding compound, has been shown to have anticancer activity in in vitro and in vivo model systems. This study investigated the effects of clioquinol on cyclin D1 gene expression in breast cancer cells. Treatment with clioquinol significantly reduced cyclin D1 protein levels in a concentration-dependent manner, effects being more pronounced in the presence of zinc. Clioquinol reduced cyclin D1 mRNA contents in cells that had been pre-treated with actinomycin D, indicating that this compound alters cyclin D1 mRNA stability, an event associated with post-transcriptional regulation. Using a cyclin D1 3'-UTR reporter construct (CCND1-3'-UTR), we confirmed that this 3'-UTR mediates the inhibitory action of clioquinol, likely through miR-302C. This study demonstrates for the first time that clioquinol targets post-transcriptional steps of cyclin D1 gene expression in cancer cells, adding new insight into our understanding of its mechanisms of anticancer action.

Topics: 3' Untranslated Regions; Antineoplastic Agents; Base Sequence; Breast Neoplasms; Cell Line, Tumor; Clioquinol; Cyclin D1; Female; Gene Expression; Humans; Molecular Sequence Data; Promoter Regions, Genetic; RNA Processing, Post-Transcriptional; RNA, Messenger; Transcription, Genetic

Formononetin is one of the main components of red clover plants, and is considered as a typical phytoestrogen. This study further investigated that formononetin inactivated IGF1/IGF1R-PI3K/Akt pathways and decreased cyclin D1 mRNA and protein expression in human breast cancer cells in vitro and in vivo. MCF-7 cells were treated with different concentrations of formononetin. The proliferation of the cells treated with formononetin was tested by MTT assay. The cell cycle in the treated cells was examined by flow cytometry. The levels of p-IGF-1 R, p-Akt, and cyclin D1 protein expression and cyclin D1 mRNA expression in the treated cells were determined by Western blot and RT-PCR, respectively. In addition, the antitumor activity of formononetin was evaluated in nude mice bearing orthotopic tumor implants. Compared with the control, formononetin inhibited the proliferation of MCF-7 cells and effectively induced cell cycle arrest. The levels of p-IGF-1 R, p-Akt, cyclin D1 protein expression, and cyclin D1 mRNA expression were also downregulated. On the other hand, formononetin also prevented the tumor growth of human breast cancer cells in nude mouse xenografts. These results show that formononetin causes cell cycle arrest at the G0/G1 phase by inactivating IGF1/IGF1R-PI3K/Akt pathways and decreasing cyclin D1 mRNA and protein expression, indicating the use of formononetin in the prevention of breast cancer carcinogenesis.

Topics: Animals; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Insulin-Like Growth Factor I; Isoflavones; Mice; Mice, Inbred BALB C; Mice, Nude; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Receptor, IGF Type 1; Signal Transduction; Tamoxifen

Drug resistance remains to be a big challenge in applying anti-HER2 monoclonal antibody Trastuzumab for treating breast cancer with HER2 overexpression. Amplification of insulin-like growth factor I receptor (IGF-IR) and deletion of tumor suppressor phosphatase and tensin homolog (PTEN) are implicated in Trastuzumab resistance, however, the underlying mechanisms have not been clearly defined. Activation of Rac1, a member of Rho GTPase family, is capable of causing cytoskeleton reorganization, regulating gene expression and promoting cell proliferation. To investigate the mechanism of Trastuzumab resistance, PTEN knockdown and IGF-IR overexpressing stable cell lines were generated in HER2 overexpression human breast cancer SKBR3 cells. Rac1 was highly activated in PTEN deficient and IGF-IR overexpressing Trastuzumab-resistant cells in a HER2-independent manner. Inactivation of Rac1 by using a Rac1 inhibitor NSC23766 or siRNA knocking down the expression of Tiam1, a guanine nucleotide exchange factor for Rac, significantly reduced Trastuzumab resistance in SKBR3 cells. Inhibition of Rac1 had no effect on the levels of phosphor-HER2 and phosphor-Akt, but significantly decreased the levels of cyclin D1 in Trastuzumab-resistant cells. Inhibition of Akt with an Akt inhibitor also significantly reduced Trastuzumab resistance. However, simultaneous inhibition of both Rac1 and Akt resulted in a significantly more decrease of Trastuzumab resistance than inactivation of Rac1 or Akt alone. These results suggest that Rac1 activation is critically involved in Trastuzumab resistance caused by PTEN deletion or IGF-IR overexpression. Simultaneous inhibition of Rac1 and Akt may represent a promising strategy in reducing Trastuzumab resistance in HER2 overexpression breast cancer.

Topics: Aminoquinolines; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Drug Resistance, Neoplasm; Female; Guanine Nucleotide Exchange Factors; Humans; PTEN Phosphohydrolase; Pyrimidines; rac1 GTP-Binding Protein; Receptor, ErbB-2; Receptor, IGF Type 1; RNA Interference; T-Lymphoma Invasion and Metastasis-inducing Protein 1; Trastuzumab

Cyclin D1 is a well-characterised cell cycle regulator with established oncogenic capabilities. Despite these properties, studies report contrasting links to tumour aggressiveness. It has previously been shown that silencing cyclin D1 increases the migratory capacity of MDA-MB-231 breast cancer cells with concomitant increase in 'inhibitor of differentiation 1' (ID1) gene expression. Id1 is known to be associated with more invasive features of cancer and with the epithelial-mesenchymal transition (EMT). Here, we sought to determine if the increase in cell motility following cyclin D1 silencing was mediated by Id1 and enhanced EMT-features. To further substantiate these findings we aimed to delineate the link between CCND1, ID1 and EMT, as well as clinical properties in primary breast cancer.. Protein and gene expression of ID1, CCND1 and EMT markers were determined in MDA-MB-231 and ZR75 cells by western blot and qPCR. Cell migration and promoter occupancy were monitored by transwell and ChIP assays, respectively. Gene expression was analysed from publicly available datasets.. The increase in cell migration following cyclin D1 silencing in MDA-MB-231 cells was abolished by Id1 siRNA treatment and we observed cyclin D1 occupancy of the Id1 promoter region. Moreover, ID1 and SNAI2 gene expression was increased following cyclin D1 knock-down, an effect reversed with Id1 siRNA treatment. Similar migratory and SNAI2 increases were noted for the ER-positive ZR75-1 cell line, but in an Id1-independent manner. In a meta-analysis of 1107 breast cancer samples, CCND1low/ID1high tumours displayed increased expression of EMT markers and were associated with reduced recurrence free survival. Finally, a greater percentage of CCND1low/ID1high tumours were found in the EMT-like 'claudin-low' subtype of breast cancer than in other subtypes.. These results indicate that increased migration of MDA-MB-231 cells following cyclin D1 silencing can be mediated by Id1 and is linked to an increase in EMT markers. Moreover, we have confirmed a relationship between cyclin D1, Id1 and EMT in primary breast cancer, supporting our in vitro findings that low cyclin D1 expression can be linked to aggressive features in subgroups of breast cancer.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cluster Analysis; Cyclin D1; Epithelial-Mesenchymal Transition; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Inhibitor of Differentiation Protein 1; Prognosis; Recurrence; Risk; Transforming Growth Factor beta

MicroRNAs (miRs) function as tumor suppressors or oncogenes in multiple tumor types. Although miR expression is tightly regulated, the molecular basis of miR regulation is poorly understood. Here, we investigated the influence of the histone demethylase Jumonji/ARID1 B (JARID1B) on miR regulation in breast tumor cells. In MCF-7 cells with stable RNAi-mediated suppression of JARID1B expression we identified altered regulation of multiple miRs including let-7e, a member of the let-7 family of tumor suppressor miRs. Chromatin immunoprecipitation analysis demonstrated JARID1B binding to the let-7e promoter region as well as removal of the of H3K4me3 histone mark associated with active gene expression. These results suggest that JARID1B epigenetically represses let-7e expression. JARID1B stimulates tumor cell proliferation by promoting the G(1) to S transition. As predicted, suppression of JARID1B resulted in an accumulation of MCF-7 cells in G(1). We confirmed that cyclin D1, which also promotes G(1) progression, is a direct target of let-7e, and we show that cyclin D1 expression is suppressed in JARID1B knockdown cells. Cyclin D1 expression and cell cycle progression were restored following inhibition of let-7e, suggesting that JARID1B repression of let-7e contributes to cyclin D1 expression and JARID1B-mediated cell cycle progression. Our results indicate that the JARID1B demethylase contributes to tumor cell proliferation through the epigenetic repression of a tumor suppressor miR.

Topics: Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cyclin D1; Epigenesis, Genetic; Female; Gene Silencing; Histones; Humans; Jumonji Domain-Containing Histone Demethylases; Lysine; Methylation; MicroRNAs; Nuclear Proteins; Repressor Proteins

To investigate the efficacy of mitomycin C (MMC) in combination with curcumin in suppressing human breast cancer in vitro and in vivo.. Human breast cancer MCF-7 cells were used. Cell viability was measured using MTT assay. The cell cycle phase was detected with flow cytometric analysis. Cell cycle-associated proteins were examined using Western blot analysis. MCF-7 breast cancer xenografts were established to monitor tumor growth and cell cycle-associated protein expression.. Curcumin inhibited MCF-7 breast cancer cell viability in a concentration-dependent manner (IC(50) value=40 μmol/L). Similarly, MMC inhibited the cell viability with an IC(50) value of 5 μmol/L. Combined treatment of MMC and curcumin showed a synergistic antiproliferative effect. In the presence of curcumin (40 μmol/L), the IC(50) value of MMC was reduced to 5 μmol/L. In MCF-7 xenografts, combined administration of curcumin (100 mg/kg) and MMC (1-2 mg/kg) for 4 weeks produced significantly greater inhibition on tumor growth than either treatment alone. The combined treatment resulted in significantly greater G(1) arrest than MMC or curcumin alone. Moreover, the cell cycle arrest was associated with inhibition of cyclin D1, cyclin E, cyclin A, cyclin-dependent kinase 2 (CDK2) and CDK4, along with the induction of the cell cycle inhibitor p21 and p27 both in MCF-7 cells and in MCF-7 xenografts. These proteins were regulated through p38 MAPK pathway.. The results suggest that the combination of MMC and curcumin inhibits MCF-7 cell proliferation and cell cycle progression in vitro and in vivo via the p38 MAPK pathway.

Topics: Animals; Antineoplastic Agents; Breast; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Curcumin; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; Drug Synergism; Female; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Nude; Mitomycin

Psoriasin (S100A7) is expressed in several epithelial malignancies including breast cancer. Although S100A7 is associated with the worst prognosis in estrogen receptor α-negative (ERα(-)) invasive breast cancers, its role in ERα-positive (ERα(+)) breast cancers is relatively unknown. We investigated the significance of S100A7 in ERα(+) breast cancer cells and observed that S100A7 overexpression in ERα(+) breast cancer cells, MCF7 and T47D, exhibited decreased migration, proliferation, and wound healing. These results were confirmed in vivo in nude mouse model system. Mice injected with S100A7-overexpressing MCF7 cells showed significant reduction in tumor size compared with mice injected with vector control cells. Further mechanistic studies revealed that S100A7 mediates the tumor-suppressive effects via a coordinated regulation of the β-catenin/TCF4 pathway and an enhanced interaction of β-catenin and E-cadherin in S100A7-overexpressing ERα(+) breast cancer cells. We observed down-regulation of β-catenin, p-GSK3β, TCF4, cyclin D1, and c-myc in S100A7-overexpressing ERα(+) breast cancer cells. In addition, we observed increased expression of GSK3β. Treatment with GSK3β inhibitor CHIR 99021 increased the expression of β-catenin and its downstream target c-myc in S100A7-overexpressing cells. Tumors derived from mice injected with S100A7-overexpressing MCF7 cells also showed reduced activation of the β-catenin/TCF4 pathway. Therefore, our studies reveal for the first time that S100A7-overexpressing ERα(+) breast cancer cells exhibit tumor suppressor capabilities through down-modulation of the β-catenin/TCF4 pathway both in vitro and in vivo. Because S100A7 has been shown to enhance tumorigenicity in ERα(-) cells, our studies suggest that S100A7 may possess differential activities in ERα(+) compared with ERα(-) cells.

Topics: Animals; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; beta Catenin; Breast Neoplasms; Cyclin D1; Down-Regulation; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Mice; Mice, Nude; Neoplasm Transplantation; Proto-Oncogene Proteins c-myc; Pyridines; Pyrimidines; S100 Calcium Binding Protein A7; S100 Proteins; Transcription Factor 4; Transcription Factors; Transplantation, Heterologous; Tumor Suppressor Proteins

Cyclin D1 regulates cell proliferation and is a candidate molecular target for breast cancer therapy. This study addresses whether Cyclin D1 is indispensable for ErbB2-associated mammary tumor initiation and progression using a breast cancer model in which this cell-cycle regulator can be genetically ablated prior to or after neoplastic transformation. Deficiency in Cyclin D1 delayed tumor onset but did not prevent the occurrence of mammary cancer in mice overexpressing wild-type ErbB2. The lack of Cyclin D1 was associated with a compensatory upregulation of Cyclin D3, which explains why the targeted downregulation of Cyclin D1 in established mammary tumors had no effect on cancer cell proliferation. Cyclin D1 and D3 are overexpressed in human breast cancer cell lines and primary invasive breast cancers, and Cyclin D3 frequently exceeded the expression of Cyclin D1 in ErbB2-positive cases. The simultaneous inhibition of both cyclins in mammary tumor cells reduced cancer cell proliferation in vitro and decreased the tumor burden in vivo. Collectively, the results of this study suggest that only the combined inhibition of Cyclin D1 and D3 might be a suitable strategy for breast cancer prevention and therapy.

Topics: Animals; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cells, Cultured; Cyclin D1; Cyclin D3; Disease Progression; Female; Humans; Immunohistochemistry; Mammary Neoplasms, Animal; Mice; Mice, Knockout; Mice, Transgenic; NIH 3T3 Cells; Receptor, ErbB-2; RNA Interference

Estrogen is involved in several physiological and pathological processes through estrogen receptor (ER)-mediated transcriptional gene regulation. miRNAs (miRs), which are noncoding RNA genes, may respond to estrogen and serve as posttranscriptional regulators in tumorigenic progression, especially in breast cancer; however, only limited information about this possibility is available. In the present study, we identified the estrogen-regulated miR-34b and investigated its functional role in breast cancer progression.. Estrogen-regulated miRNAs were identified by using a TaqMan low density array. Our in vivo Tet-On system orthotopic model revealed the tumor-suppressive ability of miR-34b. Luciferase reporter assays and chromatin immunoprecipitation assay demonstrated miR-34b were regulated by p53-ER interaction.. In this study, we identified one such estrogen downregulated miRNA, miR-34b, as an oncosuppressor that targets cyclin D1 and Jagged-1 (JAG1) in an ER+/wild-type p53 breast cancer cell line (MCF-7), as well as in ovarian and endometrial cells, but not in ER-negative or mutant p53 breast cancer cell lines (T47D, MBA-MB-361 and MDA-MB-435). There is a negative association between ERα and miR-34b expression levels in ER+ breast cancer patients. Tet-On induction of miR-34b can cause inhibition of tumor growth and cell proliferation. Also, the overexpression of miR-34b inhibited ER+ breast tumor growth in an orthotopic mammary fat pad xenograft mouse model. Further validation indicated that estrogen's inhibition of miR-34b expression was mediated by interactions between ERα and p53, not by DNA methylation regulation. The xenoestrogens diethylstilbestrol and zeranol also showed similar estrogenic effects by inhibiting miR-34b expression and by restoring the protein levels of the miR-34b targets cyclin D1 and JAG1 in MCF-7 cells.. These findings reveal that miR-34b is an oncosuppressor miRNA requiring both ER+ and wild-type p53 phenotypes in breast cancer cells. These results improve our ability to develop new therapeutic strategies to target the complex estrogenic pathway in human breast cancer progression through miRNA regulation.

Topics: Adult; Aged; Animals; Antineoplastic Agents, Hormonal; Breast Neoplasms; Calcium-Binding Proteins; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Estrogens; Female; Gene Expression; Gene Expression Profiling; Genes, Tumor Suppressor; Humans; Intercellular Signaling Peptides and Proteins; Jagged-1 Protein; Membrane Proteins; Mice; Mice, Nude; Mice, SCID; MicroRNAs; Middle Aged; Models, Biological; Neoplasm Staging; Receptors, Estrogen; Serrate-Jagged Proteins; Tamoxifen; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

Topics: Aged; Antineoplastic Agents, Hormonal; Biomarkers, Tumor; Breast Neoplasms; Cyclin D1; Female; Humans; Middle Aged; Neoplasm Staging; Neoplasms, Hormone-Dependent; Postmenopause; Receptors, Estrogen; Receptors, Progesterone; Tamoxifen

About two thirds of breast cancers in women are hormone-dependent and require estrogen for growth, its effects being mainly mediated through estrogen receptor alpha (ERalpha). Docosahexaenoic acid (DHA, 22:6n-3) and arachidonic acid (AA, 20:4n-6) have opposite effects on carcinogenesis, with DHA suppressing and AA promoting tumor growth both in vitro and in vivo. However, the mechanism is not clear. Here, we examined whether the effect is mediated through changes in ERalpha distribution. MCF-7 cells, an ERalpha-positive human breast cancer cell line, was cultured in estrogen-free medium containing 0, 10 or 60 microM DHA or AA, then were stimulated with estradiol. DHA supplementation resulted in down-regulation of ERalpha expression (particularly in the extranuclear fraction), a reduction in phosphorylated MAPK, a decrease in cyclin D1 levels and an inhibition in cell viability. In contrast, AA had no such effects. The DHA-induced decrease in ERalpha expression resulted from proteasome-dependent degradation and not from decreased ERalpha mRNA expression. We propose that breast cancer cell proliferation is inhibited by DHA through proteasome-dependent degradation of ERalpha, reduced cyclin D1 expression and inhibition of MAPK signaling.

Topics: Albumins; Breast Neoplasms; Cell Line, Tumor; Cell Nucleus; Cell Survival; Cyclin D1; Docosahexaenoic Acids; Dose-Response Relationship, Drug; Estradiol; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Signaling System; Proteasome Endopeptidase Complex

Amplification of chromosome 11q13 is commonly seen in breast carcinomas and candidate genes from this region include CCND1 and EMSY. Here, we investigate the prognostic significance of CCND1 and EMSY amplification in a large series of breast carcinomas and in BRCA1 and BRCA2 mutation positive breast cancers. Amplification of CCND1 and EMSY was assessed by fluorescent in situ hybridization. Both CCND1 and EMSY amplifications were associated with a significantly worse outcome in ER-positive patients treated with tamoxifen only, in contrast to nonamplified tumors (P = 8.55 x 10(-4) and P = 8.35 x 10(-5), respectively). In multivariable Cox models, which included standard prognostic markers, co-amplification of CCND1 and EMSY was significantly more predictive of outcome than was amplification of either gene alone or neither gene amplified in ER-positive tamoxifen-treated patients (P = 5.47 x 10(-5)). EMSY gene amplification was a significantly less common event in BRCA2 mutation carriers as compared to BRCA1 mutation carriers (9 versus 24%, respectively). In contrast, CCND1 amplification occurred at a similar frequency in both BRCA1 and BRCA2 breast cancers (22 versus 18%, respectively). In summary, co-amplification of CCND1 and EMSY identified a poor prognostic subset of ER-positive tamoxifen-treated patients. In addition, EMSY amplification occurred at a lower frequency in BRCA2 mutation carriers providing evidence to support EMSY amplification as a somatic surrogate for BRCA2 loss in sporadic breast cancer.

Topics: Biomarkers, Tumor; Breast Neoplasms; Cyclin D1; Female; Gene Amplification; Genes, BRCA1; Genes, BRCA2; Humans; In Situ Hybridization, Fluorescence; Middle Aged; Mutation; Neoplasm Proteins; Nuclear Proteins; Prognosis; Proportional Hazards Models; Receptors, Estrogen; Repressor Proteins; Selective Estrogen Receptor Modulators; Survival Analysis; Tamoxifen; Tissue Array Analysis; Treatment Outcome

Understanding the consequences of miR-145 reintroduction in human breast cancer (BC) could reveal its tumor-suppressive functions and may disclose new aspects of BC biology. Therefore, we characterized the effects of miR-145 re-expression in BC cell lines by using proliferation and apoptosis assays. As a result, we found that miR-145 exhibited a pro-apoptotic effect, which is dependent on TP53 activation, and that TP53 activation can, in turn, stimulate miR-145 expression, thus establishing a death-promoting loop between miR-145 and TP53. We also found that miR-145 can downregulate estrogen receptor-alpha (ER-alpha) protein expression through direct interaction with two complementary sites within its coding sequence. In conclusion, we described a tumor suppression function of miR-145 in BC cell lines, and we linked miR-145 to TP53 and ER-alpha. Moreover, our findings support a view that miR-145 re-expression therapy could be mainly envisioned in the specific group of patients with ER-alpha-positive and/or TP53 wild-type tumors.

Topics: Apoptosis; Breast Neoplasms; Cell Division; Cell Line, Tumor; Cell Survival; Cyclin D1; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Transfection; Tumor Suppressor Protein p53

Recently, amplification of PPFIA1, encoding a member of the liprin family located about 600 kb telomeric to CCND1 on chromosome band 11q13, was described in squamous cell carcinoma of head and neck. Because 11q13 amplification is frequent in breast cancer, and PPFIA1 has been suggested to contribute to mammary gland development, we hypothesized that PPFIA1 might also be involved in the 11q13 amplicon in breast cancer and contribute to breast cancer development. A tissue microarray containing more than 2000 human breast cancers was analyzed for gene copy numbers of PPFIA1 and CCND1 by means of fluorescence in situ hybridization. PPFIA1 amplification was found in 248/1583 (15.4%) of breast cancers. Coamplification with CCND1 was found in all (248/248, 100%) PPFIA1-amplified cancers. CCND1 amplification without PPFIA1 coamplification was found in additional 117 (4.7%) tumors. Amplification of both PPFIA1 and CCND1 were significantly associated with high-grade phenotype (P = 0.0002) but were unrelated to tumor stage (P = 0.7066) or nodal stage (P = 0.5807). No difference in patient prognosis was found between 248 CCND1/PPFIA1 coamplified tumors and 117 tumors with CCND1 amplification alone (P = 0.6419). These data show that PPFIA1 amplification occurs frequently in breast cancer. The higher incidence of CCND1 amplification when compared with PPFIA1, the lack of prognostic relevance of coamplifications, and the fact that PPFIA1 amplification was found exclusively in CCND1-amplified cancers suggest that PPFIA1 gene copy number changes represent concurrent events of CCND1 amplification rather than specific biological incidents.

Topics: Adaptor Proteins, Signal Transducing; Adult; Aged; Aged, 80 and over; Breast Neoplasms; Chromosomes, Human, Pair 11; Cyclin D1; Female; Gene Amplification; Gene Dosage; Humans; Incidence; Middle Aged; Phenotype; Prognosis; Tissue Array Analysis

The transcription factor Pit-1/Pou1f1 regulates GH and prolactin (PRL) secretion in the pituitary gland. Pit-1 expression and GH regulation by Pit-1 have also been demonstrated in mammary gland. However, no data are available on the role of Pit-1 on breast PRL. To evaluate this role, several human breast cancer cell lines were transfected with either the Pit-1 expression vector or a Pit-1 small interference RNA construct, followed by PRL mRNA and protein evaluation. In addition, transient transfection of MCF-7 cells by a reporter construct containing the proximal PRL promoter, and ChIP assays were performed. Our data indicate that Pit-1 regulates mammary PRL at transcriptional level by binding to the proximal PRL promoter. We also found that Pit-1 raises cyclin D1 expression before increasing PRL levels, suggesting a PRL-independent effect of Pit-1 on cell proliferation. By using immunohistochemistry, we found a significant correlation between Pit-1 and PRL expression in 94 human breast invasive ductal carcinomas. Considering the possible role of PRL in breast cancer disorders, the function of Pit-1 in breast should be the focus of further research.

Topics: Animals; Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Division; Cell Line, Tumor; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Genes, bcl-1; Humans; Mice; Mutagenesis, Site-Directed; Neoplasm Proteins; NIH 3T3 Cells; Prolactin; Promoter Regions, Genetic; RNA Interference; RNA, Small Interfering; Transcription Factor Pit-1; Transcription, Genetic

CCND1 encodes for the cyclin D1 protein involved in G1/S cell cycle transition. In breast cancer the mechanism of CCND1 amplification, relationship between cyclin D1 protein expression and the key clinical markers estrogen receptor (ER) and HER2 requires elucidation. Tissue microarrays of primary invasive breast cancer from 93 women were evaluated for CCND1 amplification by fluorescent in-situ hybridization and cyclin D1 protein overexpression by immunohistochemistry. CCND1 amplification was identified in 27/93 (30%) cancers and 59/93 (63%) cancers had overexpression of cyclin D1. CCND1 amplification was significantly associated with cyclin D1 protein overexpression (p < 0.001; Fisher's exact test) and both CCND1 amplification and cyclin D1 protein expression with oestrogen receptor (ER) expression (p = 0.003 and p < 0.001; Fishers exact test). Neither CCND1 amplification nor cyclinD1 expression was associated with tumor size, pathological node status or HER2 amplification, but high CCND1 amplification (Copy Number Gain (CNG) > or = 8) was associated with high tumor grade (p = 0.005; chi square 7.915, 2 df) and worse prognosis by Nottingham Prognostic Index (p = 0.001; 2 sample t-test). High CCND1 amplification (CNG > or = 8) may identify a subset of patients with poor prognosis ER-positive breast cancers who should be considered for additional therapy.

Topics: Biomarkers, Tumor; Breast Neoplasms; Cyclin D1; Female; Gene Amplification; Humans; Immunoenzyme Techniques; In Situ Hybridization, Fluorescence; Neoplasms, Hormone-Dependent; Prognosis; Receptors, Estrogen; Tissue Array Analysis

Liverwort constituents have been reported to exert a broad spectrum of biological activities. In this study, we used a bioactivity-guided separation of an extract from the liverwort species Marchantia emarginata subsp. tosana to determine its anticancer activity. A high level of the active ingredient was isolated from this liverwort and its chemical structure was identified and characterized by various spectra. It was found to be identical to a well-known compound, marchantin A, a cyclic bisbibenzyl ether. However, no anticancer activities of this compound have previously been reported. We found that marchantin A efficiently induced cell growth inhibition in human MCF-7 breast cancer cells, with an IC(50) of 4.0microg/mL. Fluorescence microscopy and a Western blot analysis indicated that marchantin A actively induced apoptosis of MCF-7 cells. The levels of cleaved caspase-8, cleaved caspase-3, cleaved caspase-9, and cleaved poly (ADP ribose) polymerase (PARP) increased. However, the level of Bid markedly decreased in a dose- and time-dependent manner. We also evaluated the anticancer activities of marchantin A on the regulation of cell cycle regulators such as p21, p27, cyclin B1, and cyclin D1. The p21 and p27 gene expressions increased markedly while cyclin B1 and D1 gene expression decreased markedly by treatment with marchantin A. Many report demonstrated that liverwort was suggested to possess potent antioxidant activity. Our results indicate that marchantin A possesses free radical-scavenging activity (EC(50)=20microg/mL). Taken together, for the first time, the compound marchantin A from liverworts demonstrated to be a potent inducer of apoptosis in MCF-7 cells.

Topics: Apoptosis; Bibenzyls; Breast Neoplasms; Caspases; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Ethers, Cyclic; Female; Free Radical Scavengers; Humans

The Notch ligand, JAG1 is associated with breast cancer recurrence. Herein, we report on a genomics approach to elucidate mechanisms downstream of JAG1 that promote breast cancer growth. In a survey of 46 breast cancer cell lines, we found that triple negative (TN; basal and mesenchymal ER-, PR-, and Her2-negative) lines express JAG1 at significantly higher levels than do HER2(+) or luminal (ER(+)) Her2(-) cell lines. In contrast to the luminal lines tested (T47D and MCF7), TN breast cancer cell lines (HCC1143 and MDA MB231) display high-level JAG1 expression and growth inhibition with RNA interference-induced JAG1 down-regulation. We used microarray profiling of TN tumor cells transfected with JAG1 siRNA to identify JAG1-regulated genes (P or=1.5). Among JAG1-regulated genes identified, cyclin D1 was found to be a direct target of NOTCH1 and NOTCH3. We show that JAG1 down-regulation reduces direct binding of Notch to the cyclin D1 promoter, reduced cyclin D1 expression and inhibition of cell cycle progression through the cyclin D1-dependant G1/S checkpoint. Furthermore, we show that cyclin D1 and JAG1 expression correlate in TN breast cancer expression datasets. These data suggest a model whereby JAG1 promotes cyclin D1-mediated proliferation of TN breast cancers.

Topics: Blotting, Western; Breast Neoplasms; Calcium-Binding Proteins; Cell Line, Tumor; Chromatin Immunoprecipitation; Cyclin D1; Female; Gene Expression; Gene Expression Regulation, Neoplastic; Humans; Intercellular Signaling Peptides and Proteins; Jagged-1 Protein; Membrane Proteins; Oligonucleotide Array Sequence Analysis; Promoter Regions, Genetic; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Notch; Receptors, Progesterone; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Serrate-Jagged Proteins; Signal Transduction; Transfection

IKKepsilon has recently been identified as a breast cancer oncogene. Elevated levels of IKKepsilon are associated with cell survival and growth. Here, we show that IKKepsilon interacts with and phosphorylates estrogen receptor alpha (ERalpha) on serine 167 in vitro and in vivo. As a result, IKKepsilon induces ERalpha transactivation activity and enhances ERalpha binding to DNA. Cyclin D1, a major target of ERalpha, is transcriptionally up-regulated by IKKepsilon in a phospho-ERalpha-Ser-167-dependent manner. Further, overexpression of IKKepsilon induces tamoxifen resistance, whereas knockdown of IKKepsilon sensitizes cells to tamoxifen-induced cell death. These data suggest that ERalpha is a bona fide substrate of IKKepsilon and IKKepsilon plays an important role in tamoxifen resistance. Thus, IKKepsilon represents a critical therapeutic target in breast cancer.

Topics: Amino Acid Substitution; Antineoplastic Agents, Hormonal; Apoptosis; Breast Neoplasms; Cell Line; Cell Line, Tumor; Cell Survival; Cyclin D1; Drug Resistance, Neoplasm; Estrogen Receptor alpha; Humans; I-kappa B Kinase; Immunoblotting; Phosphorylation; Protein Binding; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Serine; Tamoxifen; Transcriptional Activation; Transfection

Breast cancer development is related to genes regulating cell cycle progression such as CCND1, CDK7, and ESR1. We conducted a hospital-based case-control study to evaluate the role of genetic polymorphisms in these genes in breast cancer development among Korean women. Questionnaire data and samples were obtained from 864 incident breast cancer cases and 723 controls recruited from 1995 to 2002. Four single nucleotide polymorphisms (SNPs) in three genes were genotyped by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry [CCND1 Ex4-1G>A (rs9344), CDK7 Ex2-28C>T (rs2972388), ESR1 P325P Ex4-122G>C (rs1801132), and ESR1 T594T Ex8+229G>A (rs2228480)], and their associations with breast cancer risk were assessed. The odds ratios (ORs) and 95% confidence intervals (CIs) were estimated by unconditional logistic regression analysis adjusted for age, education, age at the first full-term pregnancy, and family history of breast cancer. Women carrying the CDK7 TT genotype had increased risk of breast cancer (OR, 1.4; 95% CI, 1.1-1.7). There was no significant association between breast cancer risk and the genetic polymorphisms of CCND1 and ESR1. However, when CDK7 and ESR1 P325P were combined, women carrying both the CDK7 TT and ESR1 P325P CC genotypes showed increased breast cancer risk (OR, 1.7; 95% CI, 1.1-2.5; P for trend, <0.01). In conclusion, our findings suggest that the combined genotypes of CDK7 and ESR1 are associated with breast cancer risk among Korean women.

Topics: Asian People; Breast Neoplasms; Case-Control Studies; Cyclin D1; Cyclin-Dependent Kinase-Activating Kinase; Cyclin-Dependent Kinases; Epistasis, Genetic; Estrogen Receptor alpha; Female; Genetic Predisposition to Disease; Humans; Incidence; Korea; Logistic Models; Polymorphism, Single Nucleotide

A yeast two-hybrid system was utilized to identify novel PI3K p110alpha-interacting proteins, of which receptor of activated protein kinase C1 (RACK1) was chosen for successive detailed analyses. Our aim was to investigate the function(s) of RACK1 and its involvement in mechanisms of breast carcinoma proliferation and invasion/metastasis. Experiments in breast carcinoma cell lines stably transfected with RACK1, as well as nude mouse models, showed that RACK1 promotes breast carcinoma proliferation and invasion/metastasis in vitro and in vivo. Conversely, knockdown of RACK1 by siRNA in vitro inhibited proliferation, migration, and invasion. In cell lines stably transfected with RACK1, p-AKT, cyclin D1, cyclin D3, and CD147 expression, as well as MMP2 activity, were elevated. RACK1-induced migration could be inhibited by the addition of Rho-kinase inhibitor. In 160 breast carcinoma cases, survival analyses established that RACK1 is an independent prognostic factor for poor outcome (P < 0.001). In conclusion, RACK1 is an independent prognosis-related factor and promotes breast carcinoma proliferation and invasion/metastasis in vitro and in vivo.

Topics: Adult; Aged; Animals; Basigin; Breast Neoplasms; Carcinoma; Cell Line, Tumor; Cell Movement; Cell Proliferation; Class I Phosphatidylinositol 3-Kinases; Cyclin D1; Cyclin D3; Female; GTP-Binding Proteins; Humans; Kaplan-Meier Estimate; Matrix Metalloproteinase 2; Mice; Mice, Nude; Middle Aged; Neoplasm Invasiveness; Neoplasm Proteins; Neoplasm Staging; Phosphatidylinositol 3-Kinases; Phosphorylation; Prognosis; Proportional Hazards Models; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Receptors for Activated C Kinase; Receptors, Cell Surface; rho-Associated Kinases; RNA Interference; Time Factors; Transfection; Two-Hybrid System Techniques; Xenograft Model Antitumor Assays

Alternate splicing of the cyclin D1 gene gives rise to transcripts a and b which encode two protein isoforms cyclin D1a and cyclin D1b. Cyclin D1a can substitute for estrogen to activate estrogen receptor alpha- (ERalpha) mediated transcription and can induce the proliferation of estrogen responsive tissues. However, little is known about the biological role of cyclin D1b in transcriptional regulation. In this study, we determined that cyclin D1b is incapable of inducing ERalpha-mediated transcription because it fails to recruit steroid receptor coactivator-1 (SRC-1) to ERalpha. Moreover, cyclin D1b antagonizes cyclin D1a-induced ERalpha-mediated transcription by competing with cyclin D1a for ERalpha binding. Cell proliferation assay showed that cyclin D1b repressed the ERalpha-positive breast cancer T47D cell growth. Our findings suggest that the cyclin D1b represses breast cancer cell growth by antagonizing the action of cyclin D1a on ERalpha-mediated transcription.

Topics: Alternative Splicing; Binding Sites; Breast Neoplasms; Cell Line; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Dose-Response Relationship, Drug; Estrogen Receptor alpha; Gene Expression Regulation, Neoplastic; Humans; Models, Biological; Nuclear Receptor Coactivator 1; Protein Structure, Tertiary; Transcription, Genetic

Insulin-like growth factor binding protein (IGFBP)-3 has been shown to potently inhibit proliferation of various cell types in an insulin-like growth factor (IGF)-independent manner. We have previously shown that IGFBP-3 induces apoptosis in an IGF-independent manner through the activation of caspases involved in a death receptor-mediated pathway in MCF-7 human breast cancer cells. In the present study, we present further evidence that IGFBP-3 inhibits cell proliferation through the induction of cell cycle arrest in the same cell line. Induction of IGFBP-3 in MCF-7 cells inhibited cell proliferation whereas presence of small interfering RNA against IGFBP-3 abolished cell inhibitory effect of IGFBP-3, suggesting that the observed growth inhibition is specific. Flow cytometry analysis showed that induced expression of IGFBP-3 led to an arrest of the cell cycle in G1-S phase. Western immunoblot analysis showed a significant decrease in the levels of the cell cycle-regulated proteins such as cyclin D1, cyclin D3, cyclin E, cyclin A, cyclin-dependent kinase (CDK) 2, CDK4, retinoblastoma protein (pRB), and phosph-pRB, suggesting a possible mechanism for cell cycle arrest by IGFBP-3. Northern blot analysis and real-time quantitative PCR demonstrated a significant decrease in gene expression of cyclin D1. Additional phosphorylation assay showed that IGFBP-3 decreased the phosphorylation activity of CDK2 and CDK4. These results show that cellular production of IGFBP-3 leads to G1 cell cycle arrest with inhibition of CDK2 and CDK4. Taken together, IGFBP-3 exerts its growth inhibitory action through not only induction of apoptosis but also the G1 cell cycle arrest in human breast cancer cells.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor Proteins; Ecdysterone; Female; G1 Phase; Gene Expression Regulation, Neoplastic; Humans; Insulin-Like Growth Factor Binding Protein 3; Insulin-Like Growth Factor Binding Proteins; RNA, Messenger

Fibroblast growth factor 8 (FGF-8) is expressed in a large proportion of breast cancers, whereas its level in normal mammary gland epithelium is low. Previous studies have shown that FGF-8b stimulates breast cancer cell growth in vitro and in vivo. To explore the mechanisms by which FGF-8b promotes growth, we studied its effects on cell cycle regulatory proteins and signalling pathways in mouse S115 and human MCF-7 breast cancer cells. We also studied the effect of FGF-8b on cell survival. FGF-8b induced cell cycle progression and up-regulated particularly cyclin D1 mRNA and protein in S115 cells. Silencing cyclin D1 with siRNA inhibited most but not all FGF-8b-induced proliferation. Inhibition of the FGF-8b-activated ERK/MAPK pathway decreased FGF-8b-stimulated proliferation. Blocking the constitutively active PI3K/Akt and p38 MAPK pathways also lowered FGF-8b-induced cyclin D1 expression and proliferation. Corresponding results were obtained in MCF-7 cells. In S115 and MCF-7 mouse tumours, FGF-8b increased cyclin D1 and Ki67 levels. Moreover, FGF-8b opposed staurosporine-induced S115 cell death which effect was blocked by inhibiting the PI3K/Akt pathway but not the ERK/MAPK pathway. In conclusion, our results suggest that FGF-8b increases breast cancer cell growth both by stimulating cell cycle progression and by protecting against cell death.

Topics: Animals; Breast Neoplasms; Cell Cycle; Cell Death; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Enzyme Activation; Enzyme Inhibitors; Female; Fibroblast Growth Factor 8; Humans; Mice; Mice, Nude; p38 Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Protein Isoforms; Proto-Oncogene Proteins c-akt; RNA, Small Interfering; Signal Transduction

Cyclin D1 is involved in regulating the transition of G1 to S-phase in the cell cycle through phosphorylation of the retinoblastoma susceptibility product (pRB). Amplification and overexpression of the cyclin D1 gene (CCND1) have been reported in human breast cancers and are suggested to play important roles in the pathogenesis of the disease process. Although cyclin D1 is potentially an important gene, relatively little is known about the distribution of its amplification in breast cancer cell lines. In this study, a cyclin D1 cosmid probe was isolated and used with fluorescence in situ hybridization (FISH) to identify the gene in chromosomal spreads of 12 breast cancer cell lines. Nine cell lines showed increased gene copy levels of cyclin D1, including Five cell lines had more than six copies of cyclin D1 on sister chromatids and four had more than four copies but less than six copies grouped at the chromosome 11 q13 band. Three cell lines had two "normal" chromosome 11 and one and two additional derivative chromosome 11's with three and four 11q13 sites which lacked amplification of cyclin D1 on any of these sites. Using progesterone receptor (PR) gene as an internal control, a 2.0-fold or greater increase in cyclin D1 gene signals, was observed in five of the ten cell lines by Southern hybridization, the Amplification level of cyclin D1 varied from 2.3 to 19.6-fold. Three cell lines with low amplification of cyclin D1 showed overexpression of the gene by Northern analysis. Our experiments demonstrated that FISH was more sensitive than Southern blot at demonstrating low levels of gene amplification and, additionally, permitted assessment of the distribution of cyclin D1 gene among chromosomes.

Topics: Blotting, Northern; Blotting, Southern; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Chromosomes, Human; Cosmids; Cyclin D1; DNA Probes; Female; Gene Amplification; Gene Expression Regulation, Neoplastic; Humans; In Situ Hybridization, Fluorescence; Metaphase; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

To evaluate the extent to which each estrogen receptor (ER) subtype contributes to the stimulation or to the inhibition of mammary tumor growth, we evaluated the effects of specific agonists in MC4-L2 cells, which are stimulated by 17β-estradiol (E(2)), and in mammary carcinomas of the MPA mouse breast cancer model, which are inhibited by E(2). Both express ERα and ERβ. In MC4-L2 cells, 4,4',4"-(4-propyl-(1H)-pyrazole-1,3,5-triyl)trisphenol (PPT; ERα agonist) and (4-hydroxy-phenyl)-propionitrile (DPN; ERβ agonist) stimulated cell proliferation, whereas the opposite occurred in C4-HI primary cultures. The inhibitory effect was associated with a decrease in ERα and cyclin D1 expression and an increase in progesterone receptor (PR) expression as well as in the Bax/Bcl-xl ratio. In vivo, mice carrying C4-HI or 32-2-HI tumors were treated with E(2), PPT or DPN (3 mg/kg/day) or with vehicle. PPT and DPN inhibited tumor size, as did E(2), during the first 72 h. After a few days, DPN-treated tumors started to grow again, while PPT-treated tumors remained quiescent for a longer period of time. A pronounced decrease in the mitotic index and an increase in the apoptotic index was associated with tumor regression. All treated tumors showed: (a) an increase in integrin α6 and Bax expression, (b) an increased stromal laminin redistribution, and (c) a decrease in ERα, Bcl-xl and Bcl-2 expression (P < 0.001). Apoptosis-inducing factor (Aif) expression was increased in DPN-treated tumors, while active caspase 9 was up-regulated in PPT-treated mice, demonstrating the involvement of the intrinsic apoptotic pathway in estrogen-induced regression in this model. In conclusion, our data indicate that although there may be some preferences for activation pathways by the different agonists, the stimulatory or inhibitory effects triggered by estrogens are cell-context dependent rather than ER isoform dependent.

Topics: Animals; Antineoplastic Agents, Hormonal; Apoptosis; Apoptosis Inducing Factor; bcl-2-Associated X Protein; bcl-X Protein; Breast Neoplasms; Caspase 9; Cell Proliferation; Cyclin D1; Dose-Response Relationship, Drug; Estradiol; Estrogen Receptor alpha; Estrogen Receptor beta; Female; Mice; Mice, Inbred BALB C; Nitriles; Phenols; Pyrazoles; Receptors, Progesterone; Time Factors; Tumor Burden; Tumor Cells, Cultured

Multiple different biologically and clinically relevant genes are often amplified in invasive breast cancer, including HER2, ESR1, CCND1, and MYC. So far, little is known about their role in tumor progression. To investigate their significance for tumor invasion, we compared pure ductal carcinoma in situ (DCIS) and DCIS associated with invasive cancer with regard to the amplification of these genes. Fluorescence in situ hybridization (FISH) was performed on a tissue microarray containing samples from 130 pure DCIS and 159 DCIS associated with invasive breast cancer. Of the latter patients, we analyzed the intraductal and invasive components separately. In addition, lymph node metastases of 23 patients with invasive carcinoma were included. Amplification rates of pure DCIS and DCIS associated with invasive cancer did not differ significantly (pure DCIS vs. DCIS associated with invasive cancer: HER2 22.7 vs. 24.2%, ESR1 19.0 vs. 24.1%, CCND1 10.0 vs. 14.8%, MYC 11.8 vs. 6.5%; P > 0.05). Furthermore, we observed a high concordance of the amplification status for all genes if in situ and invasive carcinoma of individual patients were compared. This applied also to the corresponding lymph node metastases. Our results indicate no significant differences between the gene amplification status of DCIS and invasive breast cancer concerning HER2, ESR1, CCND1, and MYC. Therefore, our data suggest an early role of all analyzed gene amplifications in breast cancer development but not in the initiation of invasive tumor growth.

Topics: Adult; Aged; Aged, 80 and over; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Intraductal, Noninfiltrating; Chi-Square Distribution; Cyclin D1; Estrogen Receptor alpha; Female; Gene Amplification; Gene Expression Regulation, Neoplastic; Genotype; Humans; In Situ Hybridization, Fluorescence; Lymphatic Metastasis; Middle Aged; Neoplasm Invasiveness; Phenotype; Proto-Oncogene Proteins c-myc; Receptor, ErbB-2; Tissue Array Analysis

Estrogens induce breast tumor cell proliferation by directly regulating gene expression via the estrogen receptor (ER) transcriptional activity and by affecting growth factor signaling pathways such as mitogen-activated protein kinase (MAPK) and AKT/mammalian target of rapamycin Complex1 (mTORC1) cascades. In this study we demonstrated the preclinical therapeutic efficacy of combining the aromatase inhibitor letrozole with the multi-kinase inhibitor sorafenib in aromatase-expressing breast cancer cell lines. Treatment with letrozole reduced testosterone-driven cell proliferation, by inhibiting the synthesis of estrogens. Sorafenib inhibited cell proliferation in a concentration-dependent manner; this effect was not dependent on sorafenib-mediated inhibition of Raf1, but involved the down-regulation of mTORC1 and its targets p70S6K and 4E-binding protein 1 (4E-BP1). At concentrations of 5-10 μM the growth-inhibitory effect of sorafenib was associated with the induction of apoptosis, as indicated by release of cytochrome c and Apoptosis-Inducing Factor into the cytosol, activation of caspase-9 and caspase-7, and PARP-1 cleavage. Combination of letrozole and sorafenib produced a synergistic inhibition of cell proliferation associated with an enhanced accumulation of cells in the G(0)/G(1) phase of the cell cycle and with a down-regulation of the cell cycle regulatory proteins c-myc, cyclin D1, and phospho-Rb. In addition, longer experiments (12 weeks) demonstrated that sorafenib may be effective in preventing the acquisition of resistance towards letrozole. Together, these results indicate that combination of letrozole and sorafenib might constitute a promising approach to the treatment of hormone-dependent breast cancer.

Topics: Adaptor Proteins, Signal Transducing; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Apoptosis Inducing Factor; Aromatase; Aromatase Inhibitors; Benzenesulfonates; Breast Neoplasms; Caspase 7; Caspase 9; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cytochromes c; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Drug Synergism; Estradiol; Female; Humans; Letrozole; Mechanistic Target of Rapamycin Complex 1; Multiprotein Complexes; Niacinamide; Nitriles; Phenylurea Compounds; Phosphoproteins; Phosphorylation; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Protein Kinase Inhibitors; Proteins; Proto-Oncogene Proteins c-myc; Pyridines; Retinoblastoma Protein; Ribosomal Protein S6 Kinases, 70-kDa; Sorafenib; Testosterone; Time Factors; TOR Serine-Threonine Kinases; Transfection; Triazoles

Numerous recent studies indicate that most anticancer effects of PPARγ agonists like thiazolidinediones are the result of PPARγ-independent pathways. These conclusions were obtained by several approaches including the use of thiazolidinedione derivatives like Δ2-Troglitazone (Δ2-TGZ) that does not activate PPARγ. Since biotinylation has been proposed as a mechanism able to increase the specificity of drug delivery to cancer cells which could express a high level of vitamin receptor, a biotinylated derivative of Δ2-TGZ (bΔ2-TGZ) has been synthetized. In the present article, we have studied the in vitro effects of this molecule on both hormone-dependent (MCF-7) and hormone-independent (MDA-MB-231) breast cancer cells. In both cell lines, bΔ2-TGZ was more efficient than Δ2-TGZ to decrease cell viability. bΔ2-TGZ was also more potent than Δ2-TGZ to induce the proteasomal degradation of cyclin D1 in both cell lines and those of ERα in MCF-7 cells. However, in competition experiments, the presence of free biotin in the culture medium did not decrease the antiproliferative action of bΔ2-TGZ. Besides, other compounds that had no biotin but that were substituted at the same position of the phenolic group of the chromane moiety of Δ2-TGZ decreased cell viability similarly to bΔ2-TGZ. Hence, we concluded that the increased antiproliferative action of bΔ2-TGZ was not due to biotin itself but to the functionalization of the terminal hydroxyl group. This should be taken into account for the design of new thiazolidinedione derivatives able to affect not only hormone-dependent but also hormone-independent breast cancer cells in a PPARγ-independent pathway.

Topics: Antineoplastic Agents; Biotinylation; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chromans; Cyclin D1; Dose-Response Relationship, Drug; Estrogen Receptor alpha; Estrogens; Female; Humans; Inhibitory Concentration 50; Neoplasms, Hormone-Dependent; PPAR gamma; Proteasome Endopeptidase Complex; Protein Processing, Post-Translational; Thiazolidinediones; Transfection; Troglitazone

The epidermal growth factor receptor (EGFR)-family and cyclin-D1 have been extensively studied in breast cancer; however systematic studies that examine protein expression and gene status in the same cohort of patients are lacking. Also emerging evidences suggest existence of a direct EGFR-signaling pathway, which involves cellular transport of EGFR from cell membrane to the nucleus, and transcriptional regulation of the target genes. Thus, we examined the protein expression of membrane EGFR, nuclear EGFR, cyclin-D1 and the corresponding gene status in 113 breast carcinomas by immunohistochemistry and fluorescence in situ hybridization using tissue microarrays. Membrane EGFR overexpression and EGFR gene amplification were detected in 2% cases, while nuclear EGFR was detected in 40% of cases, with 12% having high nuclear EGFR staining. Nuclear EGFR correlated with tumor size (P=0.0005), lymph node metastasis (P=0.0288), Nottingham prognostic index (P=0.0011) and estrogen receptor (ER) expression (P=0.0258) but the letter correlation was observed only in premenopausal group of patients. Strong cyclin-D1 expression and cyclin-D1 gene (CCND1) amplification were found in 64 and 13% of the cases, respectively. Cyclin-D1 expression showed positive correlation with ER (P=0.0113) and inverse correlation with Nottingham prognostic index (P=0.0309) and membrane EGFR (P=0.0201). CCND1 amplification also showed inverse correlation with membrane EGFR (P=0.0420). A strong correlation between membrane EGFR expression and gene amplification (P=0.0035), as well as cyclin-D1 overexpression and gene amplification (P=0.0362), was demonstrated. On univariate analysis cyclin-D1 expression showed a correlation with longer overall survival in the premenopausal group and nuclear EGFR correlated with shorter overall survival in whole cohort as well in the premenopausal group of patients. Multivariate analysis revealed nuclear EGFR to be an independent prognostic factor and showed 3.4 times greater mortality risk for nuclear EGFR+++ patients as compared with nuclear EGFR negative patients (hazard ratio =3.402; P=0.0026).

Topics: Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Nucleus; Cyclin D1; ErbB Receptors; Female; Humans; Immunohistochemistry; In Situ Hybridization, Fluorescence; Neoplasm Staging; Premenopause; Prognosis; Survival Rate; Tissue Array Analysis

Constitutive activation of signal transducer and activator of transcription 3 (STAT3) signaling is frequently detected in cancer, promoting its emergence as a promising target for cancer treatment. Inhibiting constitutive STAT3 signaling represents a potential therapeutic approach. We used structure-based design to develop a nonpeptide, cell-permeable, small molecule, termed as LLL12, which targets STAT3. LLL12 was found to inhibit STAT3 phosphorylation (tyrosine 705) and induce apoptosis as indicated by the increases of cleaved caspase-3 and poly (ADP-ribose) polymerase in various breast, pancreatic, and glioblastoma cancer cell lines expressing elevated levels of STAT3 phosphorylation. LLL12 could also inhibit STAT3 phosphorylation induced by interleukin-6 in MDA-MB-453 breast cancer cells. The inhibition of STAT3 by LLL12 was confirmed by the inhibition of STAT3 DNA binding activity and STAT3-dependent transcriptional luciferase activity. Downstream targets of STAT3, cyclin D1, Bcl-2, and survivin were also downregulated by LLL12 at both protein and messenger RNA levels. LLL12 is a potent inhibitor of cell viability, with half-maximal inhibitory concentrations values ranging between 0.16 and 3.09 microM, which are lower than the reported JAK2 inhibitor WP1066 and STAT3 inhibitor S3I-201 in six cancer cell lines expressing elevated levels of STAT3 phosphorylation. In addition, LLL12 inhibits colony formation and cell migration and works synergistically with doxorubicin and gemcitabine. Furthermore, LLL12 demonstrated a potent inhibitory activity on breast and glioblastoma tumor growth in a mouse xenograft model. Our results indicate that LLL12 may be a potential therapeutic agent for human cancer cells expressing constitutive STAT3 signaling.

Topics: Animals; Anthraquinones; Antineoplastic Agents; Apoptosis; Blotting, Western; Breast Neoplasms; Caspase 3; Cell Line; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Glioblastoma; Humans; Inhibitor of Apoptosis Proteins; Male; Mice; Mice, Nude; Microtubule-Associated Proteins; Neoplasms; Pancreatic Neoplasms; Phosphorylation; Poly(ADP-ribose) Polymerases; Protein Binding; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; STAT3 Transcription Factor; Sulfonamides; Survivin; Xenograft Model Antitumor Assays

Cyclin D1 belongs to a family of proteins that regulate progression through the G1-S phase of the cell cycle by binding to cyclin-dependent kinase (cdk)-4 to phosphorylate the retinoblastoma protein and release E2F transcription factors for progression through cell cycle. Several cancers, including breast, colon, and prostate, overexpress the cyclin D1 gene. However, the correlation of cyclin D1 overexpression with E2F target gene regulation or of cdk-dependent cyclin D1 activity with tumor development has not been identified. This suggests that the role of cyclin D1 in oncogenesis may be independent of its function as a cell cycle regulator. One such function is the role of cyclin D1 in cell adhesion and motility. Filamin A (FLNa), a member of the actin-binding filamin protein family, regulates signaling events involved in cell motility and invasion. FLNa has also been associated with a variety of cancers including lung cancer, prostate cancer, melanoma, human bladder cancer, and neuroblastoma. We hypothesized that elevated cyclin D1 facilitates motility in the invasive MDA-MB-231 breast cancer cell line. We show that MDA-MB-231 motility is affected by disturbing cyclin D1 levels or cyclin D1-cdk4/6 kinase activity. Using mass spectrometry, we find that cyclin D1 and FLNa coimmunoprecipitate and that lower levels of cyclin D1 are associated with decreased phosphorylation of FLNa at Ser2152 and Ser1459. We also identify many proteins related to cytoskeletal function, biomolecular synthesis, organelle biogenesis, and calcium regulation whose levels of expression change concomitant with decreased cell motility induced by decreased cyclin D1 and cyclin D1-cdk4/6 activities.

Topics: Amino Acid Sequence; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Contractile Proteins; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Filamins; Humans; Microfilament Proteins; Molecular Sequence Data; Neoplasm Invasiveness; Phosphoproteins

Amplification of 11q13 is found in approximately 15% of breast cancers. Cyclin D1 (CCND1) has been reported to be the 'driver' of this amplicon, however, multiple genes map to the smallest region of amplification of 11q13. Out of these genes, cortactin (CTTN) has been shown to be consistently overexpressed at the mRNA level in tumours harbouring 11q13 amplification. The aims of this study are to define whether CTTN is consistently co-amplified with the main core of the 11q13 amplicon, whether it is consistently overexpressed when amplified and to determine correlations between CTTN amplification and overexpression with clinicopathological features of breast cancers and survival of breast cancer patients. CTTN and CCND1 chromogenic in situ hybridisation (CISH) probes and a validated monoclonal antibody against CTTN were applied to a tissue microarray of a cohort of breast cancers from patients treated with anthracycline-based chemotherapy. CTTN and CCND1 amplifications were found in 12.3 and 12.4% of cases, respectively. All cases harbouring CTTN amplification also displayed CCND1 amplification. High expression of CTTN was found in 10.8% of cases and was associated with CTTN amplification, expression of 'basal' markers and topoisomerase IIα. Exploratory subgroup analysis of tumours devoid of 11q13 amplification revealed that high expression of CTTN in the absence of CTTN gene amplification was associated with lymph node negative disease, lack of hormone receptors and FOXA1, expression of 'basal' markers, high Ki-67 indices, p53 nuclear expression, and basal-like and triple negative phenotypes. CTTN expression and CTTN gene amplification were not associated with disease-, metastasis-free and overall survival. In conclusion, CTTN is consistently co-amplified with CCND1 and expressed at higher levels in breast cancers harbouring 11q13 amplification, suggesting that CTTN may also constitute one of the drivers of this amplicon. CTTN expression is not associated with the outcome of breast cancer patients treated with anthracycline-based chemotherapy.

Topics: Biomarkers, Tumor; Breast Neoplasms; Chemotherapy, Adjuvant; Chromogenic Compounds; Chromosomes, Human, Pair 11; Cortactin; Cyclin D1; Digoxigenin; Disease-Free Survival; Female; Gene Amplification; Humans; Immunohistochemistry; In Situ Hybridization; Kaplan-Meier Estimate; London; Mastectomy; Neoplasm Staging; Predictive Value of Tests; Retrospective Studies; Time Factors; Tissue Array Analysis; Treatment Outcome; Up-Regulation

Cadmium is an environmental contaminant that enters the body through diet or cigarette smoke. It affects multiple cellular processes, including cell proliferation, differentiation, and apoptosis. Recently, cadmium has been shown to function as an endocrine disruptor, to stimulate estrogen receptor alpha (ERalpha) activity and promote uterine and mammary gland growth in mice. Although cadmium exposure has been associated with the development of breast cancer, the mechanism of action of cadmium remains unclear. To address this deficit, we examined the effects of cadmium treatment on breast cancer cells. We found that ERalpha is required for both cadmium-induced cell growth and modulation of gene expression. We also determined that ERalpha translocates to the nucleus in response to cadmium exposure. Additionally, we provide evidence that cadmium potentiates the interaction between ERalpha and c-Jun and enhances recruitment of this transcription factor complex to the proximal promoters of cyclin D1 and c-myc, thus increasing their expression. This study provides a mechanistic link between cadmium exposure and ERalpha and demonstrates that cadmium plays an important role in the promotion of breast cancer.

Topics: Blotting, Western; Breast Neoplasms; Cadmium; Cell Line, Tumor; Cell Proliferation; Chromatin Immunoprecipitation; Cyclin D1; Estrogen Receptor alpha; Female; Humans; Immunoprecipitation; Protein Binding; Proto-Oncogene Proteins c-jun; Reverse Transcriptase Polymerase Chain Reaction

Resistance to endocrine therapy is a major clinical problem in breast cancer. The role of ERalpha splice variants in endocrine resistance is largely unknown. We observed reduced protein expression of an N-terminally truncated ERalpha46 in endocrine-resistant LCC2, LCC9, and LY2 compared to MCF-7 breast cancer cells. Transfection of LCC9 and LY2 cells with hERalpha46 partially restored growth inhibition by TAM. Overexpression of hERalpha46 in MCF-7 cells reduced estradiol (E(2))-stimulated endogenous pS2, cyclin D1, nuclear respiratory factor-1 (NRF-1), and progesterone receptor transcription. Expression of oncomiR miR-21 was lower in TAM-resistant LCC9 and LY2 cells compared to MCF-7 cells. Transfection with ERalpha46 altered the pharmacology of E(2) regulation of miR-21 expression from inhibition to stimulation, consistent with the hypothesis that hERalpha46 inhibits ERalpha activity. Established miR-21 targets PTEN and PDCD4 were reduced in ERalpha46-transfected, E(2)-treated MCF-7 cells. In conclusion, ERalpha46 appears to enhance endocrine responses by inhibiting selected ERalpha66 responses.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Drug Resistance, Neoplasm; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Nude; MicroRNAs; Protein Isoforms; PTEN Phosphohydrolase; Tamoxifen; Transcription, Genetic; Transplantation, Heterologous

Alternative splicing of precursor mRNA is a fundamental mechanism to generate multiple proteins from a single gene. Although constitutive and alternative mRNA splicing is temporally and spatially regulated, deregulation of mRNA splicing could cause development, progression, and metastasis of tumors. Through yeast two-hybrid screening of a human breast cDNA library using estrogen receptor-alpha (ERalpha) as bait, we identified a novel nuclear receptor box containing full-length protein, nuclear protein E3-3 (NPE3-3). Our results revealed that NPE3-3 associates with not only ERalpha but also with splicing factors, serine/arginine-rich protein (SRp)-30c, SRp40, and splicing factor SC-35, suggesting that NPE3-3 is likely to be involved in regulation of mRNA splicing. Accordingly, transient expression of NPE3-3 in cells resulted in expected splicing of the CD44 control minigene. We also discovered that NPE3-3-overexpressing clones produced a novel, previously unrecognized, alternatively spliced variant of ERalpha (termed ERalphaV), which had a molecular size of 37 kDa composed of only exons 1, 2, 7, and 8. ERalphaV was expressed and sequestered in the cytoplasm in MCF-7 cells stably overexpressing NPE3-3, suggesting its involvement in nongenomic hormone signaling. NPE3-3 clones exhibited up-regulation of ERK1/2 signaling, cyclin D1, and cathepsin D and enhanced tumor cell proliferation, migration, and tumorigenicity. Moreover, direct expression of the ERalphaV in breast cancer cells stimulated ERK1/2 up-regulation and cyclin D1 expression. We found that ERalphaV physically interacted with MAPK kinase (MEK)-1/2, and thus, an ERalphaV and MEK1/2 complex could lead to the activation of the ERK1/2 pathway. Interestingly, NPE3-3 was up-regulated in human breast tumors. These findings revealed a role for NPE3-3 in alternative splicing and suggest that ERalpha is a physiological target of NPE3-3, leading to a constitutive nongenomic signaling pathway in breast cancer cells.

Topics: Animals; Blotting, Western; Breast Neoplasms; Cathepsin D; Cell Line; Cell Line, Tumor; Cell Movement; Cell Proliferation; Chlorocebus aethiops; Chromatography, Liquid; COS Cells; Cyclin D1; Estrogen Receptor alpha; Humans; Immunoprecipitation; Mice; Microscopy, Confocal; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nuclear Proteins; Protein Binding; RNA Splicing; Tandem Mass Spectrometry; Two-Hybrid System Techniques

Polychlorinated biphenyls (PCBs) are environmental chemical contaminants that can produce reactive oxygen species (ROS) by autoxidation of dihydroxy-PCBs and redox-cycling. We investigate the hypothesis that PCB induced perturbations in ROS signaling regulate the entry of quiescent cells into the proliferative cycle. Quiescent MCF-10A human breast epithelial cells were incubated with 0-3 micromolar of 2-(4-chlorophenyl)benzo-1,4-quinone (4-Cl-BQ), 2, 2', 4, 4', 5, 5'-hexachlorobiphenyl (PCB 153), and Aroclor 1254 for 4 days. Cells were replated at a lower density and analyzed for cell cycle phase distributions, ROS levels, MnSOD expression, and cyclin D1 protein levels. Quiescent cells incubated with 4-Cl-BQ showed the maximal delay in entering S phase. This delay was associated with a decrease in MnSOD activity, protein and mRNA levels, and an increase in cellular ROS levels. Results from the mRNA turnover assay showed that the 4-Cl-BQ treatment selectively enhanced the degradation of the 4.2kb MnSOD transcript, while the half-life of the 1.5 kb transcript did not change. Accumulation of cyclin D1 protein levels in replated cells was suppressed in cells treated with 4-Cl-BQ. Pretreatment of quiescent cells with polyethylene glycol-conjugated superoxide dismutase and catalase suppressed 4-Cl-BQ induced increase in ROS levels, which was consistent with an increase in cyclin D1 accumulation, and entry into S phase. These results showed 4-Cl-BQ induced perturbations in ROS signaling inhibit the entry of quiescent cells into S phase.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Separation; Cyclin D1; Epithelial Cells; Flow Cytometry; Humans; Polychlorinated Biphenyls; Reactive Oxygen Species; Signal Transduction; Superoxide Dismutase

Although the ability of coactivators to enhance the expression of estrogen receptor-alpha (ERalpha) target genes is well established, the role of corepressors in regulating 17beta-estradiol (E2)-induced gene expression is poorly understood. Previous studies revealed that the silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) corepressor is required for full ERalpha transcriptional activity in MCF-7 breast cancer cells, and we report herein the E2-dependent recruitment of SMRT to the regulatory regions of the progesterone receptor (PR) and cyclin D1 genes. Individual depletion of SMRT or steroid receptor coactivator (SRC)-3 modestly decreased E2-induced PR and cyclin D1 expression; however, simultaneous depletion revealed a cooperative effect of this coactivator and corepressor on the expression of these genes. SMRT and SRC-3 bind directly in an ERalpha-independent manner, and this interaction promotes E2-dependent SRC-3 binding to ERalpha measured by co-IP and SRC-3 recruitment to the cyclin D1 gene as measured by chromatin IP assays. Moreover, SMRT stimulates the intrinsic transcriptional activity of all of the SRC family (p160) coactivators. Our data link the SMRT corepressor directly with SRC family coactivators in positive regulation of ERalpha-dependent gene expression and, taken with the positive correlation found for SMRT and SRC-3 in human breast tumors, suggest that SMRT can promote ERalpha- and SRC-3-dependent gene expression in breast cancer.

Topics: Breast Neoplasms; Cell Line, Tumor; Cyclin D1; Enhancer Elements, Genetic; Estradiol; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Humans; Nuclear Receptor Co-Repressor 2; Nuclear Receptor Coactivator 2; Nuclear Receptor Coactivator 3; Protein Binding; Protein Structure, Tertiary; Receptors, Progesterone; Transcription, Genetic; Transcriptional Activation

Defects in epigenetic regulation of gene transcription play an important role in carcinogenesis of the breast and other tissues. The two most widely studied epigenetic changes are DNA methylation and acetylation of histone proteins and inhibition of these processes inhibits growth in breast cancer cell lines. These data coupled with the evidence that fetal and neonatal exposure to oestrogenic substances may lead to epigenetic changes that predispose or protect against the development of breast cancer in later life formed the basis for this study. Three histone deacetylases, valproic acid (VPA), trichostatin A (TCA) and apicidin dose-dependently inhibited basal growth in MCF-7 and MDA-MB-231 as well as the growth promoting effects of oestradiol (E(2)) and epidermal growth factor (EGF) in MCF-7 cells. The growth inhibitory responses to the DNA methyl transferase inhibitor, 5-aza-2'deoxycytidine (decitabine) were weak. HDACi's reduced the protein levels of pro-caspase 9 and cyclin D1, whereas decitabine had no effect. Long-term genistein treatment (LTGT) of MCF-7 cells markedly reduced the basal expression of acetylated histone 3 (H3) and the effects of HDACi's on increasing the levels of acetylated H3 protein. However, this was not correlated with a reduced expression of total H3 except after a high dose of VPA. LTGT inhibited growth of MCF-7 cells and the mitogenic responses to E(2) and EGF. The growth inhibitory responses to HDACI's in the presence of E(2) and EGF was significantly reduced in LTGT cells compared to control MCF-7 cells and there was evidence that LTGT maintained the protein levels of pro-caspase 9 in the presence of HDACi's. This study provides further evidence that oestrogenic substances can induce significant epigenetic changes to alter the dynamics of growth in breast cancer cell lines.

Topics: Acetylation; Anticarcinogenic Agents; Breast Neoplasms; Caspase 9; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Epigenesis, Genetic; Female; Gene Expression; Gene Expression Regulation, Neoplastic; Genistein; Histone Deacetylase Inhibitors; Histones; Humans; Mitogens

Estrogen and progesterone are the defining hormones of normal female development, and both play critical roles in breast carcinogenesis. Cyclin D1 is a breast cancer oncogene whose amplification is linked to poor prognosis in estrogen and progesterone receptor-positive breast cancers. Here we report that cyclin D1 regulates progesterone receptor expression, consequently enhancing responses to estrogen and progesterone. Estrogen treatment of cyclin D1 transgenic mice increased progesterone receptor expression and induced mammary hyperplasias that were stimulated by progesterone and blocked by a progesterone antagonist. Progesterone receptor levels decreased in cyclin D1 knockout mice. Cyclin D1 regulated progesterone receptor expression through a novel estrogen- and cyclin D1-responsive enhancer in DNA encoding part of the 3' untranslated region of the progesterone receptor gene. Small inhibitory RNAs for cyclin D1 decreased progesterone receptor expression and estrogen receptor binding to the 3' enhancer region in human breast cancer cells. Since estrogen and progesterone regulate cyclin D1, our results suggest that cyclin D1's participation in a feed-forward loop could contribute to increased breast cancer risks associated with estrogen and progesterone combinations. Additionally, its regulation of the progesterone receptor identifies a novel role for cyclin D1 in ovarian hormone control of breast development and breast carcinogenesis.

Topics: Animals; Binding Sites; Breast Neoplasms; Cell Line, Tumor; Cyclin D1; Enhancer Elements, Genetic; Estradiol; Estrogens; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Hyperplasia; Mammary Glands, Animal; Mice; Mice, Transgenic; Progesterone; Promoter Regions, Genetic; Protein Binding; Receptors, Estrogen; Receptors, Progesterone; RNA, Small Interfering; Signal Transduction

Alteration of protein trafficking and localization is associated with several diseases, including cystic fibrosis, breast cancer, colorectal cancer, leukemia and diabetes. Specifically, aberrant nuclear localization of the epidermal growth factor receptor (EGFR), a receptor tyrosine kinase, is a poor prognostic indicator in several epithelial carcinomas. It is now appreciated that in addition to signaling from the plasma membrane, EGFR also trafficks to the nucleus, and can directly bind the promoter regions of genes encoding cyclin D1 (CCND1) and B-Myb (MYBL2). We have previously established that loss of MUC1 in an EGFR-dependent transgenic mouse model of breast cancer correlates with the loss of cyclin D1 expression. Here, we provide evidence for a novel regulatory function of MUC1 in the trafficking and nuclear activity of EGFR. We found that MUC1 and EGFR interact in the nucleus of breast cancer cells, which promotes the accumulation of chromatin-bound EGFR. Additionally, the presence of MUC1 results in significant colocalization of EGFR and phosphorylated RNA polymerase II, indicating that MUC1 influences the association of EGFR with transcriptionally active promoter regions. Importantly, we found that the loss of MUC1 expression resulted in a decrease in the interaction between EGFR and the CCND1 promoter, which translated to a significant decrease in cyclin D1 protein expression. This data offers insights into a novel regulatory mechanism of EGFR nuclear function and could have important implications for evaluating nuclear localization in cancer.

Topics: Active Transport, Cell Nucleus; Breast Neoplasms; Carcinoma; Cell Cycle Proteins; Cell Line, Tumor; Cell Nucleus; Cloning, Molecular; Cyclin D1; ErbB Receptors; Female; Humans; Mucin-1; Promoter Regions, Genetic; Protein Binding; RNA, Small Interfering; Trans-Activators

Cyclin D1 gene (CCND1) is a critical mitogen-regulated cell-cycle control element whose transcriptional modulation plays a crucial role in breast cancer growth and progression. Here we demonstrate that the non-aromatizable androgen 5-α-dihydrotestosterone (DHT) inhibits endogenous cyclin D1 expression, as evidenced by reduction of cyclin D1 mRNA and protein levels, and decrease of CCND1-promoter activity, in MCF-7 cells. The DHT-dependent inhibition of CCND1 gene activity requires the involvement and the integrity of the androgen receptor (AR) DNA-binding domain. Site directed mutagenesis, DNA affinity precipitation assay, electrophoretic mobility shift assay and chromatin immunoprecipitation analyses indicate that this inhibitory effect is ligand dependent and it is mediated by direct binding of AR to an androgen response element (CCND1-ARE) located at -570 to -556-bp upstream of the transcription start site, in the cyclin D1 proximal promoter. Moreover, AR-mediated repression of the CCND1 involves the recruitment of the atypical orphan nuclear receptor DAX1 as a component of a multiprotein repressor complex also embracing the participation of Histone Deacetylase 1. In conclusion, identification of the CCND1-ARE allows defining cyclin D1 as a specific androgen target gene in breast and might contribute to explain the molecular basis of the inhibitory role of androgens on breast cancer cells proliferation.

Topics: Androgens; Binding Sites; Breast Neoplasms; Cell Line; Cell Line, Tumor; Cell Proliferation; Cyclin D1; DAX-1 Orphan Nuclear Receptor; Dihydrotestosterone; Female; Gene Expression Regulation, Neoplastic; Humans; Promoter Regions, Genetic; Receptors, Androgen; Response Elements

Overexpression of p70S6K in breast cancer patients is associated with aggressive disease and poor prognosis. Recent studies showed that patients with breast cancer with increased p70S6K phosphorylation had poor survival and increased metastasis. The purpose of our study was to determine whether knockdown of p70S6K would inhibit cell growth, invasion, and metastasis in breast cancer. We therefore stably knocked down p70S6K expression in MDA-231, a highly metastatic breast cancer cell line, using a lentiviral short hairpin RNA (shRNA) based approach. Inhibition of p70S6K led to inhibition of cell growth, migration, and invasion in vitro. To determine the role of p70S6K in breast cancer tumorigenesis and metastasis, we used an MDA-231 orthotopic and metastatic animal model. In the orthotopic model, mice injected with MDA-231-p70S6K shRNA cells developed significantly smaller tumors than control mice injected with MDA-231 control shRNA cells (P < 0.01). No metastasis was observed in the p70S6K downregulated group, whereas lung metastasis was detected in all mice in the control group. To determine the role of p70S6K on growth and invasion, we tested downstream signaling targets by Western blot analysis. Knockdown of p70S6K inhibited phosphorylation of focal adhesion kinase, tissue transglutaminase 2, and cyclin D1 proteins, which promote cell growth, survival, and invasion. In addition, downregulation of p70S6K induced expression of PDCD4, a tumor-suppressor protein. In conclusion, we showed that p70S6K plays an important role in metastasis by regulating key proteins like cyclin D1, PDCD4, focal adhesion kinase, E-cadherin, beta-catenin, and tissue transglutaminase 2, which are essential for cell attachment, survival, invasion, and metastasis in breast cancer.

Topics: Animals; Antineoplastic Agents; beta Catenin; Breast Neoplasms; Cadherins; Carcinoma; Cyclin D1; Drug Delivery Systems; Female; Focal Adhesion Protein-Tyrosine Kinases; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Mice; Mice, Nude; Protein Glutamine gamma Glutamyltransferase 2; Protein Kinase Inhibitors; Ribosomal Protein S6 Kinases, 70-kDa; RNA, Small Interfering; Xenograft Model Antitumor Assays

Breast cancer associated with BRCA1 and BRCA2 gene mutations differs from non-BRCA tumors in several respects. We determined whether there was any difference in CCND1 (11q13) and ZNF217 (20q13) gene amplification with respect to BRCA status. Of 40 breast cancer samples examined, 15 and 9 were from BRCA1 and BRCA2 mutation carriers, respectively, and 16 from patients without mutation. Fluorescence in situ hybridization showed that eight tumors exhibited CCND1 amplification (20%; 3 BRCA1, 3 BRCA2, 2 non-BRCA). ZNF217 amplification was observed in three of 38 cases (8%; 2 BRCA1, 1 non-BRCA). There was no significant difference in CCND1 and ZNF217 amplification between BRCA1, BRCA2 and non-BRCA tumors. CCND1 amplification was associated with decreased disease-free (P = 0.045) and overall survival (P = 0.015). BRCA1 tumors with CCND1 amplification were estrogen receptor negative, in contrast to CCND1 amplified BRCA2 and non-BRCA tumors, suggesting that concurrent CCND1 amplification and estrogen and progesterone receptor negativity may predict germline BRCA1 gene mutation. All ZNF217 amplified tumors were of the medullary histological type (P = 0.002). There was no statistical correlation between CCND1 and ZNF217 amplification and estrogen receptor, progesterone receptor, and ERBB2 expression and TNM classification. CCND1 amplification did not correlate with EGFR expression.

Topics: Adenocarcinoma, Mucinous; Adult; Apoptosis Regulatory Proteins; BRCA1 Protein; BRCA2 Protein; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Lobular; Cyclin D1; ErbB Receptors; Female; Gene Amplification; Genetic Predisposition to Disease; Germ-Line Mutation; Humans; Immunoenzyme Techniques; In Situ Hybridization, Fluorescence; Middle Aged; Prognosis; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone; Trans-Activators; Young Adult

The transcription factor CCAAT/enhancer binding protein delta (C/EBPdelta, CEBPD, NFIL-6beta) has tumor suppressor function; however, the molecular mechanism(s) by which C/EBPdelta exerts its effect are largely unknown. Here, we report that C/EBPdelta induces expression of the Cdc27 (APC3) subunit of the anaphase promoting complex/cyclosome (APC/C), which results in the polyubiquitination and degradation of the prooncogenic cell cycle regulator cyclin D1, and also down-regulates cyclin B1, Skp2, and Plk-1. In C/EBPdelta knockout mouse embryo fibroblasts (MEF) Cdc27 levels were reduced, whereas cyclin D1 levels were increased even in the presence of activated GSK-3beta. Silencing of C/EBPdelta, Cdc27, or the APC/C coactivator Cdh1 (FZR1) in MCF-10A breast epithelial cells increased cyclin D1 protein expression. Like C/EBPdelta, and in contrast to cyclin D1, Cdc27 was down-regulated in several breast cancer cell lines, suggesting that Cdc27 itself may be a tumor suppressor. Cyclin D1 is a known substrate of polyubiquitination complex SKP1/CUL1/F-box (SCF), and our studies show that Cdc27 directs cyclin D1 to alternative degradation by APC/C. These findings shed light on the role and regulation of APC/C, which is critical for most cellular processes.

Topics: Animals; Apc3 Subunit, Anaphase-Promoting Complex-Cyclosome; Blotting, Western; Breast Neoplasms; CCAAT-Enhancer-Binding Protein-delta; Cell Cycle Proteins; Cell Line, Tumor; Cyclin B1; Cyclin D1; Gene Expression Regulation, Neoplastic; Immunoprecipitation; Mice; Mice, Knockout; Microscopy, Fluorescence; Polo-Like Kinase 1; Proteasome Endopeptidase Complex; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; S-Phase Kinase-Associated Proteins

Angiotensin II (Ang II) is a bioactive peptide of the renin-angiotensin system, acting not only as a vasoconstrictor but also as a growth promoter via angiotensin II type 1 receptor (AT1R) in some cancer. In this study, we examined the mechanisms by which Ang II affected the cell proliferation in AT1R-positive MCF-7 human breast cancer cells. Ang II stimulated the growth of breast cancer cells in a dose- and time-dependent manner. The maximal proliferation effect on MCF-7 cells was obtained with 10(-4) M Ang II at 24 h. Losartan (10(-5) M, an AT1R antagonist) significantly decreased the level of Ang-II-induced proliferative effects, whereas PD123319 (10(-5) M, an AT2R antagonist) had no effects. Moreover, Ang II could significantly accelerate S-phase progression, which was inhibited by losartan (10(-5) M) or LY294002 (50 microM, a PI3-kinase inhibitor). In addition, Ang II caused rapid activation of p-Akt in a dose- and time-dependent manner. 10(-4) M Ang II induced a significant increase of p-Akt in 15 min. The peak level of p-Akt could be persisted for at least 6 h. Among the signaling molecules downstream of Akt, we revealed that Ang II also significantly upregulated CyclinD1, GSK3beta, and downregulated p27. Pretreatment with losartan (10(-5) M) or LY294002 (50 microM) could significantly suppress these effects of Ang II. These findings suggest that Ang II plays a role in the growth of AT1R-positive breast cancer cells through PI3-kinase/Akt pathway activation. Therefore, targeting Ang II/AT1R signaling could be a novel therapeutic for breast cancer.

Topics: Angiotensin II; Angiotensin II Type 1 Receptor Blockers; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chromones; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Dose-Response Relationship, Drug; Enzyme Inhibitors; Female; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Imidazoles; Losartan; Morpholines; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Pyridines; Receptor, Angiotensin, Type 1; Signal Transduction; Vasoconstrictor Agents

Poor oral bioavailability limits the use of curcumin and other dietary polyphenols in the prevention and treatment of cancer. Minimally invasive strategies that can provide effective and sustained tissue concentrations of these agents will be highly valuable tools in the fight against cancer. The objective of this study was to investigate the use of an injectable sustained release microparticle formulation of curcumin as a novel approach to breast cancer chemoprevention. A biodegradable and biocompatible polymer, poly(d,l-lactide-co-glycolide), was used to fabricate curcumin microparticles. When injected s.c. in mice, a single dose of microparticles sustained curcumin levels in the blood and other tissues for nearly a month. Curcumin levels in the lungs and brain, frequent sites of breast cancer metastases, were 10- to 30-fold higher than that in the blood. Further, curcumin microparticles showed marked anticancer efficacy in nude mice bearing MDA-MB-231 xenografts compared with other controls. Repeated systemic injections of curcumin were not effective in inhibiting tumor growth. Treatment with curcumin microparticles resulted in diminished vascular endothelial growth factor expression and poorly developed tumor microvessels, indicating a significant effect on tumor angiogenesis. These results suggest that sustained delivery of chemopreventives such as curcumin using polymeric microparticles is a promising new approach to cancer chemoprevention and therapy.

Topics: Animals; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Growth Processes; Cell Line, Tumor; Curcumin; Cyclin D1; Cyclooxygenase 2; Delayed-Action Preparations; Down-Regulation; Female; Humans; Lactic Acid; Matrix Metalloproteinase 9; Mice; Mice, Inbred BALB C; Neovascularization, Pathologic; Particle Size; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Random Allocation; Vascular Endothelial Growth Factor A

Control of cell cycling and proliferation is critical to the development of neoplasia and may play a role in the pathogenesis of phyllodes tumours (PTs). This study aimed to evaluate the immunohistochemical expression of certain proteins from the G(1)/S transition of the cell cycle in a cohort of PTs, to determine their role in tumour pathogenesis and to identify any associations with patient outcome.. Sixty-five PTs (34 benign, 23 borderline and eight malignant) diagnosed at a single institution between 1990 and 2006 were analysed. Immunohistochemistry for p16, pRb, cyclin D1 and Ki67 was performed. Expression of the following markers increased significantly with tumour grade: stromal nuclear and cytoplasmic p16 (P = 0.01 and 0.002, respectively), stromal and epithelial pRb (P = 0.000,000,06 and 0.004, respectively), and stromal and epithelial Ki67 (P = 0.03 and 0.04, respectively). Epithelial pRb scores of 7 (range 0-7) were significantly associated with reduced disease-free survival (DFS) compared with scores of <7 (P = 0.0009). No relationship was found between cyclin D1 expression in either the epithelium or the stroma, and grade or DFS.. The results suggest that alterations at the G(1)/S transition of the cell cycle play an important role in the progression of PTs.

Topics: Biomarkers, Tumor; Blotting, Western; Breast Neoplasms; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Disease-Free Survival; Female; Humans; Immunohistochemistry; Ki-67 Antigen; Neoplasm Proteins; Phyllodes Tumor; Retinoblastoma Protein; Tissue Array Analysis

Rac1 GTPase regulates a variety of signaling pathways that are implicated in malignant phenotypes. Here, we show that selective inhibition of Rac1 activity by the pharmacologic inhibitor NSC23766 suppressed cell growth in a panel of human breast cancer cell lines, whereas it had little toxicity to normal mammary epithelial cells. NSC23766 elicits its cytotoxicity via two distinct mechanisms in a cell line-dependent manner: induction of G(1) cell cycle arrest in cell lines (MDA-MB-231, MCF7, and T47D) that express retinoblastoma (Rb) protein or apoptosis in Rb-deficient MDA-MB-468 cells. In MDA-MB-231 cells, Rac1 inhibition induced G(1) cell cycle arrest through downregulation of cyclin D1 and subsequent dephosphorylation/inactivation of Rb. By contrast, MDA-MB-468 cells underwent substantial apoptosis that was associated with loss of antiapoptotic proteins survivin and X-linked inhibitor of apoptosis protein (XIAP). Rac1 knockdown by RNAi interference confirmed the specificity of NSC23766 and requirement for Rac1 in the regulation of cyclin D1, survivin, and XIAP in breast cancer cells. Further, NF-kappaB, but not c-Jun NH(2)-terminal kinase or p38 pathways, mediates the survival signal from Rac1. Overall, our results indicate that Rac1 plays a central role in breast cancer cell survival through regulation of NF-kappaB-dependent gene products.

Topics: Aminoquinolines; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Down-Regulation; Epithelial Cells; Extracellular Signal-Regulated MAP Kinases; Female; G1 Phase; Humans; Inhibitor of Apoptosis Proteins; JNK Mitogen-Activated Protein Kinases; Mammary Glands, Human; MAP Kinase Signaling System; Microtubule-Associated Proteins; Neoplasm Proteins; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Pyrimidines; rac1 GTP-Binding Protein; Survivin; X-Linked Inhibitor of Apoptosis Protein

Anterior-gradient 2 (AGR2) is an estrogen-responsive secreted protein. Its upregulation has been well documented in a number of cancers, particularly breast cancer, for which mixed data exist on the prognostic implications of AGR2 expression. Although emerging evidence indicates that AGR2 is associated with poor prognosis, its function and impact on cancer-relevant pathways have not been elucidated in breast cancer.. To investigate the biologic role of AGR2 in breast cancer, AGR2 was transiently knocked down, by using siRNA, in T47 D and ZR-75-1 (estrogen receptor-alpha (ER)-positive) and MDA-MB-231 and SK-BR-3 (ER-negative) human breast cancer cell lines. The impact of silencing AGR2 was evaluated in both anchorage-dependent and anchorage-independent growth (soft agar, spheroid) assays. Cell-cycle profiles in ER-positive cell lines were determined with BrdU incorporation, and cell death was measured with Annexin V, JC-1, and F7-26 staining. After transiently silencing AGR2 or stimulating with recombinant AGR2, modulation of key regulators of growth and survival pathways was assessed with Western blot. Combination studies of AGR2 knockdown with the antiestrogens tamoxifen and fulvestrant were carried out and assessed at the level of anchorage-dependent growth inhibition and target modulation (cyclin D1, ER).. AGR2 knockdown inhibited growth in anchorage-dependent and anchorage-independent assays, with a more-pronounced effect in ER-positive cell lines. Cyclin D1 levels and BrdU incorporation were reduced with AGR2 knockdown. Conversely, cyclin D1 was induced with recombinant AGR2. AGR2 knockdown induced cell death in ZR-75-1 and T47 D cells, and also downregulated survivin and c-Myc. Evidence of AGR2-ER crosstalk was demonstrated by a reduction of ER at the protein level after transiently silencing AGR2. AGR2 knockdown in combination with fulvestrant or tamoxifen did not preclude the efficacy of the antiestrogens, but enhanced it. In addition, p-Src, implicated in tamoxifen resistance, was downregulated with AGR2 knockdown.. Transiently silencing AGR2 in ER-positive breast cancer cell lines inhibited cell growth and cell-cycle progression and induced cell death. Breast cancer drivers (ER and cyclin D1) as well as cancer-signaling nodes (pSrc, c-Myc, and survivin) were demonstrated to be downstream of AGR2. Collectively, the data presented support the utility of anti-AGR2 therapy in ER-positive breast cancers because of its impact on cancer-relevant pathways.

Topics: Animals; Apoptosis; Blotting, Western; Breast Neoplasms; Cell Adhesion; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colony-Forming Units Assay; Cyclin D1; Estradiol; Estrogen Antagonists; Estrogen Receptor alpha; Female; Flow Cytometry; Fulvestrant; Humans; Immunization; Inhibitor of Apoptosis Proteins; Membrane Potential, Mitochondrial; Microtubule-Associated Proteins; Mucoproteins; Oncogene Proteins; Proteins; Rats; Rats, Inbred Lew; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Survivin; Tamoxifen

Cyclin D1 is a mediator of cell-cycle control that is frequently overexpressed in primary ductal breast carcinomas, but its role is controversial. A polymorphism in the CCND1 gene, G870A, results in an aberrantly spliced protein (cyclin D1b) lacking the Thr-286 phosphorylation site necessary for nuclear export. Studies of murine fibroblasts have shown that although overexpression of canonical cyclin D1 (cyclin D1a) alone is not sufficient to drive malignant transformation, expression of nuclear cyclin D1b is oncogenic. Our objectives were to determine whether cyclin D1b is expressed in human breast carcinomas and to characterize the relationship of this protein to both cyclin D1a and clinical outcome in breast cancer patients.. We performed a prospective cohort study of women with early-stage breast cancer and analyzed cyclin D1a and D1b expression in primary breast tumor sections. Expression was tested for correlation with other breast cancer prognostic factors and clinical outcome, including recurrence or death.. A total of 118 patients were included in this analysis, with a median follow-up of 44 months. Cyclin D1b was expressed in 26% of tumors and cyclin D1a was overexpressed in 27%; co-expression occurred in 4%. Cyclin D1a and/or D1b expression were not significantly associated with estrogen or progesterone receptor negativity, Her2 overexpression, young age, lymph node positivity, high tumor grade, nor large tumor size. The risk of recurrence was higher in those co-expressing D1a and D1b compared to the expression of either alone (relative risk=5.3, 95% confidence interval 1.27 to 22.1, p=0.02). The hazard ratio for those with co-expression compared with those without was 6.05 (p=0.04).. Expression of cyclin D1b occurs in primary human breast carcinomas and its coexpression with cyclin D1a may be a marker for increased recurrence risk, independently of other factors.

Topics: Adult; Aged; Aged, 80 and over; Breast Neoplasms; Cohort Studies; Cyclin D1; Female; Follow-Up Studies; Humans; Immunohistochemistry; Middle Aged; Neoplasm Recurrence, Local; Neoplasm Staging; Prognosis; Prospective Studies; Protein Isoforms; Risk Factors; RNA, Messenger

Luciferase reporter constructs and transient co-transfection approaches demonstrate that elevated expression of RORalpha1 augments 17-beta-estradiol (E(2))-induced transcriptional activation of the full-length ERalpha, but not truncated ERalpha constructs (ABCD or CDEF), in MCF-7 breast cancer and HEK293 embryonic kidney cells, and that physiologic concentrations of MLT inhibit the individual and combined transcriptional activity of ERalpha by RORalpha1 and E(2). Gel mobility shift and co-immunoprecipitation (IP)/pull-down assays demonstrate that RORalpha1 and ERalpha do not interact directly at the DNA-binding level or as heterodimers, however, RORalpha1 augments E(2)-induced pS2 and cyclin D1 mRNA expression while MLT inhibits RORalpha1/E(2)-induced expression of pS2 and cyclin D1 in MCF-7 cells.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Estradiol; Estrogen Receptor alpha; Humans; Melatonin; Mitosis; Nuclear Receptor Subfamily 1, Group F, Member 1; Protein Multimerization; Protein Structure, Quaternary; Receptor Cross-Talk; Response Elements; Signal Transduction; Transcription, Genetic; Transcriptional Activation; Trefoil Factor-1; Tumor Suppressor Proteins

BZL101 is an aqueous extract from the Scutellaria barbata plant shown to have anticancer properties in a variety of human cancers. In order to determine its efficacy on human reproductive cancers, we assessed the responses of two human breast cancer cell lines, estrogen sensitive MCF7 and estrogen insensitive MDA-MB-231, and of two human prostate cancer cell lines, androgen sensitive LNCaP and androgen insensitive PC3 which are human cell lines that represent early and late stage reproductive cancers. BZL101 inhibited reproductive cancer growth in all cell lines by regulating expression levels of key cell cycle components that differ with respect to the cancer cell phenotypes. In early stage estrogen sensitive MCF7 cells, BZL101 induced a G₁ cell cycle arrest and ablated expression of key G₁ cell cycle regulators Cyclin D1, CDK2 and CDK4, as well as growth factor stimulatory pathways and estrogen receptor-α expression. Transfection of luciferase reporter plasmids revealed that the loss of CDK2, CDK4 and estrogen receptor-α transcript expression resulted from the BZL-dependent ablation of promoter activities. BZL101 growth arrests early stage androgen sensitive LNCaP cells in the G₂/M phase with corresponding decreases in Cyclin B1, CDK1 and androgen receptor expression. In late stage hormone insensitive breast (MDA-MB-231) and prostate (PC3) cancer cells, BZL101 induced an S phase arrest with corresponding ablations in Cyclin A2 and CDK2 expression. Our results demonstrate that BZL101 exerts phenotype specific anti-proliferative gene expression responses in human breast and prostate cancer cells, which will be valuable in the potential development of BZL-based therapeutic strategies for human reproductive cancers.

Topics: Antineoplastic Agents, Phytogenic; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin A2; Cyclin B1; Cyclin D1; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Female; Flow Cytometry; G1 Phase; Gene Expression; Humans; Male; Phytotherapy; Plant Extracts; Prostatic Neoplasms; Receptors, Androgen; Receptors, Estrogen; Receptors, Growth Factor; Reverse Transcriptase Polymerase Chain Reaction; Scutellaria

Previous studies have shown alterations in the cell cycle regulatory proteins in breast carcinomas. However, the results of these studies remain controversial. Cyclin D1 (CCND1) and p27(KIP1) (CDKN1B) are two essential regulators of cell cycle progression. This study aimed to investigate the associations of CCND1 A870G and CDKN1B C79T polymorphisms with breast cancer risk.. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to determine the genotype and allelic frequencies of polymorphisms. Seventy-eight breast cancer patients and 84 age-matched healthy controls were included in the study.. Frequencies of CT genotype and T allele of CDKN1B were found to be higher in breast cancer patients than in controls (p=0.013, OR: 1.514 95% CI: 1.086-2.114.15; p=0.007, OR=1.496; 95% CI: 1.111-2.014, respectively). The frequency of AA genotype of CCND1 was decreased in hormone receptor- (estrogen and progesterone receptors) negative patients with breast cancer (p<0.049, OR=0.286; 95% CI: 0.071-1.142). Even though CDKN1B polymorphism appears to be an important predictive factor for breast cancer risk and CCND1 polymorphism may be a prognostic biomarker for breast cancer, further investigations with larger study groups are needed to fully elucidate the role of CCND1 and CDKN1B polymorphisms in the development and prognosis of breast cancer.

Topics: Adult; Aged; Alleles; Breast Neoplasms; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Female; Genetic Predisposition to Disease; Genotype; Humans; Intracellular Signaling Peptides and Proteins; Middle Aged; Neoplasm Staging; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Polymorphism, Single Nucleotide

eIF4E is over-expressed in many tumours, including a high proportion of breast cancers. eIF4E is an oncogene, and signalling pathways which promote eIF4E activity represent potential targets for therapeutic intervention in cancer. MNKs phosphorylate eIF4E on serine 209, a modification that can be required for eIF4E-dependent cell transformation. There is therefore a clear requirement to determine the role of MNKs in the proliferation and survival of cells from the major human tumours, such as breast cancer. Phosphorylated eIF4E protein was readily detectable in some breast tumour samples, but was below the limits of detection in others. Of 6 breast cancer cell lines representing the major sub-types of breast cancer, phosphorylated eIF4E was readily detectable in 5 of them, with MCF-7 cells displaying markedly lower levels. Long term colony forming assays demonstrated that all the five lines with high levels of phosphorylated eIF4E were highly sensitive to a MNK inhibitor. In short term assays, a range of responses was revealed. MCF-7 cells were insensitive in both assays. The anti-proliferative effects of the MNK inhibitor in breast cancer cells are primarily cytostatic, rather than cytotoxic, and are potentially due to the inhibition of cyclin D1 synthesis. Our data provide evidence that the sensitivity of breast cancer cells to MNK inhibition may correlate with baseline levels of eIF4E phosphorylation, and suggest that a proportion of breast cancers could be sensitive to inhibiting MNK kinase activity, and that the presence of phosphorylated eIF4E could provide a biomarker for the identification of responsive tumours.

Topics: Aniline Compounds; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Eukaryotic Initiation Factor-4E; Female; Humans; Intracellular Signaling Peptides and Proteins; Phosphorylation; Protein Serine-Threonine Kinases; Purines

Topics: Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Non-Small-Cell Lung; Clinical Trials as Topic; Cyclin D1; ErbB Receptors; Female; Humans; Lung Neoplasms; Mutation; Neoplasms; Proto-Oncogene Proteins; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins p21(ras); ras Proteins; Research Design; Retinoid X Receptors; Vascular Endothelial Growth Factor A

To compare the morphologic and immunohistochemical properties of breast carcinomas from Chinese and Australian women in order to define possible biologic differences between these carcinomas.. Three hundred cases of breast carcinomas were assessed for histologic and immunophenotypic characteristics from the pathology archives of the Changhai and St. Vincent's Hospitals.. The Chinese women had proportionally more grade 2 and 3 tumors, whereas Australian women had a higher proportion of grade 1 tumor. There was a higher proportion of younger patients with a larger tumor and patients with lymph node involvement in the Chinese group as compared with Australian women. There was no difference in rate of estrogen receptor positive tumors between the 2 groups. p53 Expression was statistically more common with less cyclin D1 expression in Chinese as compared with Australian women.. This study indicates that both inherent tumor biology and stage at presentation influence adversely affect the outcome of breast carcinoma in Chinese compared as with Australian women.

Topics: Asian People; Australia; Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Ductal, Breast; China; Cyclin D1; Female; Humans; Immunohistochemistry; Middle Aged; Tumor Suppressor Protein p53; White People

Prognostic factors in patients who are diagnosed with T4 breast carcinomas are widely awaited. We here evaluated the clinical role of some molecular alterations involved in tumorigenesis in a well-characterized cohort of T4 breast cancer patients with a long follow-up period.. A consecutive series of 53 patients with T4 breast carcinoma was enrolled between 1992 and 2001 in Sardinia, and observed up for a median of 125 months. Archival paraffin-embedded tissue sections were used for immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) analyses, in order to assess alterations in expression levels of survivin, p53, and pERK1-2 proteins as well as in amplification of CyclinD1 and h-prune genes. The Kaplan-Meier and Cox regression methods were used for survival assessment and statistical analysis.. Overall, patients carrying increased expression of pERK1-2 (p = 0.027) and survivin (p = 0.008) proteins as well as amplification of h-prune gene (p = 0.045) presented a statistically-significant poorer overall survival in comparison with cases found negative for such alterations. After multivariate analysis, the pathological response to primary chemotherapy and the survivin overexpression in primary carcinoma represented the main parameters with a role as independent prognostic factors in our series.. Although retrospective, our study identified some molecular parameters with a significant impact on prediction of the response to therapy or prognosis among T4 breast cancer patients. Further large prospective studies are needed in order to validate the use of such markers for the management of these patients.

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Breast Neoplasms; Carrier Proteins; Cyclin D1; Female; Follow-Up Studies; Gene Amplification; Gene Expression Regulation, Neoplastic; Humans; Immunoenzyme Techniques; In Situ Hybridization, Fluorescence; Inhibitor of Apoptosis Proteins; Italy; Microtubule-Associated Proteins; Middle Aged; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neoplasm Staging; Phosphoric Monoester Hydrolases; Retrospective Studies; Survival Rate; Survivin; Treatment Outcome; Tumor Suppressor Protein p53

EGF induces the translocation of EGF receptor (EGFR) from the cell surface to the nucleus where EGFR activates gene transcription through its binding to an AT-rich sequence (ATRS) of the target gene promoter. However, how EGFR, without a DNA-binding domain, can bind to the gene promoter is unclear. In the present study, we show that RNA helicase A (RHA) is an important mediator for EGFR-induced gene transactivation. EGF stimulates the interaction of EGFR with RHA in the nucleus of cancer cells. The EGFR/RHA complex then associates with the target gene promoter through binding of RHA to the ATRS of the target gene promoter to activate its transcription. Knockdown of RHA expression in cancer cells abrogates the binding of EGFR to the target gene promoter, thereby reducing EGF/EGFR-induced gene expression. In addition, interruption of EGFR-RHA interaction decreases the EGFR-induced promoter activity. Consistently, we observed a positive correlation of the nuclear expression of EGFR, RHA, and cyclin D1 in human breast cancer samples. These results indicate that RHA is a DNA-binding partner for EGFR-mediated transcriptional activation in the nucleus.

Topics: Active Transport, Cell Nucleus; Breast Neoplasms; Cell Nucleus; Cyclin D1; DNA; ErbB Receptors; Gene Expression Regulation; HeLa Cells; Humans; Promoter Regions, Genetic; Protein Binding; RNA Helicases; Transcriptional Activation

Recent reports have shown that t-DARPP (truncated isoform of DARPP-32) can mediate trastuzumab resistance in breast cancer cell models. In this study, we evaluated expression of t-DARPP in human primary breast tumors, and investigated the role of t-DARPP in regulating growth and proliferation in breast cancer cells.. Quantitative real time RT-PCR analysis using primers specific for t-DARPP demonstrated overexpression of t-DARPP in 36% of breast cancers (13/36) as opposed to absent to very low t-DARPP expression in normal breast tissue (p < 0.05). The mRNA overexpression of t-DARPP was overwhelmingly observed in ductal carcinomas, including invasive ductal carcinomas and intraductal carcinomas, rather than other types of breast cancers. The immunohistochemistry analysis of DARPP-32/t-DARPP protein(s) expression in breast cancer tissue microarray that contained 59 tumors and matched normal tissues when available indicated overexpression in 35.5% of primary breast tumors that were more frequent in invasive ductal carcinomas (43.7%; 21/48). In vitro studies showed that stable overexpression of t-DARPP in MCF-7 cells positively regulated proliferation and anchorage-dependent and -independent growth. Furthermore, this effect was concomitant with induction of phosphorylation of AKT(ser473) and its downstream target phospho(ser9) GSK3β, and increased Cyclin D1 and C-Myc protein levels. The knockdown of endogenous t-DARPP in HCC1569 cells led to a marked decrease in phosphorylation of AKTs(ser473) and GSK3β(ser9). The use of PI3K inhibitor LY294002 or Akt siRNA abrogated the t-DARPP-mediated phosphorylation of AKT(ser473) and led to a significant reduction in cell growth.. Our findings underscore the potential role of t-DARPP in regulating cell growth and proliferation through PI3 kinase-dependent mechanism.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chromones; Cyclin D1; Dopamine and cAMP-Regulated Phosphoprotein 32; Female; Humans; Immunoblotting; Immunohistochemistry; Morpholines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Polymerase Chain Reaction; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-myc; RNA, Small Interfering

IκB Kinase ε (IKKε) is a member of the IKK family which plays an important role in the activation of nuclear factor-κB (NF-κB). Overexpressed in over 30% of breast cancers, IKKε has been recently identified as a potential breast cancer oncogene. The purpose of this study is to examine the therapeutic potential of IKKε siRNA on human breast cancer cells.. Eight siRNAs targeting different regions of the IKKε mRNA were designed, and the silencing effect was screened by quantitative real time RT-PCR. The biological effects of synthetic siRNAs on human breast cancer cells were investigated by examining the cell proliferation, migration, invasion, focus formation, anchorage-independent growth(via soft agar assay), cell cycle arrest, apoptosis (via annexing binding), NF-κB basal level, and NF-κB related gene expressions upon the IKKε silencing.. Silencing of IKKε in human breast cancer cells resulted in decrease of focus formation potential and clonogenicity as well as in vitro cell migration/invasion capabilities. Moreover, knockdown of IKKε suppressed cell proliferation. Cell cycle assay showed that the anti-proliferation effect of IKKε siRNA was mediated by arresting cells in G(0)/G(1) phase, which was caused by down-regulation of cyclin D(1). Furthermore, we demonstrated that silencing of IKKε inhibited the NF-κB basal activity as well as the Bcl-2 expression. Significant apoptosis was not observed in breast cancer cells upon the silencing of IKKε. The present study provided the first evidence that silencing IKKε using synthetic siRNA could inhibit the invasiveness properties and proliferation of breast cancer cells.. Our results suggested that silencing IKKε using synthetic siRNA may offer a novel therapeutic strategy for breast cancer.

Topics: Apoptosis; Breast Neoplasms; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Down-Regulation; Female; Humans; I-kappa B Kinase; Neoplasm Invasiveness; NF-kappa B; Proto-Oncogene Proteins c-bcl-2; RNA Interference; RNA, Small Interfering; Signal Transduction

The DNA damage response (DDR) activates downstream pathways including cell cycle checkpoints. The cyclin D1 gene is overexpressed or amplified in many human cancers and is required for gastrointestinal, breast, and skin tumors in murine models. A common polymorphism in the human cyclin D1 gene is alternatively spliced, resulting in cyclin D1a and D1b proteins that differ in their carboxyl terminus. Cyclin D1 overexpression enhances DNA damage-induced apoptosis. The role of cyclin D1 and the alternative splice form in regulating the DDR is not well understood. Herein cyclin D1a overexpression enhanced the DDR as characterized by induction of γH2AX phosphorylation, the assembly of DNA repair foci, specific recruitment of DNA repair factors to chromatin, and G(2)-M arrest. Cyclin D1 deletion in fibroblasts or small interfering RNA-mediated reduction of endogenous cyclin D1 in colon cancer cells reduced the 5-fluorouracil-mediated DDR. Mechanistic studies showed that cyclin D1a, like DNA repair factors, elicited the DDR when stably associated with chromatin.

Topics: Alternative Splicing; Animals; Antimetabolites, Antineoplastic; Blotting, Western; Breast Neoplasms; Cells, Cultured; Chromatin; Colonic Neoplasms; Comet Assay; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; DNA Damage; DNA Repair; Embryo, Mammalian; Fibroblasts; Fluorescent Antibody Technique; Fluorouracil; Gene Expression Regulation, Neoplastic; Histones; Humans; Immunoprecipitation; Mice; Phosphorylation; Protein Isoforms

The oncogene cyclin D1 is highly expressed in many breast cancers and, despite its proliferation-activating properties, it has been linked to a less malignant phenotype. To clarify this observation, we focused on two key components of malignant behavior, migration and proliferation, and observed that quiescent G(0)/G(1) cells display an increased migratory capacity compared to cycling cells. We also found that the down-regulation of cyclin D1 in actively cycling cells significantly increased migration while also decreasing proliferation. When analyzing a large set of premenopausal breast cancers, we observed an inverse proliferation-independent link between cyclin D1 and tumor size and recurrence, suggesting that this protein might abrogate infiltrative malignant behavior in vivo. Finally, gene expression analysis after cyclin D1 down-regulation by siRNA confirmed changes in processes associated with migration and enrichment of our gene set in a metastatic poor prognosis signature. This novel function of cyclin D1 illustrates the interplay between tumor proliferation and migration and may explain the attenuation of malignant behavior in breast cancers with high cyclin D1 levels.

Topics: Biomarkers, Tumor; Breast Neoplasms; Carcinoma; Cell Movement; Cell Proliferation; Cyclin D1; Down-Regulation; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Microarray Analysis; Neoplasm Invasiveness; Oncogenes; Prognosis; RNA, Small Interfering; Tumor Cells, Cultured; Up-Regulation

To investigate the role that E-cadherin (E-cad) plays on cell adhesion and proliferation of human breast carcinoma.. E-cad expression vector was transfected into an E-cad-negative human breast carcinoma MDA-MB-231 cells. G418 was used to screen positive clones. E-cad, β-catenin (β-cat) and cyclin D1 expressions of these clones were confirmed by Western blot. Their cell-cell and cell-matrix adhesion abilities were detected. E-cad/β-catenin interaction was confirmed by immunoprecipitation. Cell proliferation was evaluated by MTT. Cell apoptosis was analyzed by flow cytometry. Direct two-step immunocytochemistry was used to detect the localization of β-cat.. E-cad(+) cell strains Ecad-231-7 and Ecad-231-9 were established. When cultured in ultra-low-binding dishes Ecad-231 cells grow in suspension while Ecad-231-7 and Ecad-231-9 cells grow in large clamps. When co-cultured with HCT116 cells, the average adhesion rates at 30 min are 39.0%, 60.0% and 59.5% for MDA-MB-231, Ecad-231-7 and Ecad-231-9 respectively. The average detachment rates by EDTA for 5 min are 37.4%, 4.2% and 7.4% respectively. So E-cad expression enhanced hemotypic and heterotypic cell-cell adhesion and cell-matrix adhesion. Forced exogenously expressed E-cad could combine with endogenous β-cat, whereas down stream cyclin D1 expression was significantly decreased, as evidenced by Western blot. The rates of cell apoptosis of MDA-MB-231, Ecad-231-7 and Ecad-231-9 were 1.8%, 2.0% and 2.1%. Expression of E-cad had no obvious effect on the apoptosis of tumor cells with regular culture. β-cat increased in the cytoplasma.. Two monoclonal tumor cell strains (Ecad-231-7 and Ecad-231-9) stably expressing E-cad were successfully established. E-cad could enhance adhesion and inhibit proliferation of human breast carcinoma cells through a pathway involving β-cat and cyclin D1.

Topics: Apoptosis; beta Catenin; Breast Neoplasms; Cadherins; Cell Adhesion; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Genetic Vectors; Humans; Plasmids; Transfection

Topics: Antineoplastic Agents; Breast Neoplasms; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Female; Humans; Receptors, Estrogen; Retinoblastoma Protein; Signal Transduction

To investigate the therapeutic effect of methylselenocysteine (MSC) combined with tamoxifen in MCF-7 breast cancer xenograft and the underlying mechanisms. MCF-7 breast cancer xenograft was established in ovariectomized female athymic nude mice and treated with tamoxifen and/or MSC. Tumor size was measured twice a week. Immunohistochemistry and TUNEL assays were used to measure ERalpha expression, ERalpha target genes (progesterone receptor (PR) and cyclin D1 expression), Ki-67 index, apoptosis and microvessel density. Combined treatment with tamoxifen and MSC synergistically inhibited tumor growth compared to MSC alone and tamoxifen alone. MSC alone or MSC + tamoxifen significantly reduced ERalpha, PR and cyclin D1, Ki67 index and microvessel density while increasing apoptosis in tumor tissues. These findings demonstrate synergistic growth inhibition of ERalpha positive breast cancer xenografts by combination of tamoxifen with organic selenium compounds. Organic selenium may provide added benefit when combined with tamoxifen in adjuvant therapy or prevention.

Topics: Adenocarcinoma; Angiogenesis Inhibitors; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Breast Neoplasms; Cell Division; Cyclin D1; Cysteine; Drug Synergism; Estradiol; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Nude; Neoplasm Proteins; Neoplasms, Hormone-Dependent; Neovascularization, Pathologic; Organoselenium Compounds; Random Allocation; Receptors, Progesterone; Selenocysteine; Specific Pathogen-Free Organisms; Tamoxifen; Xenograft Model Antitumor Assays

To investigate the expression and association of ER, Ki-67 and cyclinD1 in usual ductal hyperplasia(UDH), atypical ductal hyperplasia (ADH) and ductal carcinoma in situ(DCIS) in the breast. The study included 56 cases of pre-cancerous lesions which were surgically excised at Qi Lu Hospital of Shangdong University. Immunohistochemistry was used to determine the expression of ER, Ki-67 and cyclinD1 and double-labelling immunofluorescence technique was used to observe the coexpression of ER and Ki-67. The expression and distribution of ER-positive cells were significantly different in UDH, ADH and DCIS. The ER-positive cells were much more in UDH than in normal TDLUs (terminal duct lobular units). The distribution of ER-positive cells interspersed amid ER-negative cells within UDH. However , the ER positive cells showed marked increases in ADH and low grade nuclear DCIS (P < 0.05), distributing in almost all constituent cells. The expression of ki-67 and cyclinD1 were significantly different between UDH and DCIS (P < 0.05) , and a positive correlation was found between expression of Ki-67 and morphological classification of pre-cancerous lesions (r = 0.3522, P < 0.05) as well as cyclinD1 (r = 0.3901, P < 0.05). Double-labelling immunofluorescence showed that there was no coexpression of ER and Ki-67 in normal breast tissue. The coexpression of the two markers was found in ADH and increased in DCIS. Overexpression of ER, Ki-67 and cyclinD1 significantly accompanies the transition of normal cells and UDH to ADH and DCIS. The coexpression of ER and ki-67 may present the early change in carcinogenesis of breast cancer.

Topics: Breast; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Intraductal, Noninfiltrating; Cyclin D1; Female; Fluorescent Antibody Technique, Indirect; Humans; Immunoenzyme Techniques; Ki-67 Antigen; Lymphatic Metastasis; Microscopy, Fluorescence; Precancerous Conditions; Prognosis; Receptors, Estrogen

The breast cancer susceptibility gene 1 (BRCA1) is mutated in approximately 50% of hereditary breast cancers, and its expression is decreased in 30-40% of sporadic breast cancers, suggesting a general role in breast cancer development. BRCA1 physically and functionally interacts with estrogen receptor-alpha (ERalpha) and several transcriptional regulators. We investigated the relationship between cellular BRCA1 levels and tamoxifen sensitivity. Decreasing BRCA1 expression in breast cancer cells by small interfering RNA alleviated tamoxifen-mediated growth inhibition and abolished tamoxifen suppression of several endogenous ER-targeted genes. ER-stimulated transcription and cytoplasmic signaling was increased without detectable changes in ER or ER coregulator expression. Co-immunoprecipitation studies showed that with BRCA1 knockdown, tamoxifen-bound ERalpha was inappropriately associated with coactivators, and not effectively with corepressors. Chromatin immunoprecipitation studies demonstrated that with tamoxifen, BRCA1 knockdown did not change ERalpha promoter occupancy, but resulted in increased coactivator and decreased corepressor recruitment onto the endogenous cyclin D1 promoter. Our results suggest that decreased BRCA1 levels modify ERalpha-mediated transcription and regulation of cell proliferation in part by altering ERalpha-coregulator association. In the presence of tamoxifen, decreased BRCA1 expression results in increased coactivator and decreased corepressor recruitment on ER-regulated gene promoters.

Topics: Antineoplastic Agents, Hormonal; BRCA1 Protein; Breast Neoplasms; Cell Line, Tumor; Cyclin D1; Drug Resistance, Neoplasm; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Humans; Mutation; Promoter Regions, Genetic; RNA, Small Interfering; Signal Transduction; Tamoxifen; Transcription, Genetic

SEPT9_v1, the largest transcript of the septin gene family member, SEPT9, encodes a septin isoform implicated in the tumorigenic transformation of mammary epithelial cells. High levels of SEPT9_v1 expression also have been observed in both breast cancer cell lines, primary breast cancers as well as other solid tumor malignancies. We found a novel interaction between SEPT9_v1 and the c-Jun-N-terminal kinase (JNK), a mitogen-activated protein kinase important in cellular stress responses, cell proliferation, and cell survival. We found that up-regulation of SEPT9_v1 stabilizes JNK by delaying its degradation, thereby activating the JNK transcriptome. C-jun kinase assays in mammary epithelial cells expressing SEPT9_v1, compared to controls, exhibited increased JNK/c-Jun transcriptional activity. This increase was associated with increased levels of cyclin D1, a critical component of the proliferative response required for progression through G(1) of the cell cycle in many cell types. These findings demonstrate the first link between a septin protein and the JNK signaling pathway. Importantly, it suggests a novel functional role of SEPT9_v1 in driving cellular proliferation of mammary epithelial cells, a hallmark feature of oncogenesis that is directly relevant to breast cancer.

Topics: Breast Neoplasms; Cell Division; Cell Line, Transformed; Cell Line, Tumor; Cyclin D1; Epithelial Cells; Female; Gene Expression Regulation, Neoplastic; GTP Phosphohydrolases; Humans; JNK Mitogen-Activated Protein Kinases; Neoplasm Proteins; Protein Isoforms; Protein Structure, Tertiary; Recombinant Fusion Proteins; Septins; Sequence Deletion; Signal Transduction; Transduction, Genetic; Up-Regulation

Each of the three members of the p160 steroid receptor coactivator (SRC) family of coactivators (SRC-1, SRC-2 and SRC-3) stimulates estrogen receptor (ER)-alpha function in trans-activation assays. Consequently, we sought to elucidate their contributions to the ER-regulated processes of cell proliferation, apoptosis, and the expression of ERalpha target genes in MCF-7 breast cancer cells. The small interfering RNA depletion of SRC-2 or SRC-3 but not SRC-1 inhibited growth of MCF-7 cells, and this was reflected in decreased cell cycle progression and increased apoptosis in SRC-2- or SRC-3-depleted cells as well as a reduction in ERalpha transcriptional activity measured on a synthetic reporter gene. However, only SRC-3 depletion blocked estradiol stimulated cell proliferation. Depletion of SRC-1 did not affect these events, and together this reveals functional differences between each of the three SRC family coactivators. Regulation of the endogenous ERalpha target gene, c-myc was not affected by depletion of any of the p160 coactivators although depletion of each of them decreased pS2 mRNA expression in estradiol-treated MCF-7 cells. Moreover, progesterone receptor and cyclin D1 gene expression were decreased in SRC-3 small interfering RNA-treated cells. Expression of mRNA and protein levels for the antiapoptotic gene, Bcl-2 was dependent on SRC-3 expression, whereas Bcl-2 protein but not mRNA expression also was sensitive to SRC-1 depletion. Together these data indicate that the closely related p160 coactivators are not functionally redundant in breast cancer cells because they play gene-specific roles in regulating mRNA and protein expression, and they therefore are likely to make unique contributions to breast tumorigenesis.

Topics: Apoptosis; Blotting, Western; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Enzyme-Linked Immunosorbent Assay; Estrogen Receptor alpha; Flow Cytometry; Histone Acetyltransferases; Humans; Nuclear Receptor Coactivator 1; Nuclear Receptor Coactivator 2; Nuclear Receptor Coactivator 3; Receptors, Progesterone; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Trans-Activators; Transcription Factors; Transcription, Genetic; Transfection

8-Prenylnaringenin (8PN), one of the strongest plant-derived oestrogen receptors (ERs) ligand, has been suggested to have potential cancer chemo-preventive activities and anti-angiogenic properties. Because published data suggest that ERs serve as nodal point that allows interactions between hormones and growth factors mediated pathways, we decided to investigate the effects exerted by 8PN on Epidermal growth factor (EGF)-elicited pathways in breast cancer cells. Here we show that in ER positive MCF-7 cells, 8PN interferes with EGF induced cell proliferation by strongly inhibiting activation of PI(3)K/Akt pathway, without affecting EGFR expression or tyrosine phosphorylation, and exerting a synergistic activation of Erk1/2 phosphorylation. Moreover, we demonstrate that 8PN is a direct inhibitor of PI(3)K activity as it is shown by in vitro experiments with the purified enzyme and by its inability to impair serine phosphorylation of a constitutive active form of Akt. These findings suggest that inhibition of PI(3)K is a novel mechanism which contributes to 8PN activity to inhibit cancer cell survival and EGF induced proliferation.

Topics: Breast Neoplasms; Cell Line, Tumor; Cyclin D1; Enzyme Activation; Epidermal Growth Factor; Extracellular Signal-Regulated MAP Kinases; Female; Flavanones; Humans; Molecular Structure; Phosphatidylinositol 3-Kinases; Phytoestrogens; Receptors, Estrogen; Signal Transduction

Because signal transducer and activator of transcription 3 (STAT3) is constitutively activated in most human solid tumors and is involved in the proliferation, angiogenesis, immune evasion, and antiapoptosis of cancer cells, researchers have focused on STAT3 as a target for cancer therapy. We screened for natural compounds that inhibit the activity of STAT3 using a dual-luciferase assay. Cryptotanshinone was identified as a potent STAT3 inhibitor. Cryptotanshinone rapidly inhibited STAT3 Tyr705 phosphorylation in DU145 prostate cancer cells and the growth of the cells through 96 hours of the treatment. Inhibition of STAT3 Tyr705 phosphorylation in DU145 cells decreased the expression of STAT3 downstream target proteins such as cyclin D1, survivin, and Bcl-xL. To investigate the cryptotanshinone inhibitory mechanism in DU145 cells, we analyzed proteins upstream of STAT3. Although phosphorylation of Janus-activated kinase (JAK) 2 was inhibited by 7 micromol/L cryptotanshinone at 24 hours, inhibition of STAT3 Tyr705 phosphorylation occurred within 30 minutes and the activity of the other proteins was not affected. These results suggest that inhibition of STAT3 phosphorylation is caused by a JAK2-independent mechanism, with suppression of JAK2 phosphorylation as a secondary effect of cryptotanshinone treatment. Continuing experiments revealed the possibility that cryptotanshinone might directly bind to STAT3 molecules. Cryptotanshinone was colocalized with STAT3 molecules in the cytoplasm and inhibited the formation of STAT3 dimers. Computational modeling showed that cryptotanshinone could bind to the SH2 domain of STAT3. These results suggest that cryptotanshinone is a potent anticancer agent targeting the activation STAT3 protein. It is the first report that cryptotanshinone has antitumor activity through the inhibition of STAT3.

Topics: bcl-X Protein; Breast Neoplasms; Cell Growth Processes; Cell Line, Tumor; Cyclin D1; Dimerization; Down-Regulation; Drugs, Chinese Herbal; HCT116 Cells; HeLa Cells; Humans; Inhibitor of Apoptosis Proteins; Luciferases; Male; Microtubule-Associated Proteins; Models, Molecular; Phenanthrenes; Phosphorylation; Prostatic Neoplasms; Protein-Tyrosine Kinases; STAT3 Transcription Factor; Stomach Neoplasms; Survivin

Association studies have been widely used to search for common low-penetrance susceptibility alleles to breast cancer in general. However, breast cancer is a heterogeneous disease and it has been suggested that it may be possible to identify additional susceptibility alleles by restricting analyses to particular subtypes. We used data on 710 single nucleotide polymorphisms (SNP) in 120 candidate genes from a large candidate gene association study of up to 4,470 cases and 4,560 controls to compare the results of analyses of "overall" breast cancer with subgroup analyses based on the major clinicopathologic characteristics of breast cancer (stage, grade, morphology, and hormone receptor status). No SNP was highly significant in overall effects analysis. Subgroup analysis resulted in substantial reordering of ranks of SNPs, as assessed by the magnitude of the test statistics, and some associations that were not significant for an overall effect were detected in subgroups at a nominal 5% level adjusted for multiple testing. The most significant association of CCND1 SNP rs3212879 with estrogen receptor-negative tumor types (P = 0.001) did not reach genome-wide significance levels. These results show that it may be possible to detect associations using subgroup analysis that are missed in overall effects analysis. If the associations we found can be replicated in independent studies, they may provide important insights into disease mechanisms in breast cancer.

Topics: Aged; Alleles; Breast Neoplasms; Case-Control Studies; Cyclin D1; Female; Genetic Predisposition to Disease; Genetic Variation; Humans; Middle Aged; Neoplasm Staging; Polymorphism, Single Nucleotide; Surveys and Questionnaires

Lapatinib, a selective orally available inhibitor of epidermal growth factor receptor (EGFR) and ErbB2 receptor tyrosine kinases, is a promising agent for the treatment of breast cancer. We examined the effect of lapatinib on the development of mammary tumors in MMTV-erbB2 transgenic mice, which express wild-type ErbB2 under the control of the mouse mammary tumor virus promoter and spontaneously develop estrogen receptor (ER)-negative and ErbB2-positive mammary tumors by 14 months of age. Mice were treated from age 3 months to age 15 months with vehicle (n = 17) or lapatinib (30 or 75 mg/kg body weight; n = 16 mice per group) by oral gavage twice daily (6 d/wk). All statistical tests were two-sided. By 328 days after the start of treatment, all 17 (100%) of the vehicle-treated mice vs five (31%) of the 16 mice treated with high-dose lapatinib developed mammary tumors (P < .001). Among MMTV-erbB2 mice treated for 5 months (n = 20 mice per group), those treated with lapatinib had fewer premalignant lesions and noninvasive cancers in their mammary glands than those treated with vehicle (P = .02). Lapatinib also effectively blocked epidermal growth factor-induced signaling through the EGFR and ErbB2 receptors, suppressed cyclin D1 and epiregulin mRNA expression, and stimulated p27 mRNA expression in human mammary epithelial cells and in mammary epithelial cells from mice treated for 5 months with high-dose lapatinib. Thus, cyclin D1, epiregulin, and p27 may represent useful biomarkers of lapatinib response in patients. These data suggest that lapatinib is a promising agent for the prevention of ER-negative breast cancer.

Topics: Animals; Antineoplastic Agents; Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Cyclin D1; Epidermal Growth Factor; Epiregulin; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Lapatinib; Mammary Neoplasms, Experimental; Mice; Mice, Transgenic; Precancerous Conditions; Protein Kinase Inhibitors; Quinazolines; Receptor, ErbB-2; Receptors, Estrogen; RNA, Messenger; Signal Transduction

Overexpression of the human epidermal growth factor receptor 2 (HER2) in breast tumors is associated with bad prognosis. Therefore, it is highly relevant to further improve understanding of the regulatory mechanisms of HER2 expression. In addition to gene amplification, transcriptional regulation plays a crucial role in HER2 overexpression. In this study, we analyzed 3 polymorphisms E2F2_-5368_A>G, CCND1_870_A>G and CCND3_-677_C>T located in genes involved in cell cycle regulation in the GENICA population-based and age-matched breast cancer case-control study from Germany. We genotyped 1,021 cases and 1,015 controls by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Statistical analyses were performed by conditional logistic regression. We observed no differences in genotype frequencies between breast cancer cases and controls. Subgroup analysis showed associations between carriers of the E2F2_-5368_G allele (OR: 0.60, 95% CI: 0.42-0.85), carriers of the CCND1_870_G allele (OR: 0.66, 95% CI: 0.45-0.96) and carriers of the CCND3_-677_T allele (OR: 1.72, 95% CI: 1.20-2.49) and HER2 expression in breast tumors. This finding points to an association of an increased expression of these cell cycle regulators with lower expression of HER2. An explanation for this observation might be that low expression of E2F2, CCND1 and CCND3 decrease levels of factors down-regulating HER2. We conclude that the analyzed polymorphisms located in E2F2, CCND1 and CCND3 are potential markers for HER2 status of breast tumors.

Topics: Binding Sites; Biomarkers, Tumor; Breast Neoplasms; Case-Control Studies; Cyclin D1; Cyclin D3; Cyclins; DNA Mutational Analysis; E2F2 Transcription Factor; Female; Genotype; Germany; Humans; Immunoenzyme Techniques; Middle Aged; Neoplasm Staging; Polymorphism, Genetic; Prognosis; Receptor, ErbB-2; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Transcription Factors

Specific inhibitors of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) have been developed that efficiently inhibit the oncogenic RAF-MEK-ERK pathway. We used a systems-based approach to identify breast cancer subtypes particularly susceptible to MEK inhibitors and to understand molecular mechanisms conferring resistance to such compounds. Basal-type breast cancer cells were found to be particularly susceptible to growth inhibition by small-molecule MEK inhibitors. Activation of the phosphatidylinositol 3-kinase (PI3K) pathway in response to MEK inhibition through a negative MEK-epidermal growth factor receptor-PI3K feedback loop was found to limit efficacy. Interruption of this feedback mechanism by targeting MEK and PI3K produced synergistic effects, including induction of apoptosis and, in some cell lines, cell cycle arrest and protection from apoptosis induced by proapoptotic agents. These findings enhance our understanding of the interconnectivity of oncogenic signal transduction circuits and have implications for the design of future clinical trials of MEK inhibitors in breast cancer by guiding patient selection and suggesting rational combination therapies.

Topics: Breast Neoplasms; Camptothecin; Cell Line, Tumor; Cyclin D1; Drug Synergism; ErbB Receptors; Feedback, Physiological; G1 Phase; Humans; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase Kinases; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors

In order to evaluate the importance of estrogen production in tumor and surrounding tissues, we measured mRNA expression levels of 5 enzymes participating to estrogen synthesis in situ and 4 breast cancer-related proteins in 27 pairs of tumor and non-malignant tissues. Steroid sulfatase (STS) mRNA was more frequently detected in tumor tissues rather than in their non-malignant counterparts. Estrogen sulfotransferase (EST) was constantly expressed with high level not only in tumor tissues but also in their surrounding non-malignant counterparts. In contrast, mRNA expression levels of aromatase, and 17beta-hydroxysteroid dehydrogenase type I and II were relatively low and detected only in small proportion of the patients. We also measured the mRNA expression levels of the same nine genes in tumor tissues of 197 breast cancer patients, and analyzed relationship between the mRNA expression level and the clinicopathological parameters. The mRNA expression levels of STS, aromatase and erbB2 in tumor tissues increased as breast cancer progressed. The tumoral mRNA expression levels of STS, estrogen receptor beta, and erbB2 in patients with recurrence were higher than those in patients without recurrence. Upregulation of STS expression plays an important role in tumor progression of human breast cancer and is considered to be responsible for estrogen production in tumor and surrounding tissues.

Topics: 17-Hydroxysteroid Dehydrogenases; Adult; Aged; Aromatase; Breast Neoplasms; Cyclin D1; Estradiol Dehydrogenases; Estrogen Receptor alpha; Estrogen Receptor beta; Estrogens; Female; Humans; Middle Aged; Receptor, ErbB-2; RNA, Messenger; Steryl-Sulfatase; Sulfotransferases

The tumorigenic origin and progression capacity of isolated tumor cells in breast cancer sentinel lymph nodes are uncertain. True lymph node metastases are often associated with aberrant protein expression by means of immunohistochemistry. Therefore, evaluation of isolated tumor cells by immunohistochemistry for proteins often overexpressed in breast cancer such as cyclin D1 and p53 could provide relevant information with regard to the malignant potential of these cells. Of 383 consecutive patients with primary invasive breast cancer and sentinel lymph node involvement, 40 had isolated tumor cells in the sentinel lymph nodes, from which 16 sentinel lymph nodes contained isolated tumor cells (n = 16) in newly cut sections that could successfully be immunostained for cyclin D1 and p53. Immunohistochemistry was performed on the primary tumor as well as on the isolated tumor cells in the sentinel lymph nodes. Similarly stained sections of patients with sentinel lymph nodes micro- (n = 15) and macrometastases (n = 15) served as controls. Sentinel lymph node isolated tumor cells as a group showed significantly lower expression of cyclin D1 and p53 compared with micro- and macrometastases. Comparing cyclin D1 expression of the primary tumor with the corresponding sentinel lymph node metastases showed an increased expression in micro- and macrometastases (P = .08 and P = .0025, respectively) compared with the corresponding primary tumor. In patients with sentinel lymph node isolated tumor cells, however, there was no substantial difference in cyclin D1 expression between the primary tumor and the corresponding isolated tumor cells. This supports the hypothesis that some of these cells may be displaced benign cells or concern tumor cells with limited malignant potential compared with micro- and macrometastases.

Topics: Breast Neoplasms; Cyclin D1; Female; Humans; Immunohistochemistry; Lymph Nodes; Lymphatic Metastasis; Retrospective Studies; Sentinel Lymph Node Biopsy; Tumor Suppressor Protein p53

Uncontrolled growth of cancer cells can be related to dysfunctional cell cycle control, including entry into S-phase, initiating cell division. Cyclin CCND3 and CCNE1 along with CDK2 and CDK6 regulate this checkpoint, and genetic changes, detectable by fluorescence in situ hybridization, are hypothesized to increase the aggressiveness of breast cancer, thereby influencing patient survival. Genomic change was investigated in 106 primary breast cancer samples, where the combined gene copy number changes in one of these four cell cycle regulatory factors was observed in 22% of the 98 tumors of successful analysis, distributed with 15 deletions and 7 amplifications. A trend towards decreased survival was observed with the aberrations, suggesting a prognostic potential of this set of markers, which was supported by an association with tumor grade. For validation of the new set of FISH probes for the G1/S-phase cell cycle factors, two additional markers, frequently amplified in breast cancers, were included in this study: The G1/S phase control gene CCND1 and the proliferation marker MYC. Both markers were amplified in 14% and deleted in 5% of the cases. This is the first report of genomic deletions of MYC in breast cancer.

Topics: Breast Neoplasms; Cyclin D1; Female; G1 Phase; Gene Amplification; Humans; In Situ Hybridization, Fluorescence; Middle Aged; Prognosis; Proto-Oncogene Proteins c-myc; S Phase; Sequence Deletion; Survival Rate; Tissue Array Analysis

Aberrant expression of cyclin D1 protein is a common feature of breast cancer. However, the CCND1 gene encodes two gene products, cyclin D1a and cyclin D1b, which have discrete mechanisms of regulation and impact on cell behavior. A polymorphism at nucleotide 870 in the CCND1 gene, rs603965, influences the relative production of the encoded proteins and can impart increased risk for tumor development. Here, the impact of both the G/A870 polymorphism and cyclin D1b protein production on breast cancer risk, disease phenotype and patient outcome was analysed. In a large multiethnic case-control study, the G/A870 polymorphism conferred no significant risk for breast cancer overall or by stage or estrogen receptor (ER) status. However, the cyclin D1b protein was found to be upregulated in breast cancer, independent of cyclin D1a levels, and exhibited heterogeneous levels in breast cancer specimens. High cyclin D1a expression inversely correlated with the Ki67 proliferation marker and was not associated with clinical outcome. In contrast, elevated cyclin D1b expression was independently associated with adverse outcomes, including recurrence, distant metastasis and decreased survival. Interestingly, cyclin D1b was particularly associated with poor outcome in the context of ER-negative breast cancer. Thus, specific cyclin D1 isoforms are associated with discrete forms of breast cancer and high cyclin D1b protein levels hold prognostic potential.

Topics: Breast Neoplasms; Cyclin D1; Genes, erbB-2; Humans; Immunohistochemistry; Ki-67 Antigen; Polymorphism, Genetic; Prognosis; Protein Isoforms; Receptors, Estrogen

To characterize the molecular genetic profiles of grade 3 invasive ductal carcinomas of no special type using high-resolution microarray-based comparative genomic hybridization (aCGH) and to identify recurrent amplicons harboring putative therapeutic targets associated with luminal, HER-2, and basal-like tumor phenotypes.. Ninety-five grade 3 invasive ductal carcinomas of no special type were classified into luminal, HER-2, and basal-like subgroups using a previously validated immunohistochemical panel. Tumor samples were microdissected and subjected to aCGH using a tiling path 32K BAC array platform. Selected regions of recurrent amplification were validated by means of in situ hybridization. Expression of genes pertaining to selected amplicons was investigated using quantitative real-time PCR and gene silencing was done using previously validated short hairpin RNA constructs.. We show that basal-like and HER-2 tumors are characterized by "sawtooth" and "firestorm" genetic patterns, respectively, whereas luminal cancers were more heterogeneous. Apart from confirming known amplifications associated with basal-like (1q21, 10p, and 12p), luminal (8p12, 11q13, and 11q14), and HER-2 (17q12) cancers, we identified previously unreported recurrent amplifications associated with each molecular subgroup: 19q12 in basal-like, 1q32.1 in luminal, and 14q12 in HER-2 cancers. PPM1D gene amplification (17q23.2) was found in 20% and 8% of HER-2 and luminal cancers, respectively. Silencing of PPM1D by short hairpin RNA resulted in selective loss of viability in tumor cell lines harboring the 17q23.2 amplification.. Our results show the power of aCGH analysis in unraveling the genetic profiles of specific subgroups of cancer and for the identification of novel therapeutic targets.

Topics: Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Line, Tumor; Cyclin D1; Estrogen Receptor alpha; Gene Amplification; Gene Dosage; Gene Expression Profiling; Gene Silencing; Genes, erbB-1; Genes, erbB-2; Humans; Neoplasm Staging; Phosphoprotein Phosphatases; Protein Phosphatase 2C

Co-amplification at chromosomes 8p11-8p12 and 11q12-11q14 occurs often in breast tumors, suggesting possible cooperation between genes in these regions in oncogenesis. We used high-resolution array comparative genomic hybridization (array CGH) to map the minimal amplified regions. The 8p and 11q amplicons are complex and consist of at least four amplicon cores at each site. Candidate oncogenes mapping to these regions were identified by combining copy number and RNA and protein expression analyses. These studies also suggested that CCND1 at 11q13 induced expression of ZNF703 mapping at 8p12, which was subsequently shown to be mediated by the Rb/E2F pathway. Nine candidate oncogenes from 8p12 and four from 11q13 were further evaluated for oncogenic function. None of the genes individually promoted colony formation in soft agar or collaborated with each other functionally. On the other hand, FGFR1 and DDHD2 at 8p12 cooperated functionally with MYC, whereas CCND1 and ZNF703 cooperated with a dominant negative form of TP53. These observations highlight the complexity and functional consequences of the genomic rearrangements that occur in these breast cancer amplicons, including transcriptional cross-talk between genes in the 8p and 11q amplicons, as well as their cooperation with major pathways of tumorigenesis.

Topics: Breast Neoplasms; Carrier Proteins; Cell Line, Tumor; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 8; Comparative Genomic Hybridization; Cyclin D1; Epistasis, Genetic; Female; Gene Amplification; Gene Dosage; Gene Expression Regulation, Neoplastic; Genetic Predisposition to Disease; Humans; Neoplastic Stem Cells; Oncogene Protein p55(v-myc); Oncogenes; Phospholipases; Receptor, Fibroblast Growth Factor, Type 1; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Signal Transduction; Transfection; Tumor Suppressor Protein p53

Breast cancer development is a complex pathobiological process involving sequential genetic alterations in normal epithelial cells that results in uncontrolled growth in a permissive microenvironment. Accordingly, physiologically relevant models of human breast cancer that recapitulate these events are needed to study cancer biology and evaluate therapeutic agents. Here, we report the generation and utilization of the human breast cancer in mouse (HIM) model, which is composed of genetically engineered primary human breast epithelial organoids and activated human breast stromal cells. By using this approach, we have defined key genetic events required to drive the development of human preneoplastic lesions as well as invasive adenocarcinomas that are histologically similar to those in patients. Tumor development in the HIM model proceeds through defined histological stages of hyperplasia, DCIS to invasive carcinoma. Moreover, HIM tumors display characteristic responses to targeted therapies, such as HER2 inhibitors, further validating the utility of these models in preclinical compound testing. The HIM model is an experimentally tractable human in vivo system that holds great potential for advancing our basic understanding of cancer biology and for the discovery and testing of targeted therapies.

Topics: Animals; Apoptosis; Breast Neoplasms; Cell Transformation, Neoplastic; Cyclin D1; Disease Models, Animal; Disease Progression; Gene Expression Regulation, Neoplastic; Humans; Mice; Neoplasm Invasiveness; Phosphatidylinositol 3-Kinases; ras Proteins; Receptor, ErbB-2; RNA Interference; Simian virus 40; Telomerase; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

We studied the expression levels of cyclins B1, D1, and E1 and the implications of cyclin overexpression for patient outcomes in distinct breast cancer subtypes defined by clinical variables and transcriptional profiling.. The expression levels of cyclins B1, D1, and E1 were quantified in 779 breast tumors and 53 cell lines using reverse phase protein arrays and/or transcriptional profiling.. Whereas cyclin E1 overexpression was a specific marker of triple-negative and basal-like tumors, cyclin B1 overexpression occurred in poor prognosis hormone receptor-positive, luminal B and basal-like breast cancers. Cyclin D1 overexpression occurred in luminal and normal-like cancers. Breast cancer subgroups defined by integrated expression of cyclins B1, D1, and E1 correlated significantly (P < 0.000001) with tumor subtypes defined by transcriptional profiling and clinical criteria. Across three hormone receptor-positive data sets, cyclin B1 was the dominant cyclin associated with poor prognosis in univariate and multivariate analyses. Although CCNE1 was present in significantly higher copy numbers in basal-like versus other subtypes (ANOVA P < 0.001), CCNB1 gene copy number did not show gain in breast cancer. Instead, cyclin B1 expression was increased in tumors with co-occurrence of TP53 mutations and MYC amplification, a combination that seems to characterize basal-like and luminal B tumors. CCNB1 gene expression was significantly correlated with PLK, CENPE, and AURKB gene expression.. Cyclins B1, D1, and E1 have distinct expressions in different breast cancer subtypes. Novel PLK, CENPE, and AURKB inhibitors should be assessed for therapeutic utility in poor prognosis cyclin B1-overexpressing breast cancers.

Topics: Breast; Breast Neoplasms; Cell Line, Tumor; Class I Phosphatidylinositol 3-Kinases; Cyclin B; Cyclin B1; Cyclin D1; Cyclin E; DNA Mutational Analysis; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Mutation; Oligonucleotide Array Sequence Analysis; Oncogene Proteins; Phosphatidylinositol 3-Kinases; Predictive Value of Tests; Prognosis; Proportional Hazards Models; Proteomics; Receptors, Estrogen; Receptors, Progesterone; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Survival Analysis

Silibinin is a nontoxic flavonoid reported to have anticancer properties. In this study, we show that silibinin exhibits antiproliferative activity on MCF-7 breast cancer cells. Exposure to silibinin leads to a concentration-dependent decrease in global protein synthesis associated with reduced levels of eukaryotic initiation factor 4F complex. Moreover, polysome profile analysis of silibinin-treated cells shows a decrease in polysome content and translation of cyclin D1 mRNA. Silibinin exerts its effects on translation initiation by inhibiting the mammalian target of rapamycin signaling pathway by acting upstream of TSC2. Our results show that silibinin blocks mammalian target of rapamycin signaling with a concomitant reduction in translation initiation, thus providing a possible molecular mechanism of how silibinin can inhibit growth of transformed cells.

Topics: Antineoplastic Agents; Antioxidants; Blotting, Western; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Cyclin D1; Dose-Response Relationship, Drug; Eukaryotic Initiation Factor-4E; Eukaryotic Initiation Factor-4F; Fluorescent Antibody Technique; Humans; Mechanistic Target of Rapamycin Complex 1; Multiprotein Complexes; Protein Biosynthesis; Protein Transport; Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Silybin; Silymarin; TOR Serine-Threonine Kinases; Transcription Factors; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins

During estrogen-induced proliferation, c-Myc and cyclin D1 initiate independent pathways that activate cyclin E1-Cdk2 by sequestration and/or downregulation of the CDK inhibitor p21(Waf1/Cip1), without significant increases in cyclin E1 protein levels. In contrast, cyclin E2 undergoes a marked increase in expression, which occurs within 9 to 12 h of estrogen treatment of antiestrogen-pretreated MCF-7 breast cancer cells. Both E cyclins are important to estrogen action, as small interfering RNA (siRNA)-mediated knockdown of either cyclin E1 or cyclin E2 attenuated estrogen-mediated proliferation. Inducible expression of cyclin D1 upregulated cyclin E2, while siRNA-mediated knockdown of cyclin D1 attenuated estrogen effects on cyclin E2. However, manipulation of c-Myc levels did not profoundly affect cyclin E2. Cyclin E2 induction by estrogen was accompanied by recruitment of E2F1 to the cyclin E1 and E2 promoters, and cyclin D1 induction was sufficient for E2F1 recruitment. siRNA-mediated knockdown of the chromatin remodelling factor CHD8 prevented cyclin E2 upregulation. Together, these data indicate that cyclin E2-Cdk2 activation by estrogen occurs via E2F- and CHD8-mediated transcription of cyclin E2 downstream of cyclin D1. This contrasts with the predominant regulation of cyclin E1-Cdk2 activity via CDK inhibitor association downstream of both c-Myc and cyclin D1 and indicates that cyclins E1 and E2 are not always coordinately regulated.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; Cyclins; DNA-Binding Proteins; E2F Transcription Factors; Enzyme Activation; Estrogens; Female; Gene Expression Regulation, Neoplastic; Humans; Mice; Oncogene Proteins; Promoter Regions, Genetic; Proto-Oncogene Proteins c-myc; RNA, Small Interfering; Transcription Factors

The aim of the present study was to examine parity and age at first childbirth, in relation to the risk of specific breast cancer subgroups. A prospective cohort, The Malmö Diet and Cancer Study, including 17,035 women were followed with linkage to Swedish Cancer Registry until December 31, 2004. A total of 622 incident breast cancers were diagnosed during follow-up and were evaluated regarding invasiveness, tumour size, axillary lymph node status, Nottingham grade, tumour proliferation (Ki67), HER2, cyclin D1 and p27. The tumours were also examined for WHO type and hormone receptor status. Nulliparity was associated with an overall increased risk of breast cancer, although not statistically significant (the relative risk was 1.39 with a 95% confidence interval of 0.92-2.08). Nulliparity was also associated with large tumours (>20 mm) (1.89: 0.91-3.91), high Ki67 levels (1.95: 0.93-4.10), high cyclin D1 levels (2.15: 0.88-5.27), grade III (2.93: 1.29-6.64) and HER2 positive tumours (3.24: 1.02-10.25). High parity was not statistically significantly associated with any specific breast cancer subgroup. Older age at first childbirth (>30) was associated with a slightly increased risk of breast cancer (1.39: 0.94-2.07). There was a statistically significant association between late first childbirth and lobular type (2.51: 1.01-6.28), grade III tumours (2.67: 1.19-6.02), high levels of cyclin D1 (2.69: 1.18-6.12) and low levels of p27 (2.23: 1.15-4.35). We conclude that nulliparity and late first childbirth are associated with relatively more aggressive breast cancer subgroups.

Topics: Adult; Age Factors; Breast Neoplasms; Carcinoma in Situ; Carcinoma, Lobular; Cell Proliferation; Cyclin D1; Estrogen Replacement Therapy; Female; Follow-Up Studies; Humans; Lymph Nodes; Lymphatic Metastasis; Menopause; Middle Aged; Neoplasm Invasiveness; Parity; Pregnancy; Prognosis; Prospective Studies; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone; Risk Factors; Young Adult

There is current evidence implicating the Wnt/beta-catenin/TCF pathway in breast cancer. We investigated the effect of para- and meta-positional isomers of nitric oxide-releasing aspirin (NO-ASA), and aspirin (ASA) on MCF-7 human breast cancer cell growth and beta-catenin/TCF signaling. The p- and m-NO-ASA isomers strongly inhibited cell growth and beta-catenin/TCF transcriptional activity compared to ASA; the IC50s for growth inhibition were 57+/-4, 193+/-10 and >5000microM, and for transcriptional inhibition they were 12+/-1.8, 75+/-6.5 and >5000microM for p-, m-NO-ASA and ASA, respectively. p-NO-ASA reduced the expression of Wnt/beta-catenin downstream target gene cyclin D1, and total cellular beta-catenin levels. COX-2 expression was induced by p-NO-ASA, protein kinase C inhibitors reversed this induction. p-NO-ASA blocked the cell cycle transition at S to G2/M phase. These studies suggest a targeted chemopreventive/chemotherapeutic potential for NO-ASA against breast cancer.

Topics: Anticarcinogenic Agents; Aspirin; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; beta Catenin; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclooxygenase 2; DNA-Binding Proteins; Down-Regulation; Enzyme Induction; Female; Genes, Reporter; Humans; Inhibitory Concentration 50; Isomerism; Signal Transduction; Transcription Factor 4; Transcription Factors; Transcription, Genetic; Wnt Proteins

The proliferative capacity of cancer cells is regulated by factors intrinsic to cancer cells and by secreted factors in the microenvironment. Here, we investigated the proto-oncogenic potential of the chemokine receptor, CCR5, in MCF-7 breast cancer cell lines. At physiological levels, CCL5, a ligand for CCR5, enhanced MCF-7.CCR5 proliferation. Treatment with the mTOR inhibitor, rapamycin, inhibited this CCL5-inducible proliferation. Because mTOR directly modulates mRNA translation, we investigated whether CCL5 activation of CCR5 leads to increased translation. CCL5 induced the formation of the eIF4F translation initiation complex through an mTOR-dependent process. Indeed, CCL5 initiated mRNA translation, shown by an increase in high-molecular-weight polysomes. Specifically, we show that CCL5 mediated a rapid up-regulation of protein expression for cyclin D1, c-Myc and Dad-1, without affecting their mRNA levels. Taken together, we describe a mechanism by which CCL5 influences translation of rapamycin-sensitive mRNAs, thereby providing CCR5-positive breast cancer cells with a proliferative advantage.

Topics: Apoptosis Regulatory Proteins; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Chemokine CCL5; Cyclin D1; Eukaryotic Initiation Factor-4F; Female; Humans; Membrane Proteins; Polyribosomes; Protein Biosynthesis; Protein Kinases; Proto-Oncogene Proteins c-myc; Receptors, CCR5; RNA, Messenger; TOR Serine-Threonine Kinases

ErbB2/HER2/Neu-overexpressing breast cancers are characterized by poor survival due to high proliferation and metastasis rates and identifying downstream targets of ErbB2 should facilitate developing novel therapies for this disease. Gene expression profiling revealed the transcriptional regulator LIM-only protein 4 (LMO4) is upregulated during ErbB2-induced mouse mammary gland tumorigenesis. Although LMO4 is frequently overexpressed in breast cancer and LMO4-overexpressing mice develop mammary epithelial tumors, the mechanisms involved are unknown. In this study, we report that LMO4 is a downstream target of ErbB2 and PI3K in ErbB2-dependent breast cancer cells. Furthermore, LMO4 silencing reduces proliferation of these cells, inducing a G2/M arrest that was associated with decreased cullin-3, an E3-ubiquitin ligase component important for mitosis. Loss of LMO4 subsequently results in reduced Cyclin D1 and Cyclin E. Further supporting a role for LMO4 in modulating proliferation by regulating cullin-3 expression, we found that LMO4 expression oscillates throughout the cell cycle with maximum expression occurring during G2/M and these changes precede oscillations in cullin-3 levels. LMO4 levels are also highest in high-grade/less differentiated breast cancers, which are characteristically highly proliferative. We conclude that LMO4 is a novel cell cycle regulator with a key role in mediating ErbB2-induced proliferation, a hallmark of ErbB2-positive disease.

Topics: Adaptor Proteins, Signal Transducing; Animals; Breast Neoplasms; Cell Cycle; Cell Division; Cell Line, Tumor; Cell Proliferation; Cullin Proteins; Cyclin D1; G2 Phase; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; LIM Domain Proteins; Mice; Neuregulin-1; Phosphatidylinositol 3-Kinases; Receptor, ErbB-2; RNA, Messenger; Signal Transduction; Transcription Factors; Up-Regulation

More than 60% of conventional drugs are derived from natural compounds, some of the most effective pharmaceuticals (e.g. aspirin, quinine and various antibiotics) originate from plants or microbes, and large numbers of potentially valuable natural substances remain to be discovered. Plants with considerable medicinal potential include members of the genus Acalypha. Notably, extracts of A. platyphilla, A. fruticosa, A. siamensis, A. guatemalensis and A. wilkesiana have been recently shown to have antioxidant, antimicrobial and cytotoxic effects. In the study presented here we investigated the anti-inflammatory, anti-proliferative and pro-apoptotic activities of A. alopecuroidea, which is endemic in parts of Central America and is traditionally used by the Mopan- and Itza-Maya in the form of decoctions to treat skin conditions, and as a tea to treat stomach and urinary complaints. We demonstrate here that extracts of A. alopecuroidea can inhibit TNFalpha-induced E-selectin production, providing a mechanistic validation of its traditional use against inflammatory diseases. Furthermore, a fraction of A. alopecuroidea root extracts purified by solid phase extraction and separated by HPLC displayed strong cell cycle inhibitory activity by down-regulating and inactivating two proto-oncogenes (cyclin D1 and Cdc25A), and simultaneously inducing cyclin A, thereby disturbing orchestrated cell cycle arrest, and thus (presumably) triggering caspase 3-dependent apoptosis. The results of this study indicate that there are high prospects for purifying an active principle from A. alopecuroidea for further in vivo and preclinical studies.

Topics: Anti-Inflammatory Agents; Antineoplastic Agents, Phytogenic; Apoptosis; Breast Neoplasms; Caspase 3; cdc25 Phosphatases; Cell Cycle; Cell Proliferation; Cell Survival; Checkpoint Kinase 2; Chromatin Assembly and Disassembly; Cyclin A; Cyclin D1; Dose-Response Relationship, Drug; E-Selectin; Endothelial Cells; Euphorbiaceae; Female; HL-60 Cells; Humans; Inflorescence; Leukemia, Promyelocytic, Acute; Mutation; Plant Leaves; Plant Shoots; Protein Serine-Threonine Kinases; Time Factors; Transfection; Tumor Necrosis Factor-alpha; Tumor Suppressor Protein p53

Pescadillo, which has been found to be involved in the process of ribosomal biogenesis, has been demonstrated to play a role in embryonic development, DNA replication, and gene transcription. While deregulation of ribosomal biogenesis was also found to contribute to carcinogenesis, and proteins that regulate ribosomal biogenesis are commonly overexpressed in primary tumors, little is known about the clinical significance and biological function of pescadillo in human breast cancer. In the current study, we found that the expression of pescadillo was markedly up-regulated in human breast cancer cells and tissues at both mRNA and protein levels. Immunohistochemical analysis revealed that pescadillo expression in clinical stage I-IV primary breast cancer tissues was statistically significantly higher than that in normal breast tissues (P < 0.05). Furthermore, we demonstrated that knockdown pescadillo with RNAis inhibited cell proliferation and the colony-forming ability of the cells. Anchorage-independent growth ability assay indicated that ablation of pescadillo led to the reduction of breast cancer cells tumorigenicity in vitro. Moreover, depletion of endogenous pescadillo resulted in decreased expression of cell cycle protein cyclin D1 and up-regulation of cyclin-dependent kinase inhibitor p27(Kip1), as well as attenuated protein kinase B (Akt)/glycogen synthase kinase 3 beta (GSK-3beta) signaling. Taken together, our results suggest that pescadillo might play a role in promoting the proliferation and carcinogenesis of human breast cancer, and thereby might be a potential target for human breast cancer treatment.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Down-Regulation; Female; Humans; Intracellular Signaling Peptides and Proteins; Proteins; RNA-Binding Proteins

Topics: Adult; Biomarkers, Tumor; Breast Neoplasms; Cyclin D1; Female; Genes, BRCA2; Humans; Immunohistochemistry; Middle Aged; Mutation; Tissue Array Analysis

Epidemiologic evidence suggests reduced breast cancer mortality in users of American Ginseng (AG) (Panax quinquefolium). We hypothesized that AG extract decreases proliferation of human breast cancer cells via an anti-inflammatory effect applicable to the prevention of breast and other cancers.. A defined lyophilized aqueous extract of AG (LEAG) was dissolved in DMSO 1mg/mL, and serially diluted in saline. The cell lines MDA MB 231 and MCF7 were stimulated with the phorbol ester PDBu and treated with 100-500 mcg/mL LEAG. Proliferation was measured by MDA assay. Induced COX-2 expression was assayed by ELISA. Activation of NFkappaB by phosphorylation of the p65 subunit was quantified by CASE (cellular activation of signaling ELISA).. Both cell lines had reduced proliferation when treated with LEAG. PDBu stimulation of MDA MB 231 increased expression of the COX-2 protein 20-fold at 48 hours (P<0.005). COX-2 protein expression remained at baseline concentrations in PDBu- treated MDA MB 231 cells exposed to 100 mcg/mL LEAG. The CASE assay showed a 4-fold increase in p65 activation 24 hours after PDBu treatment in normal medium, while phosphorylated p65 dropped below baseline in the cells treated with PDBu plus LEAG.. In MDA MB 231, COX-2 was inducible with PDBu. This induced COX-2 expression was blocked by 100 microgram/mL LEAG in a time course consistent with the decline in the activated p65 subunit of NFkappaB. These results provide an anti-inflammatory mechanism for a possible anti-cancer effect of American Ginseng.

Topics: Adenocarcinoma; Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclooxygenase 2; Humans; NF-kappa B; Panax; Plant Extracts

Loss of cyclin-dependent kinase (CDK) 10 expression may be an important mechanism of tamoxifen resistance and the 5' CpG island associated with the CDK10 gene has been suggested to be a target for aberrant methylation in breast cancer.. The methylation status of CDK10, RASSF1A (Ras association domain family 1A) and DAL-1 (differentially expressed in adenocarcinoma of the lung) was determined by means of methylation-specific PCR (MSP) in the formalin-fixed, paraffin-embedded (FFPE) surgical specimens of 96 breast carcinoma patients. Reverse transcription kinetic PCR (RT-kPCR) was used for assessment of the expression of CDK10.. The unmethylated form of CDK10, RASSF1A and DAL-1 was detected in all the samples analyzed. Methylation of the CDK10 5' region was not found in any of the 96 breast cancer samples. RASSF1A methylation was detected in 75 out of 96 (78%) and DAL-1 in 9 out of 15 (60%) breast cancer samples, respectively. Consistent with the methylation results, the expression of CDK10 was detected in all 96 samples.. CDK10 is not a target for aberrant DNA methylation in breast cancer.

Topics: Breast Neoplasms; Cyclin D1; Cyclin-Dependent Kinases; DNA Methylation; Estrogen Receptor alpha; Female; Humans; RNA, Messenger

In this issue of Cancer Cell, Zhang et al. reports that the iron-dependent 2-oxoglutarate dioxygenase or prolyl hydroxylase EglN2 induces Cyclin D1 levels, egging on breast tumorigenesis. Their observations through loss of function studies suggest the potential for drug-like molecules inhibiting EglN to serve as new cancer therapeutics.

Topics: Animals; Breast Neoplasms; Cyclin D1; Dioxygenases; Female; Humans; Mammary Neoplasms, Experimental; RNA, Messenger

2-Oxoglutarate-dependent dioxygenases, including the EglN prolyl hydroxylases that regulate HIF, can be inhibited with drug-like molecules. EglN2 is estrogen inducible in breast carcinoma cells and the lone Drosophila EglN interacts genetically with Cyclin D1. Although EglN2 is a nonessential gene, we found that EglN2 inactivation decreases Cyclin D1 levels and suppresses mammary gland proliferation in vivo. Regulation of Cyclin D1 is a specific attribute of EglN2 among the EglN proteins and is HIF independent. Loss of EglN2 catalytic activity inhibits estrogen-dependent breast cancer tumorigenesis and can be rescued by exogenous Cyclin D1. EglN2 depletion also impairs the fitness of lung, brain, and hematopoietic cancer lines. These findings support the exploration of EglN2 inhibitors as therapeutics for estrogen-dependent breast cancer and other malignancies.

Topics: Animals; Breast Neoplasms; Cell Culture Techniques; Cell Line, Tumor; Cyclin D1; Dioxygenases; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; HeLa Cells; Humans; Immunoblotting; Mice; Mice, Transgenic; Procollagen-Proline Dioxygenase; Reverse Transcriptase Polymerase Chain Reaction; Transfection

Pin-1 has been shown to regulate several phases of the cell cycle and is strikingly overexpressed in many human cancers. Vascular endothelial growth factor (VEGF)-C is a potent lymphangiogenic factor produced by tumor and stromal cells. However, little is known about the roles of Pin-1 and VEGF-C in breast carcinoma. p53 protein and cyclin D1 overexpressions have been shown to play a role as prognostic factors in many human cancers. To better understand the roles of Pin-1 and VEGF-C in breast carcinoma, we evaluated the immunohistochemical expression of Pin-1 and VEGF-C in relationship with p53 protein or cyclin D1 overexpression and clinicopathological parameters in 128 mammary infiltrating duct carcinomas. There was a positive expression in 100% of Pin-1, 88% of VEGF-C, 35% of p53 protein, and 66% of cyclin D1 in the breast carcinoma. Correlation of the positive expression of Pin-1 with tumor grade (p<0.01) and lymph node metastasis or cyclin D1 overexpression (p<0.05, respectively) was statistically significant. Significant correlation was observed between VEGF-C and tumor grade, lymph node metastasis or clinical stage (p<0.01, respectively). These results indicate that elevated Pin-1 or VEGF-C expression is more common in infiltrating duct carcinomas with poor prognostic characteristics and is partly associated with an unfavorable outcome. Given the role of cyclin D1 overexpression in oncogenesis of breast, these results suggest that overexpression of Pin-1 and VEGF-C may promote tumor progression and metastasis.

Topics: Adult; Aged; Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Line, Tumor; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Lymphatic Metastasis; Middle Aged; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; Prognosis; Tumor Suppressor Protein p53; Vascular Endothelial Growth Factor C

Loss of retinoblastoma protein expression and overexpression of cyclin D1 have been implicated in the development and progression of some cancers. Paget's disease of the vulva (PDV) and Paget's disease of the breast (PDB) are uncommon conditions and the pathogenesis of these diseases is still unclear. The aim was to examine the expression of the retinoblastoma and cyclin D1 proteins in PDV and PDB and to correlate any differences between PDV and PDB, and in the presence or absence of an underlying carcinoma.. Seventy-two archival cases of PDV including 10 with invasive disease and 36 cases of PDB were evaluated immunohistochemically for the expression of cyclin D1 and retinoblastoma protein. Forty-four percent (32/72) of cases of PDV showed loss of expression of the retinoblastoma protein, compared with 67% (24/36) of PDB cases. Fifty-nine percent (41/69) of PDV overexpressed cyclin D1. In PDB, 8% (3/34) overexpressed cyclin D1. There were no significant differences in the expression of retinoblastoma and cyclin D1 in PDV cases with or without underlying invasive disease. There were significant differences between the expression of retinoblastoma (P = 0.03) and cyclin D1 (P < 0.001) in PDV compared with PDB.. The differences in the expression of cyclin D1 and retinoblastoma may indicate the differences in the pathogenesis of PDV and PDB.

Topics: Breast Neoplasms; Chi-Square Distribution; Cyclin D1; Female; Humans; Immunohistochemistry; Paget Disease, Extramammary; Paget's Disease, Mammary; Retinoblastoma Protein; Vulvar Neoplasms

The molecular links between breast cancer and obesity have been studied for many years. Obesity significantly increases the incidence rate and chance of morbidity of breast cancer. Leptin, mainly secreted by adipocytes, plays an important role in breast cancer development. Leptin expression is up-regulated in obesity and it can promote breast cancer cell growth. Zeranol is used as an anabolic growth promoter to stimulate cattle growth in the U.S. beef industry. (-)-Gossypol, a natural polyphenolic compound extracted from cottonseed, is an anticancer chemopreventive agent.. Zeranol, leptin and (-)-gossypol were used to investigate MCF-7 Adr cell growth.. Leptin enhanced the sensitivity of MCF-7 Adr cells to zeranol and increased cell growth. Exposure to zeranol may lead to initiation of transformation of normal breast cells to breast preneoplastic cells.. It is suggested that obese individuals may be at greater risk of developing zeranol-induced breast cancer.

Topics: Anticarcinogenic Agents; Breast Neoplasms; Cell Growth Processes; Cell Line, Tumor; Cell Transformation, Neoplastic; Cyclin D1; Drug Synergism; Gossypol; Humans; Leptin; Recombinant Proteins; RNA, Messenger; Tumor Suppressor Protein p53; Zeranol

Cyclins D1 and E play an important role in breast carcinogenesis. High cyclin E expression is common in hormone receptor negative and high grade aggressive breast cancer, whereas cyclin D1 in hormone receptor positive and low grade breast cancer. Experimental data has suggested that cyclin D1 and E mediate cell proliferation by different mechanisms in estrogen receptor (ER) positive and negative breast cancer. To test this hypotheses in large breast cancer material and to clarify the histopathological correlations of cyclin E and D1, especially the association with proliferation, we analyzed cyclin E and D1 immunohistochemical expression on breast tumour microarrays consisting of 1348 invasive breast cancers. High cyclin D1 expression was associated with high grade (P<0.0005), high cyclin A (P<0.0005) and Ki67 (P<0.0005) expression among ER positive but with low grade (P=0.05) and low Ki67 (P=0.01) expression among ER negative breast cancers. Cyclin E and D1 expression correlated positively in ER positive (P<0.0005) but had a negative correlation in ER negative tumours (P=0.004). Cyclin E associated with high grade among all tumours (P<0.0005). In conclusion, the findings of this study show that cyclin D1 has separate roles, and proliferation is driven by different mechanisms in ER positive and negative breast cancers.

Topics: Breast Neoplasms; Cell Division; Cyclin D1; Cyclin E; Female; Finland; Gene Expression Regulation, Neoplastic; Genes, BRCA1; Genes, BRCA2; Humans; Neoplasm Invasiveness; Oligonucleotide Array Sequence Analysis; Prognosis; Receptors, Estrogen; Receptors, Progesterone

17beta-Estradiol (E2) acts through the estrogen receptor alpha (ERalpha) to stimulate breast cancer proliferation. Here, we investigated the functional relationship between ERalpha and signal transducer and activator of transcription (STAT)5b activity in ER+ MCF-7 and T47D human breast cancer cells after specific knockdown of STAT5b. STAT5b small interfering RNA (siRNA) inhibited E2-induced bromodeoxyuridine (BrdU) incorporation in both cell lines, as well as the E2-induced increase in MCF-7 cell number, cyclin D1 and c-myc mRNA, and cyclin D1 protein expression, indicating that STAT5b is required for E2-stimulated breast cancer proliferation. E2 treatment stimulated STAT5b tyrosine phosphorylation at the activating tyrosine Y699, resulting in increased STAT5-mediated transcriptional activity, which was inhibited by a Y669F STAT5b mutant. E2-induced STAT5-mediated transcriptional activity was inhibited by overexpressing a kinase-defective epidermal growth factor receptor (EGFR), or the EGFR tyrosine kinase inhibitor tyrphostin AG1478, indicating a requirement for EGFR kinase activity. Both E2-induced STAT5b tyrosine phosphorylation and STAT5-mediated transcription were also inhibited by the ER antagonist ICI 182,780 and the c-Src inhibitor PP2, indicating additional requirements for the ER and c-Src kinase activity. EGFR and c-Src kinase activities were also required for E2-induced cyclin D1 and c-myc mRNA. Together, these studies demonstrate positive cross talk between ER, c-Src, EGFR, and STAT5b in ER+ breast cancer cells. Increased EGFR and c-Src signaling is associated with tamoxifen resistance in ER+ breast cancer cells. Here we show that constitutively active STAT5b not only increased basal DNA synthesis, but also conferred tamoxifen resistance. Because STAT5b plays an integral role in E2-stimulated proliferation and tamoxifen resistance, it may be an effective therapeutic target in ER+ breast tumors.

Topics: Breast Neoplasms; Cell Count; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Drug Resistance, Neoplasm; ErbB Receptors; Estradiol; Estrogen Receptor alpha; Gene Expression Regulation, Neoplastic; Genes, Neoplasm; Humans; Phosphorylation; Phosphotyrosine; Proto-Oncogene Proteins pp60(c-src); Signal Transduction; STAT5 Transcription Factor; Tamoxifen; Transcription, Genetic

Increased Akt phosphorylation was reported in cancer cell lines and tumor tissues of patients exposed to rapamycin, a response likely contributing to the attenuated antitumor activity of rapamycin. It is, therefore, necessary to develop and validate combination strategies to reverse rapamycin-induced Akt signaling. We now report that Akt activation in response to rapamycin is abrogated by 17-allylamino-17-demethoxygeldanamycin (17-AAG), a heat shock protein 90 (HSP90) inhibitor. Rapamycin/17-AAG combination results in an enhanced antiproliferative activity in both MCF-7 and MDA-MB-231 breast cancer cells. In combination 17-AAG confers potent suppression of Raf-MEK-extracellular signal-regulated kinase signaling, a pathway that is otherwise not inhibited by rapamycin individually. Importantly, 17-AAG cooperates with rapamycin to block the phosphorylation of the mammalian target of rapamycin at Ser2448, as well as its downstream effectors ribosomal p70 S6 kinase and eukaryotic initiation factor 4E binding protein 1, which is accompanied by a substantial reduction in cyclins D1 and E. The potency of rapamycin/17-AAG combination is not affected by the activation of insulin-like growth factor 1 receptor signaling, which has been previously shown to diminish the antiproliferative activity of rapamycin. Rapamycin/17-AAG combination alleviates the induction of HSP90 protein, a heat shock response frequently associated with 17-AAG monotherapy. Our findings establish a mechanistic rationale for a combination approach using rapamycin and 17-AAG in the treatment of breast cancer.

Topics: Benzoquinones; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin E; Extracellular Signal-Regulated MAP Kinases; Female; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Protein Kinases; Proto-Oncogene Proteins c-akt; raf Kinases; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Fulvestrant is an estrogen receptor (ER) antagonist that binds, blocks and degrades the estrogen receptor and is currently used in adjuvant treatment in postmenopausal women with ER-positive breast cancer as an alternative for tamoxifen. As an antagonist, it may induce or aggravate climacteric symptoms. In order to alleviate these symptoms, one could consider hormone therapy. The objective of this study was to analyze the effect of fulvestrant alone or in combination with different steroids in human breast cancer cells in vitro, and to demonstrate whether these steroids will compromise the efficacy of fulvestrant in ER-positive breast cancer cells.. We performed experiments in vitro with various hormone therapy preparations (estradiol (E2), dihydrodydrogesterone (DHD) and tibolone) at a concentration of 10(-6) mol/l alone or combined with fulvestrant in different breast cancer cell lines, ER-positive and ER-negative. After an incubation of 144 h, proliferation and apoptosis were measured. The first was measured by quantification of the expression of cyclin D1 mRNA, the latter by the Nicoletti fragmentation assay.. This in vitro study revealed clear differences in results when various hormone therapy preparations, alone or combined with fulvestrant, are added to ER-positive and ER-negative breast cancer cell lines.. Our study demonstrated that fulvestrant, an ER antagonist used in the treatment of ER-positive breast cancer, combined with E2 and DHD or in combination with tibolone, is not compromised in its efficacy in inducing apoptosis in ER-positive breast cancer cell lines in vitro.

Topics: Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Dydrogesterone; Estradiol; Estrogen Antagonists; Estrogen Receptor Modulators; Estrogens; Female; Fulvestrant; Hormone Replacement Therapy; Humans; In Vitro Techniques; Norpregnenes; Progestins; Receptors, Estrogen; RNA, Messenger

P16(INK4a) is a tumor suppressor gene frequently inactivated by aberrant promoter hypermethylation. In this study, p16(INK4a) methylation was evaluated in intraductal proliferative lesions of the breast, using real-time quantitative polymerase chain reaction (MethyLight) and methylation-sensitive restriction endonuclease polymerase chain reaction. Immunohistochemistry was performed to compare and validate the methylation analysis. P16(INK4a) methylation associated with oncogene cyclinD1 expression, detected through the use of in situ hybridization and immunohistochemistry, was likewise characterized. P16(INK4a) methylation displayed varying significance among different types of intraductal proliferative lesions. Both the positive rate and the median quantitative methylation value increased with the evolution of intraductal proliferative lesions through the use of quantitative and qualitative assays. P16(INK4a) methylation was positively correlated to cyclinD1 overexpression. This study demonstrated that p16(INK4a) methylation served as the silencing mechanism of p16(INK4a) protein expression and played a crucial role in the intraductal proliferative lesions' progression. In the differential diagnosis of intraductal proliferative lesions, quantitative DNA methylation analysis of p16(INK4a) by MethyLight may be used as a surrogate, especially to distinguish atypical ductal hyperplasia from usual ductal hyperplasia and low-grade ductal carcinoma in situ. Furthermore, this study discovered that flat epithelial atypia do not share similar molecular profiles of p16(INK4a) epigenetic modification with atypical ductal hyperplasia and low-grade ductal carcinoma in situ.

Topics: Breast; Breast Neoplasms; CpG Islands; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; DNA Methylation; Female; Humans

Decreased expression of specific microRNAs (miRNAs) occurs in human tumors, which suggests a function for miRNAs in tumor suppression. Herein, levels of the miR-17-5p/miR-20a miRNA cluster were inversely correlated to cyclin D1 abundance in human breast tumors and cell lines. MiR-17/20 suppressed breast cancer cell proliferation and tumor colony formation by negatively regulating cyclin D1 translation via a conserved 3' untranslated region miRNA-binding site, thereby inhibiting serum-induced S phase entry. The cell cycle effect of miR-17/20 was abrogated by cyclin D1 siRNA and in cyclin D1-deficient breast cancer cells. Mammary epithelial cell-targeted cyclin D1 expression induced miR-17-5p and miR-20a expression in vivo, and cyclin D1 bound the miR-17/20 cluster promoter regulatory region. In summary, these studies identify a novel cyclin D1/miR-17/20 regulatory feedback loop through which cyclin D1 induces miR-17-5p/miR-20a. In turn, miR-17/20 limits the proliferative function of cyclin D1, thus linking expression of a specific miRNA cluster to the regulation of oncogenesis.

Topics: 3' Untranslated Regions; Animals; Base Sequence; Binding Sites; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Conserved Sequence; Cyclin D1; Down-Regulation; Feedback, Physiological; G1 Phase; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Inbred C57BL; MicroRNAs; Molecular Sequence Data; Promoter Regions, Genetic

We have shown previously that hypoxia activates the cyclin D1 promoter via the Jak2/STAT5b pathway in breast cancer cells. Most solid tumors contain hypoxic components and overexpression of cyclin D1. The purpose of the present study was to investigate the molecular mechanism by which momilactone B exerts its inhibitory effects on breast cancer cells. Momilactone B, extracted from Korean rice hulls, suppressed hypoxia-induced increases in phospho-STAT5, STAT5b, cyclin D1, and cdk4 protein levels in human breast cancer cells. STAT5b expression was inhibited by siRNA experiments leading to decreased cyclin D1. The effects of momilactone B on cell growth and apoptosis-related gene expression were investigated in breast cancer cells under hypoxic conditions (2% O2). Bax and p21 expression was found to be up-regulated, whereas ppRb and bcl-2 were down-regulated in momilactone B-treated cells under hypoxic conditions. However, the p53 protein level did not change. Flow cytometry with Annexin-FITC staining showed that the number of apoptotic cells increased in hypoxic cells treated with momilactone B compared with untreated hypoxic cells. Furthermore, caspase activity increased upon treatment with momilactone B under hypoxic conditions. These results indicate that momilactone B inhibits the growth of breast cancer cells, regulates the expression of apoptosis-related genes, and induces apoptosis through STAT5b and a caspase-3 dependent pathway. We suggest that momilactone B accelerates hypoxia-induced apoptosis of human breast cancer cells through STAT5b, and may represent an effective chemopreventive or therapeutic agent against breast cancer.

Topics: Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Breast Neoplasms; Cell Hypoxia; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p21; Diterpenes; Electrophoretic Mobility Shift Assay; Female; Flow Cytometry; Gene Expression; Humans; Lactones; Proto-Oncogene Proteins c-bcl-2; RNA, Small Interfering; STAT5 Transcription Factor

It is well established that statins display potent anticancer activity in several types of proliferating tumor cells. However, how to promote the sensitivity of statins to mammary cancer is yet to be completely deciphered. The purpose of this study was to investigate whether breast cancer susceptibility gene 1 (BRCA1) overexpression sensitizes mammary cancer cells to statins. MCF-7 cells, which have only one wild-type BRCA1 allele, were transfected with pcDNA3-beta-HA-hsBRCA1 plasmids via liposomes to reconstitute BRCA1 overexpression human breast cancer cell line, and tumoral xenografts with BRCA1 overexpression were subsequently established in BALB/c nude mice. Then, the inhibitory activity of lovastatin on cancer cells and tumoral xenografts, and the underlying mechanism involving in cell-cycle regulatory proteins were analyzed. The proliferative ability of MCF-7 cells treated with lovastatin was reduced compared to normal, and further decreased in the presence of excess BRCA1, detected by methyl thiazolyl tetrazolium and flow cytometry techniques in vitro or by 5-bromodeoxyuridine incorporation in vivo. Additionally, the mRNA and protein expression of cyclin D1, cyclin-dependent kinase 4 (CDK4) and retinoblastoma protein (pRb), was further down-regulated under exposure to lovastatin in condition of BRCA1 overexpression, but the expression of p21WAF1/CIP1, a cyclin-dependent kinase inhibitor (CDKI), was further up-regulated, both in vitro and in vivo detected with quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis. Moreover, we found the further reduced volume of tumoral xenografts treated with lovastatin in the presence of BRCA1 overexpression. Our results suggest that BRCA1 overexpression sensitizes cancer cells to lovastatin via regulation of cyclin D1-CDK4-p21WAF1/CIP1 pathway, which will provide an innovative experimental framework to study control of breast cancer cell proliferation.

Topics: Animals; Antineoplastic Agents; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p21; Female; Flow Cytometry; Humans; Lovastatin; Mice; Mice, Nude; Retinoblastoma Protein; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transfection; Ubiquitin-Protein Ligases

Stress granules aid cell survival in response to environmental stressors by acting as sites of translational repression. We report an unanticipated link between stress granules and the serine/threonine kinase RSK2. In stressed breast cells, endogenous RSK2 colocalizes in granules with TIA-1 and poly(A)-binding protein 1, and the sequestration of RSK2 and TIA-1 exhibits codependency. The RSK2 N-terminal kinase domain controls the direct interaction with the prion-related domain of TIA-1. Silencing RSK2 decreases cell survival in response to stress. Mitogen releases RSK2 from the stress granules and permits its nuclear import via a nucleocytoplasmic shuttling sequence in the C-terminal domain. Nuclear accumulation is dependent on TIA-1. Surprisingly, nuclear localization of RSK2 is sufficient to enhance proliferation through induction of cyclin D1, in the absence of other active signaling pathways. Hence, RSK2 is a pivotal factor linking the stress response to survival and proliferation.

Topics: Animals; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Cyclin D1; Cytoplasmic Granules; Female; Humans; Oxidative Stress; Poly(A)-Binding Proteins; Prions; Ribosomal Protein S6 Kinases, 90-kDa; T-Cell Intracellular Antigen-1

Breast cancers can be classified using whole genome expression into distinct subtypes that show differences in prognosis. One of these groups, the basal-like subtype, is poorly differentiated, highly metastatic, genomically unstable, and contains specific genetic alterations such as the loss of tumour protein 53 (TP53). The loss of the retinoblastoma tumour suppressor encoded by the RB1 locus is a well-characterised occurrence in many tumour types; however, its role in breast cancer is less clear with many reports demonstrating a loss of heterozygosity that does not correlate with a loss of RB1 protein expression.. We used gene expression analysis for tumour subtyping and polymorphic markers located at the RB1 locus to assess the frequency of loss of heterozygosity in 88 primary human breast carcinomas and their normal tissue genomic DNA samples.. RB1 loss of heterozygosity was observed at an overall frequency of 39%, with a high frequency in basal-like (72%) and luminal B (62%) tumours. These tumours also concurrently showed low expression of RB1 mRNA. p16INK4a was highly expressed in basal-like tumours, presumably due to a previously reported feedback loop caused by RB1 loss. An RB1 loss of heterozygosity signature was developed and shown to be highly prognostic, and was potentially a predictive marker of response to neoadjuvant chemotherapy.. These results suggest that the functional loss of RB1 is common in basal-like tumours, which may play a key role in dictating their aggressive biology and unique therapeutic responses.

Topics: Breast Neoplasms; Carcinoma; Cell Division; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Drug Resistance, Neoplasm; Female; Gene Expression Profiling; Genes, bcl-1; Genes, p16; Genes, Retinoblastoma; Humans; Loss of Heterozygosity; Neoadjuvant Therapy; Neoplasm Proteins; Oligonucleotide Array Sequence Analysis; Retinoblastoma Protein; RNA, Messenger; RNA, Neoplasm

Numerous epidemiological studies have documented that obesity is a risk factor for breast cancer especially in post-menopausal women. However, the molecular basis of this association is not well known. In contrast to leptin, plasma levels of adiponectin, another major adipokine, are decreased in obese subjects. Therefore, we and others hypothesized that adiponectin may be a paracrine factor negatively controlling mammary tumor development. We recently demonstrated growth inhibition of the estrogen-sensitive breast cancer MCF-7 cell line by adiponectin. The purpose of the present study was to determine whether this anti-proliferative effect of adiponectin also applies to the MDA-MB 231 estrogen-insensitive breast epithelial cancer cell line. Our results demonstrate that i) the adiponectin-specific receptors AdipoR1 and R2 are expressed in these cells, and ii) the subphysiological concentrations of recombinant adiponectin inhibit MDA-MB 231 cell growth and concomitantly enhance the expression of Bax and p53, two pro-apoptotic genes. Moreover, the invalidation of AdipoR1 and R2 mRNA experiments demonstrated that the anti-proliferative and pro-apoptotic effects of adiponectin were partially mediated via AdipoR1 and R2. We describe, for the first time, that AdipoR mRNA expression was down-regulated by adiponectin and leptin in MDA-MB 231 cells. Taken altogether, these results strongly suggest that the two adipokines should be considered as i) additional factors of breast cancer risk, and ii) may therefore be potential targets in breast cancer therapy.

Topics: Adiponectin; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Genes, p53; Humans; Receptors, Adiponectin; RNA, Messenger; Signal Transduction

The amplification event occurring at chromosome locus 11q13, reported in several different cancers, includes a number of potential oncogenes. We have previously reported amplification of one such oncogene, namely CCND1, to be correlated with an adverse effect of tamoxifen in premenopausal breast cancer patients. Over-expression of cyclin D1 protein, however, confers tamoxifen resistance but not a tamoxifen-induced adverse effect. Potentially, co-amplification of an additional 11q13 gene, with a resulting protein over-expression, is required to cause an agonistic effect. Moreover, during 11q13 amplification a deletion of the distal 11q region has been described. In order to assess the potential impact of the deletion we examined a selected marker for this event.. Array comparative genomic hybridization analysis was employed to identify and confirm changes in the gene expression of a number of different genes mapping to the 11q chromosomal region, associated with CCND1 amplification. The subsequent protein expression of these candidate genes was then examined in a clinical material of 500 primary breast cancers from premenopausal patients who were randomly assigned to either tamoxifen or no adjuvant treatment. The protein expression was also compared with gene expression data in a subset of 56 breast cancer samples.. Cortactin and FADD (Fas-associated death domain) over-expression was linked to CCND1 amplification, determined by fluorescence in situ hybridization, but was not associated with a diminished effect of tamoxifen. However, deletion of distal chromosome 11q, defined as downregulation of the marker Chk1 (checkpoint kinase 1), was associated with an impaired tamoxifen response, and interestingly with low proliferative breast cancer of low grade. For Pak1 (p21-activated kinase 1) and cyclin D1 the protein expression corresponded to the gene expression data.. The results indicate that many 11q13 associated gene products are over-expressed in conjunction with cyclin D1 but not linked to an agonistic effect of tamoxifen. Finally, the deletion of distal 11q, linked to 11q13 amplification, might be an important event affecting breast cancer outcome and tamoxifen response.

Topics: Adult; Antineoplastic Agents, Hormonal; Breast Neoplasms; Carcinoma; Checkpoint Kinase 1; Chromosomes, Human, Pair 11; Comparative Genomic Hybridization; Cortactin; Cyclin D1; Drug Resistance, Neoplasm; Estrogen Receptor Modulators; Fas-Associated Death Domain Protein; Female; Gene Amplification; Gene Expression Profiling; Humans; Neoplasm Invasiveness; Neoplasm Proteins; p21-Activated Kinases; Premenopause; Protein Kinases; Randomized Controlled Trials as Topic; Sequence Deletion; Tamoxifen

to determine signaling pathways in breast cancers from patients aged 35 years old or younger and patients aged more than 35 years old.. this was a cross-sectional, comparative study of female breast cancer patients who were recruited and divided into two age groups, i.e. 35 years or younger and more than 35 years old. Specimens were obtained by biopsy or surgical removal of the tumors and were confirmed by histopathological examination. The expression of ER, IGF-1R, Her-2, MAPK, and cyclin D1 were measured using immunohistochemistry.. ninety-three patients were recruited from September 2004 to December 2005. Forty-three patients were 35 years or younger. More than 90% of the patients within the two groups showed invasive ductal carcinomas and more than half of these tumors were grade 2. Immunohistochemical staining was successfully done in 90 patients. ER-alpha expression was negative in 33 breast cancers (78.6%) from patients less than 35 years old and 32 cancers (66.7%) of older patients. The expressions of IGF-1R, Her-2, MAPK, and cyclin D1 were positive, respectively in 17 (40.5%), 11 (26.2%), 28 (66.7%), and 7 (16.7%) cancers within the group of patients 35 years old or younger, and, respectively in 18 (37.5%), 11 (22.9%), 37 (77.1%), and 9 (18.8%) of cancers from patients more than 35 years old.. there were no statistically significant differences in the expression of any of the biomarkers between the two groups. In all patients, ER was negative in 72.2% cases and MAPK was positive in 76.7% cases. Patients aged 35 years or younger showed similar ER, IGF-1R, Her-2, MAPK, and cyclin D1 expressions compared to cancers from patients more than 35 years old. These were predominantly ER-negative, suggesting that estrogen does not play a dominant role in their growth. The frequent expression of MAPK in these cancers raises the possibility that growth factors play a dominant role in their growth.

Topics: Adult; Age Factors; Biomarkers, Tumor; Breast Neoplasms; Cross-Sectional Studies; Cyclin D1; Female; Humans; Immunohistochemistry; Indonesia; Mitogen-Activated Protein Kinase Kinases; Receptor, ErbB-2; Receptor, IGF Type 1; Receptors, Estrogen; Receptors, Progesterone; Signal Transduction; Time Factors

The basal-like subtype of breast cancer is associated with invasiveness, high rates of postsurgical recurrence, and poor prognosis. Aside from inactivation of the BRCA1 tumor-suppressor gene, little is known concerning the mechanisms that cause basal breast cancer or the mechanisms responsible for its invasiveness. Here, we show that the heterogeneous mouse mammary tumor virus-cyclin D1-Cdk2 (MMTV-D1K2) transgenic mouse mammary tumors contain regions of spindle-shaped cells expressing both luminal and myoepithelial markers. Cell lines cultured from these tumors exhibit the same luminal/myoepithelial mixed-lineage phenotype that is associated with human basal-like breast cancer and express a number of myoepithelial markers including cytokeratin 14, P-cadherin, alpha smooth muscle actin, and nestin. The MMTV-D1K2 tumor-derived cell lines form highly invasive tumors when injected into mouse mammary glands. Invasion is associated with E-cadherin localization to the cytoplasm or loss of E-cadherin expression. Cytoplasmic E-cadherin correlates with lack of colony formation in vitro and beta-catenin and p120(ctn) localization to the cytoplasm. The data suggest that the invasiveness of these cell lines results from a combination of factors including mislocalization of E-cadherin, beta-catenin, and p120(ctn) to the cytoplasm. Nestin expression and E-cadherin mislocalization were also observed in human basal-like breast cancer cell lines, suggesting that these results are relevant to human tumors. Together, these results suggest that abnormal Cdk2 activation may contribute to the formation of basal-like breast cancers.

Topics: Animals; beta Catenin; Breast Neoplasms; Cadherins; Catenins; Cell Adhesion Molecules; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase 2; Delta Catenin; Female; Humans; Immunoblotting; Immunohistochemistry; Intermediate Filament Proteins; Mammary Neoplasms, Experimental; Mammary Tumor Virus, Mouse; Metalloproteins; Mice; Mice, Transgenic; Microscopy, Fluorescence; Neoplasm Invasiveness; Nerve Tissue Proteins; Nestin; Phosphoproteins; Protein Transport; Stress Fibers; Zyxin

To rapidly detect molecular alterations in different malignancies and investigate the possible role of Tp53, C-myc, and CCND1 genes in development of tumors in human organs and their adjacent normal tissues, as well as the possible relation between well- and poorly-differentiated tumors.. A tissue array consisting of seven different tumors was generated. The tissue array included 120 points of esophagus, 120 points of stomach, 80 points of rectum, 60 points of thyroid gland, 100 points of mammary gland, 80 points of liver, and 80 points of colon. Expressions of Tp53, C-myc, and CCND1 were determined by RNA in situ hybridization. 3' terminal digoxin-labeled anti-sense single stranded oligonucleotide and locked nucleic acid modifying probe were used.. The expression level of Tp53 gene was higher in six different carcinoma tissue samples than in paracancerous tissue samples with the exception in colon carcinoma tissue samples (P < 0.05). The expression level of CCND1 gene was significantly different in different carcinoma tissue samples with the exception in esophagus and colon carcinoma tissue samples. The expression level of C-myc gene was different in esophagus carcinoma tissue samples (chi2 = 18.495, P = 0.000), stomach carcinoma tissue samples (chi2 = 23.750, P = 0.000), and thyroid gland tissue samples (chi2 = 10.999, P = 0.004). The intensity of signals was also different in different carcinoma tissue samples and paracancerous tissue samples.. Over-expression of the Tp53, CCND1, and C-myc genes appears to play a role in development of human cancer by regulating the expression of mRNA. Tp53, CCND1 and C-myc genes are significantly correlated with the development of different carcinomas.

Topics: Breast Neoplasms; China; Colonic Neoplasms; Cyclin D1; Esophageal Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Proto-Oncogene Proteins c-myc; Rectal Neoplasms; RNA, Messenger; Stomach Neoplasms; Thyroid Neoplasms; Tissue Array Analysis; Tumor Suppressor Protein p53

The current study expands upon previous work using a database of 710 patients treated with neoadjuvant chemotherapy. First, we studied phenotypic characteristics of tumors before and after chemotherapy using the following factors: the mitotic index of the Scarff-Bloom-Richardson grade, Ki-67, cyclin D1, and cyclo-oxygenase-2. Second, the predictive value of these factors on response was assessed. Third, we measured the prognostic impact of these markers post-therapy in comparison with clinical and pathological responses according to the Chevallier and Sataloff classifications. Patients were treated using different neoadjuvant chemotherapy combinations, mainly in successive prospective phase II trials. They received a median number of six cycles (range, 1-9). After neoadjuvant chemotherapy, patients underwent surgery and radiotherapy. In cases of important residual disease, some received additional courses of chemotherapy. In addition, menopausal patients with hormone receptor-positive tumors received tamoxifen for 5 years. According to our analysis, we found significant variations before and after neoadjuvant chemotherapy only for cyclin D1 and the mitotic index. Concerning the predictive value of biomarkers for response, Ki-67 and the mitotic index were predictive on univariate analysis, both for objective clinical and pathological complete responses. Because these two factors were correlated, no multivariate analyses were conducted. We then assessed the prognostic impact of the biopathological factors. When the factors were measured before chemotherapy, all were prognostic. When evaluated after chemotherapy, the mitotic index, objective clinical response, and pathological complete response were prognostic. Because these factors were correlated, no multivariate model was done. The main clinical fact is that there were significant correlations between clinical and pathological responses and variations in the biological factors studied.

Topics: Adult; Aged; Aged, 80 and over; Breast Neoplasms; Chemotherapy, Adjuvant; Cyclin D1; Cyclooxygenase 2; Female; Humans; Immunohistochemistry; Ki-67 Antigen; Middle Aged; Mitotic Index; Prognosis

The significant advance in the development of molecular-targeting drugs has made an evaluation of Her-2, EGFR, and cyclin D1 an important clinical issue in breast cancer patients. This study compared the Her-2, EGFR, and cyclin D1 status of primary tumors as well as their matching lymph node metastases using immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH) in 73 breast cancer patients. Her-2, EGFR, and cyclin D1 protein showed a concordance between the primary lesion and the metastatic regional lymph nodes in 82%, 90%, and 63%, respectively. CISH also revealed 92%, 93%, and 85% concordance in the gene amplification status of Her-2, EGFR, and cyclin D1, showing a reasonable agreement between primary tumors and metastatic regional lymph nodes. Although a statistically significant agreement was found in Her-2 expression, a relatively high discordance rate (18%) raises a little concern. Our findings suggest that the Her-2 status can be reliably assessed on primary tumor but a possible difference can be found in Her-2, EGFR, and cyclin D1 status between the primary and the metastatic sites and this possibility should be concerned in patients considering molecular targeted therapy or patients with progress of disease.

Topics: Adult; Aged; Breast Neoplasms; Chromogenic Compounds; Cyclin D1; ErbB Receptors; Female; Humans; Immunohistochemistry; In Situ Hybridization; Lymph Nodes; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Recurrence, Local; Receptor, ErbB-2; Survival Analysis

The latest clinical trials indicate better performance of aromatase inhibitors, compared to tamoxifen, in adjuvant hormonotherapy of breast carcinoma. The identification of molecular markers, predicting resistance to tamoxifen, could help to identify patients, which are most likely to benefit from aromatase inhibitors in up-front adjuvant hormonotherapy.. Tissue microarrays were constructed from archival paraffin blocks of primary tumors of 179 patients with estrogen receptor positive operable breast carcinoma in stage I-III, subsequently treated with tamoxifen for five years or until relapse, with at least 7 years follow up available. The amplifications of Her-2 and cyclin D1 genes were evaluated by fluorescence in-situ hybridization. The level of progesterone receptor (PR) and Ki67 were estimated by immunohistochemistry.. 54 of above patients recurred during follow up. In univariate analysis of disease free survival, the presence of more than three nodal metastases (RR=4,5 p<0,001), grade 3 (RR=2,3 p=0,035), cyclin D1 (RR=3,06 p<0,001) and Her-2 (RR=3,06 p<0,001) amplifications were identified as significant risk factors, together with the negativity of PR (RR=2,1 p=0,013). In multivariate analysis, only clinical stage III (RR=2,6 p=0,003), cyclin D1 (RR=2,7 p=0,001) and Her-2 (RR=2,1 p=0,014) amplifications proved significant. In 77 patients who received adjuvant chemotherapy no statistically significant risk factor was identified. In multivariate analysis of 102 patients without adjuvant chemotherapy only stage III (RR=6,9 p=0,001) and Her-2 amplification (RR=4,5 p=0,001) were confirmed.. The advanced clinical stage, cyclin D1 and Her-2 gene amplifications represent factors, predicting the failure of adjuvant tamoxifen treatment, but their predictive value is much lower in patients receiving adjuvant chemotherapy. This fact indicates, they can reflect the common biological aggressiveness of tumor and need not to be tamoxifen specific.

Topics: Aged; Antineoplastic Agents, Hormonal; Breast Neoplasms; Chemotherapy, Adjuvant; Cyclin D1; Disease-Free Survival; Drug Resistance, Neoplasm; Female; Humans; Middle Aged; Neoplasm Recurrence, Local; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone; Survival Analysis; Tamoxifen; Tissue Array Analysis

The ErbB2 (Her2/neu epidermal growth receptor family) oncogene is overexpressed in 30% to 40% of human breast cancers. Cyclin D1 is the regulatory subunit of the holoenzyme that phosphorylates and inactivates the retinoblastoma (pRb) tumor suppressor and is an essential downstream target of ErbB2-induced tumor growth. Herein, we demonstrate that ErbB2 induces the activity of the Notch signaling pathway. ErbB2 induction of DNA synthesis, contact-independent growth, and mammosphere induction required Notch1. ErbB2-induced cyclin D1 and cyclin D1 expression was suficient to induce Notch1 activity, and conversely, genetic deletion of Notch1 in mammary epithelial cells using foxed Notch (Notch(fl/fl)) mice demonstrated that cyclin D1 is induced by Notch1. Genetic deletion of cyclin D1 or small interfering RNA (siRNA) to cyclin D1-reduced Notch1 activity and reintroduction of cyclin D1 into cyclin D1-deficient cells restored Notch1 activity through the inhibition of Numb, an endogenous inhibitor of Notch1 activity. Thus, cyclin D1 functions downstream as a genetic target of Notch1, amplifies Notch1 activity by repressing Numb, and identifies a novel pathway by which ErbB2 induces Notch1 activity via the induction of cyclin D1.

Topics: Amyloid Precursor Protein Secretases; Animals; Breast Neoplasms; Cell Line, Tumor; Cyclin D1; DNA, Neoplasm; Female; Humans; Mice; Neuregulin-1; Receptor, ErbB-2; Receptor, Notch1; Signal Transduction; Tumor Stem Cell Assay

The activating protein-1 (AP-1) transcription factor transduces growth signals through signal transduction pathways to the nucleus, leading to the expression of genes involved in growth and malignant transformation in many cell types. We have previously shown that overexpression of a dominant negative form of the cJun proto-oncogene, a cJun dominant negative mutant (Tam67), blocks AP-1 transcriptional activity, induces a G(1) cell cycle block and inhibits breast cancer cell growth in vitro and in vivo. We found that AP-1 blockade by Tam67 in MCF-7 breast cancer cells downregulates cyclin D1 transcriptional activity by at least two mechanisms: by suppressing transcription at the known AP-1 binding site (-934/-928) and by suppressing growth factor-induced expression through suppressing E2F activation at the E2F-responsive site (-726/-719). AP-1 blockade also led to reduced expression of E2F1 and E2F2, but not E2F4, at the mRNA and protein levels. Chromatin immunoprecipitation and supershift assays demonstrated that AP-1 blockade caused decreased binding of E2F1 protein to the E2F site in the cyclin D1 promoter. We also found that Tam67 suppressed the expression of the E2F1 dimerizing partner, DP1 and E2F-upregulated cell cycle genes (cyclins E, A, B and D3) and enhanced the expression of E2F-downregulated cell cycle genes (cyclins G(2) and I). Reduced expression of other E2F-regulated genes was also seen with AP-1 blockade and E2F suppression. Thus, the AP-1 factor regulates the expression of cyclin D and E2F (the latter in turn regulates E2F-downstream genes), leading to cell cycle progression and breast cancer cell proliferation.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Chromatin Immunoprecipitation; Cyclin D1; Cyclins; Down-Regulation; E2F Transcription Factors; E2F1 Transcription Factor; E2F2 Transcription Factor; E2F4 Transcription Factor; Female; Gene Expression Regulation, Neoplastic; Humans; Peptide Fragments; Promoter Regions, Genetic; Proto-Oncogene Mas; Proto-Oncogene Proteins c-jun; RNA, Messenger; Transcription Factor AP-1; Transcription Factor DP1

Brn-3b transcription factor enhances proliferation of neuroblastoma (NB) and breast cancer cell lines in vitro and increases the rate and size of in vivo tumour growth, whereas reducing Brn-3b slows growth, both in vitro and in vivo. Brn-3b is elevated in >65% of breast cancer biopsies, and here we demonstrate that Brn-3b is also elevated in NB tumours. We show a significant correlation between Brn-3b and cyclin D1 (CD1) in breast cancers and NB tumours and cell lines. Brn-3b directly transactivates the CD1 promoter in co-transfection experiments, whereas electrophoretic mobility shift assay and chromatin immunoprecipitation assays demonstrate that Brn-3b protein binds to an octamer sequence located in the proximal CD1 promoter. Site-directed mutagenesis of this sequence resulted in loss of transactivation of the CD1 promoter by Brn-3b. Thus, Brn-3b may act to alter growth properties of breast cancer and NB cells by enhancing CD1 expression in these cells.

Topics: Breast Neoplasms; Cell Line, Tumor; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Neuroblastoma; RNA, Messenger; Transcription Factor Brn-3B; Transcriptional Activation; Tumor Cells, Cultured; Up-Regulation

Cyclin D1 overexpression has been associated with poor prognosis and resistance to therapy in human breast cancer. Thus, the development of therapeutic agents that selectively target cyclin D1 activity is of clinical interest. This study demonstrates that 12-oxo-phytodienoic acid (OPDA), a phytohormone with critical functions in growth and development in plants, induces growth arrest in MDA-MB-231 and T47D breast cancer cells. In response to OPDA treatment, the human breast cancer cell lines exhibit a progressive decline in cyclin D1 expression, which is tightly associated with the accumulation of hypophosphorylated form of the retinoblastoma protein (Rb) and G1 arrest. The decrease in cyclin D1 protein expression accompanies a dramatic decline in nuclear but not membranous beta-catenin expression and activation of glycogen synthase kinase-3-beta (GSK3beta) caused by inhibition of its serine-9 phosphorylation. The proteasome inhibitor MG132 blocks OPDA-mediated decrease in cyclin D1. In addition, the overexpression of T286A, a cyclin D1 mutant which is refractory to phosphorylation by GSK3beta and proteosomal degradation, is resistant to OPDA-mediated Rb dephosphorylation as well as G(1) cell cycle arrest. Thus, our results demonstrate that degradation of cyclin D1 protein is a key event in OPDA induced growth inhibition in breast cancer cells. These data provide the basic foundation for future efforts to develop OPDA-based approaches in the prevention and treatment of breast cancer and other types of cancer.

Topics: Antineoplastic Agents; beta Catenin; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Fatty Acids, Unsaturated; Female; Flow Cytometry; Humans; Immunohistochemistry; Retinoblastoma Protein

Despite strong evidence regarding the role of CCND1 amplification and protein overexpression in breast carcinoma, the associations between CCND1 amplification/cyclin D1 overexpression and clinicopathological variables and clinical outcome remain controversial.. (1) to correlate cyclin D1 expression with gene amplification; (2) to analyse the correlations between CCND1 amplification and overexpression with clinicopathological features and patients' outcome in invasive breast cancer; (3) to define the prevalence and clinical significance of cyclin D1 overexpression and CCND1 amplification in ER positive breast carcinomas (4) to define the prevalence of cyclin D1 overexpression and CCND1 amplification in breast cancers with basal-like immunophenotype.. CCND1 amplification and protein expression were assessed on a tissue microarray containing 880 unselected invasive breast cancer cases, by means of chromogenic in situ hybridisation using the Spotlight CCND1 amplification probe and immunohistochemistry, using the rabbit monoclonal antibody SP4.. A total of 59/613 tumours (9.6%) showed CCND1 amplification and 224/514 (43.6%) showed strong cyclin D1 expression. A strong positive correlation between CCND1 amplification and higher levels of cyclin D1 expression was found (P < 0.001). Basal-like cancers showed infrequent CCND1 amplification and cyclin D1 overexpression (P < 0.001). Both CCND1 amplification and cyclin D1 expression were associated with positive ER status. CCND1 gene amplification was an independent prognostic factor for patients with ER positive breast cancer.. Our results demonstrate a strong correlation between CCND1 amplification and its protein expression in breast cancer. However, protein expression is more pervasive than gene amplification and associated with ER expression.

Topics: Biomarkers, Tumor; Breast Neoplasms; Cyclin D; Cyclin D1; Cyclins; Female; Gene Amplification; Humans; In Situ Hybridization; Kaplan-Meier Estimate; Middle Aged; Proteomics; Tissue Array Analysis

There is currently substantial interest in the regulation of cell function by mammalian target of rapamycin (mTOR), especially effects linked to the rapamycin-sensitive mTOR complex 1 (mTORC1). Rapamycin induces G(1) arrest and blocks proliferation of many tumor cells, suggesting that the inhibition of mTORC1 signaling may be useful in cancer therapy. In MCF7 breast adenocarcinoma cells, rapamycin decreases levels of cyclin D1, without affecting cytoplasmic levels of its mRNA. In some cell-types, rapamycin does not affect cyclin D1 levels, whereas the starvation for leucine (which impairs mTORC1 signaling more profoundly than rapamycin) does. This pattern correlates with the behavior of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1, an mTORC1 target that regulates translation initiation). siRNA-mediated knock-down of 4E-BP1 abrogates the effect of rapamycin on cyclin D1 expression and increases the polysomal association of the cyclin D1 mRNA. Our data identify 4E-BP1 as a key regulator of cyclin D1 expression, indicate that this effect is not mediated through the changes in cytoplasmic levels of cyclin D1 mRNA and suggest that, in some cell types, interfering with the amino acid input to mTORC1, rather than using rapamycin, may inhibit proliferation.

Topics: Adaptor Proteins, Signal Transducing; Antibiotics, Antineoplastic; Breast Neoplasms; Cell Cycle Proteins; Cell Line; Cell Line, Tumor; Cyclin D1; Eukaryotic Initiation Factor-4E; Gene Expression Regulation, Neoplastic; Humans; Mechanistic Target of Rapamycin Complex 1; Multiprotein Complexes; Phosphoproteins; Proteins; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factors

Accumulating evidence indicates that heregulins, EGF (epidermal growth factor)-like ligands, promote breast cancer cell proliferation and are involved in the progression of breast cancer towards an aggressive and invasive phenotype. However, there is limited information regarding the molecular mechanisms that mediate these effects. We have recently established that HRG (heregulin beta1) promotes breast cancer cell proliferation and migration via cross-talk with EGFR (EGF receptor) that involves the activation of the small GTPase Rac1. In the present paper we report that Rac1 is an essential player for mediating the induction of cyclin D1 and p21(Cip1) by HRG in breast cancer cells. Inhibition of Rac function by expressing either the Rac-GAP (GTPase-activating protein) beta2-chimaerin or the dominant-negative Rac mutant N17Rac1, or Rac1 depletion using RNAi (RNA interference), abolished the cyclin D1 and p21(Cip1) induction by HRG. Interestingly, the proliferative effect of HRG was impaired not only when the expression of Rac1 or cyclin D1 was inhibited, but also when cells were depleted of p21(Cip1) using RNAi. Inhibition of EGFR, PI3K (phosphoinositide 3-kinase; kinases required for Rac activation by HRG) or MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase] also blocked the up-regulation of cyclin D1 and p21(Cip1) by HRG. In addition, we found that HRG activates NF-kappaB (nuclear factor kappaB) in a Rac1- and MEK-dependent fashion, and inhibition of NF-kappaB abrogates cyclin D1/p21(Cip1) induction and proliferation by HRG. Taken together, these findings establish a central role for Rac1 in the control of HRG-induced breast cancer cell-cycle progression and proliferation through up-regulating the expression of cyclin D1 and p21(Cip1).

Topics: Base Sequence; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Extracellular Signal-Regulated MAP Kinases; Humans; Nerve Tissue Proteins; Neuregulin-1; rac1 GTP-Binding Protein; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering

Wogonin is a plant monoflavonoid which has been reported to inhibit cell growth and/or induce apoptosis in various tumors. Herein, we investigated the in vitro and in vivo anticancer effects and associated mechanisms of wogonin in human breast cancer. Effects of wogonin were examined in estrogen receptor (ER)-positive and -negative human breast cancer cells in culture for proliferation, cell cycle progression, and apoptosis. The in vivo effect of oral wogonin was examined on tumor xenograft growth in athymic nude mice. The molecular changes associated with the biological effects of wogonin were analyzed by immunoblotting. Cell growth was attenuated by wogonin (50-200 microM), independently of its ER status, in a time- and concentration-dependent manner. Apoptosis was enhanced and accompanied by upregulation of PARP and Caspase 3 cleavages as well as proapoptotic Bax protein. Akt activity was suppressed and reduced phosphorylation of its substrates, GSK-3beta and p27, was observed. Suppression of Cyclin D1 expression suggested the downregulation of the Akt-mediated canonical Wnt signaling pathway. ER expression was downregulated in ER-positive cells, while c-ErbB2 expression and its activity were suppressed in ER-negative SK-BR-3 cells. Wogonin feeding to mice showed inhibition of tumor growth of T47D and MDA-MB-231 xenografts by up to 88% without any toxicity after 4 weeks of treatment. As wogonin was effective both in vitro and in vivo, our novel findings open the possibility of wogonin as an effective therapeutic and/or chemopreventive agent against both ER-positive and -negative breast cancers, particularly against the more aggressive and hormonal therapy-resistant ER-negative types.

Topics: Animals; Apoptosis; Breast Neoplasms; Caspases; Cell Proliferation; Cyclin D1; Drugs, Chinese Herbal; Estrogen Receptor alpha; Female; Flavanones; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Receptor, ErbB-2; Signal Transduction; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

We aimed to investigate the role of CCND1 G870A polymorphism genetic and transcriptomic effects susceptibility in association with breast cancer carcinogenesis and clinical prognosis. A case-control study was conducted with the enrollment of 992 sporadic breast cancer patients and the corresponding 960 normal controls from routine mammographic or sonographic screening for breast cancer between 1995 and 2003 in Taiwan. The 167 fragment spanning the G870A polymorphism in exon 4-intron 4 boundary was amplified to identify genotype of CCND1 (G870A) polymorphism. Competitive RT-PCR were further performed to investigate alternative transcript in four different specimens in association with immunohistochemistry markers. The results showed that AG and AA subgroup were at increased risk for developing breast cancer compared with the GG genotype by 19% (OR 1.19 (0.85-1.67)) and by 34% (OR 1.34 (0.04-1.74)), respectively. A870 allele revealed a recessive tendency while GG and AA/AG subgroup was compared (OR 1.35 (1.07-1.70)). AA genotype also had a higher risk in premenopausal women than postmenopausal ones. The recurrence-free survival was longer in patients with GG+AG than that in patients with AA (P = 0.034). A870 allele produced more transcript b both in malignant. There were significant correlations between several immunohistochemistry markers (such as Ki-67) and cyclin D1 or CDk4. We concluded CCND1 G870A polymorphism make significant contribution to breast cancer in the country with the preponderance of breast cancer in young women. The role of G870A polymorphism in alternative transcript was not only implicated in CCND1 alternative splicing but also correlated with immunohistochemistry markers.

Topics: Adult; Alleles; Breast Neoplasms; Case-Control Studies; Cyclin D1; Exons; Genetic Predisposition to Disease; Humans; Immunohistochemistry; Introns; Middle Aged; Polymorphism, Genetic; Prognosis; RNA, Messenger; Taiwan

Determination of Her2, epidermal growth factor receptor (EGFR) and cyclin D1 status is now of major clinical importance due to the development of molecule-targeting drugs in anticancer therapy. Immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH) are the most simple and convenient methods for evaluating gene alterations and their protein consequences. The purpose of the present study was to investigate the status of Her2, EGFR and cyclin D1 on both IHC and CISH in 95 primary breast carcinomas. There was substantial consistency between the IHC and CISH results of Her2 and EGFR, showing fair agreement between protein overexpression and gene amplification. However, cyclin D amplification was not related to protein overexpression. Moreover, there was no correlation between Her2, EGFR and cyclin D1. Her2 protein overexpression and amplification were positively associated with histological grade, nuclear grade and inversely correlated with the expression of estrogen receptor (ER) and progesterone receptor (PR). In ER-negative and postmenopausal patients, EGFR gene amplification was strongly associated with worse recurrence-free survival (P = 0.0087, P = 0.0149, respectively). Overall, the present findings suggest that EGFR gene amplification is important in predicting prognosis and this should be evaluated in breast carcinoma in addition to Her2 status in routine pathological practice.

Topics: Adult; Aged; Biomarkers, Tumor; Breast Neoplasms; Chromogenic Compounds; Cyclin D1; ErbB Receptors; Female; Gene Amplification; Gene Expression; Gene Expression Profiling; Genes, bcl-1; Genes, erbB-1; Genes, erbB-2; Humans; Immunohistochemistry; In Situ Hybridization; Middle Aged; Receptor, ErbB-2

Estrogen and progesterone are key regulators of normal breast epithelial cell proliferation and differentiation. They are also involved in the initiation and progression of breast tumorigenesis. Several experimental studies have demonstrated that steroid hormones affect cell cycle proteins associated with tumor initiation and progression. Hormone replacement therapy (HT) is widely used to alleviate climacteric symptoms among postmenopausal women. Little is known, however, about cell cycle protein regulation during hormonal treatment of human breast tissue (HBT). In this study we aimed to evaluate the effects of 17beta-estradiol (E(2)) and medroxyprogesterone acetate (MPA) on cultured HBTs representing samples from reduction mammoplasty of premenopausal (pre-HBT) and postmenopausal (postm-HBT) women, and from peritumoral tissue (peritum-HBT) after breast tumor surgery among postmenopausal patients. Explants of HBT were cultured for 14 days in medium supplemented with E(2), MPA or E(2) + MPA. Expression of cyclin D1, p21 and p27 was assessed by immunohistochemical staining of explants cultured for 2 and 14 days. Further, Ki-67 staining was performed to evaluate correlation between proliferation and cell cycle regulatory protein expression. Our results showed that HBTs studied were positive for ERalpha, ERbeta and PR (> or =10% of the cells stained). The level of p21 was lower (p < 0.001) in pre-HBT than in postm-HBT, whereas p27 levels were higher (p < 0.05) in pre-HBT than in postm- and peritum-HBT. The level of Ki67 positive cells was higher in pre- HBT than in post-HBT. Interestingly, level of p21 positive cells showed an opposite pattern. Treatment with E(2) increased the relative number of cyclin D1-staining cells and decreased that of p27-staining cells in postm-HBT (p < 0.05), but not in pre-HBT. All hormone regimens (E(2), MPA, E(2) + MPA) increased the number of p21-positive cells in postm-HBTs at 14 days and E(2) even at 2 days. In pre-HBT p21 staining was increased (p < 0.05) in explants cultured with E(2) for 14 days but no response was observed in cyclin D1 and p27. The number of cyclin D1-staining cells was clearly higher (p < 0.05) in peritum-HBT than in non-tumorous pre- or postm-HBT, but the response cyclin D1 to all hormonal treatments in peritum-HBT was the same as in postm-HBT. Moreover, we found that E(2), MPA, E(2) + MPA in vitro increased numbers of Ki67 positive cells in post-HBTs at 14 days and E(2) also in pre-HBT. Stimulated proli

Topics: Adult; Aged; Breast; Breast Neoplasms; Cell Cycle Proteins; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Estradiol; Female; Humans; Medroxyprogesterone Acetate; Middle Aged; Organ Culture Techniques; Postmenopause; Premenopause

The HER2 codon Ile655Val and Cyclin D1 (CCND1) G870A polymorphisms were analyzed in a hospital-based Malaysian population using PCR-RFLP method. Peripheral blood samples were collected from 230 breast cancer patients, and 200 normal and healthy women who had no history of breast disease or breast cancer. We evaluated the association between HER2 or CCND1 polymorphisms and breast cancer risk, and clinico-pathological parameters in the population. The genotype and allele frequencies of HER2 (P=0.163 vs P=0.0622) and CCND1 (P=0.377 vs P=0.284) polymorphisms were not significantly different between the breast cancer cases and normal subjects, respectively. Women who were Ile/Val heterozygotes (OR=1.48; 95% CI, 0.91-2.43), Val/Val homozygotes (OR=1.93; 95% CI, 0.51-7.77) and carriers of Val allele genotype (OR=1.53; 95% CI, 0.95-2.45) were not significantly associated with increased breast cancer risk. Similarly, women who were homozygous (OR=1.34; 95% CI, 0.77-2.34) or heterozygous (OR=0.98; 95% CI, 0.60-1.60) for A allele, or carriers of A allele genotype (OR=1.10; 95% CI, 0.70-1.73) were not associated with breast cancer risk. Analysis on clinico-pathological parameters showed that Val allele genotype was significantly correlated with nodal metastases but A allele genotype was not associated with any of the variables. Our findings suggest that the polymorphic alleles of HER2 and CCND1 may not play an important role as genetic markers for breast cancer risk, but presence of Val allele may be useful for tumor prognosis.

Topics: Adult; Aged; Breast Neoplasms; Cyclin D1; Female; Genetic Predisposition to Disease; Genotype; Humans; Lymphatic Metastasis; Middle Aged; Polymorphism, Genetic; Receptor, ErbB-2

De novo or acquired resistance to tamoxifen is a major clinical challenge for the management of estrogen receptor (ER)-positive breast cancers. Although cyclin D1 overexpression is associated with a better outcome for breast cancer patients, its overexpression is also linked to tamoxifen resistance. We previously reported that the beneficial effect of cyclin D1 correlates with its ability to repress the antiapoptotic transcription factor signal transducer and activator of transcription 3 (STAT3). In contrast, molecular pathways linking overexpression of cyclin D1 to tamoxifen resistance have not been established. In the current study, the effect of tamoxifen on the growth of genetically matched high or low cyclin D1-expressing breast cancer cells was characterized and the interactions between cyclin D1, ER, and STAT3 in response to tamoxifen treatment were determined. We show that repression of STAT3 by cyclin D1 inhibits cell growth on Matrigel and in tumors in vivo; however, treatment with tamoxifen abolishes cyclin D1-mediated repression of STAT3 and growth suppression. We show that tamoxifen induces a redistribution of cyclin D1 from STAT3 to the ER, which results in the activation of both STAT3 and the ER. These results offer a molecular mechanism for the dual effect of cyclin D1 overexpression in breast cancer and support the notion that the level of cyclin D1 expression and activated STAT3 are important markers to predict response to tamoxifen treatment.

Topics: Animals; Antineoplastic Agents, Hormonal; Breast Neoplasms; Cell Growth Processes; Cyclin D1; Drug Resistance, Neoplasm; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Receptors, Estrogen; STAT3 Transcription Factor; Tamoxifen; Up-Regulation; Xenograft Model Antitumor Assays

In the course of a project aimed to clarify the molecular mechanisms by which phorbol 12-myristate 13-acetate (PMA)-activated forms of protein kinase C (PKC) promote growth arrest in an MCF-7 cell line, we found that the PKCdelta inhibitor Rottlerin was able by itself to block cell proliferation. In the current study, we investigated further the antiproliferative response to Rottlerin. Western blotting analysis of cytoplasmic/nuclear extracts showed that the drug did not prevent either extracellular signal-regulated kinase (ERK) activation by PMA or Akt phosphorylation, but did interfere with the NFkappaB activation process (both basal and PMA-stimulated), by lowering the levels of phospho-IkappaBalpha and preventing p65 nuclear migration. The growth arrest evoked by Rottlerin was not mediated by cell-cycle inhibitors p21 and p27 but was accompanied by a dramatic fall in the cyclin-D1 protein, the levels of which were not altered by the pan-PKC inhibitor GF 109203X, thus excluding a PKC-mediated mechanism in the Rottlerin effect. The parallel drop in cyclin-D1 mRNA suggested a down-regulation of the gene caused by the inhibition of nuclear factor-kappa B (NFkappaB), which occurs via a PKC-, Akt-, ERK- and mitochondrial uncoupling-independent mechanism. We provide preliminary evidence that the interference on the NFkappaB activation process likely occurs at the level of calcium/calmodulin-dependent protein kinase II (CaMKII), a known Rottlerin target. Indeed the drug prevented calcium-induced CaMKII autophosphorylation which, in turn, led to decreased NFkappaB activation.

Topics: Acetophenones; Benzopyrans; Breast Neoplasms; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Indoles; Maleimides; NF-kappa B; Proto-Oncogene Proteins c-akt; Signal Transduction

We studied by real-time PCR cyclin D1 and thymidylate synthase (TS) mRNA in plasma as possible markers of clinical outcome in breast cancer. We observed poor outcome in patients with presence of cyclin D1 mRNA in good-prognosis groups, such as negative vascular invasion. Presence of both markers was associated with non-response to treatment after relapse. In patients treated with tamoxifen, a trend to significant relation between poor outcome and cyclin D1 mRNA was found. Cyclin D1 mRNA in plasma could identify patients with poor overall survival in good-prognosis groups and patients non-responsive to tamoxifen.

Topics: Breast Neoplasms; Cyclin D1; Disease-Free Survival; Female; Humans; Prognosis; Prospective Studies; RNA, Messenger; Survival Analysis; Tamoxifen; Thymidylate Synthase; Treatment Outcome

Sulindac has been reported to be effective in suppressing tumor growth through the induction of p21WAF1/cip1 in human, animal models of colon cancer and colon cancer cells. In this study, we treated human breast cancer cell line MCF-7 and lung cancer cell line A549 as well as colon cancer cell line SW620 with sulindac to observe the effects of sulindac in other tissue sites. In all cell lines, proliferation was significantly inhibited by sulindac after 24 and 72 h of treatment. Apoptosis was induced by sulindac in both lung cancer cells and colon cancer cells but was not induced in breast cancer cells. Western blots showed that p21 protein level were induced by sulindac in lung cancer cells and colon cancer cells, but not in breast cancer cells. However, the suppression of beta-catenin, a key mediator of Wnt signaling pathway, was seen in all three cell lines with sulindac administration. Further studies revealed that transcriptional activities of beta-catenin were significantly inhibited by sulindac and that the inhibition was sulindac dosage-dependent. The transcriptional targets of beta-catenin, c-myc, cyclin D1 and cdk 4 were also dramatically downregulated. In conclusion, our data demonstrated that the efficacy of sulindac in the inhibition of cell proliferation (rather than the induction of apoptosis) might be through the suppression of beta-catenin pathway in human cancer cells.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; beta Catenin; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p21; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; Luciferases; Lung Neoplasms; Proto-Oncogene Proteins c-myc; Sulindac; Transcription, Genetic; Transfection

Recent research indicates that inflammatory factors play important roles in the initiation and progression of cancers, including breast cancer. Daintain/allograft inflammatory factor-1 (AIF-1) is a crucial mediator in the inflammatory response, but it has not yet been reported whether daintain/AIF-1 is involved in the development of breast cancers. In this study, immunohistochemical analysis found strong positive expression of daintain/AIF-1 in breast ductal tumor epithelia, but only weakly positive or negative expression in the adjacent histologically normal ductal epithelia. Then, the effect of daintian/AIF-1 on the proliferation of the breast cancer cell line MDA-MB-231 was explored via transduction of the daintian/AIF-1 gene into the cells, and via inhibition of the expression of daintain/AIF-1 through short interference RNA. The results demonstrated that up-regulation and down-regulation of daintain/AIF-1 expressions promoted and inhibited the proliferation of MDA-MB-231, respectively. More interestingly, daintain/AIF-1 overexpression facilitated tumor growth in female nude mice. Furthermore, we found that daintain/AIF-1 overexpression up-regulated the expression of cyclin D1 and enhanced the transcriptional activity of nuclear factor-kappa B (NF-kappaB), a regulator of cyclin D1 expression. In contrast, the down-regulation of daintain/AIF-1 expression decreased cyclin D1 expression and inhibited the transcriptional activity of NF-kappaB. These results strongly suggest that daintain/AIF-1 can promote the growth of breast tumors via activating NF-kappaB signaling, which consequently up-regulates the expression of cyclin D1, implying that daintain/AIF-1 may be a novel target molecule for the prognosis and therapy of breast cancer.

Topics: Animals; Breast Neoplasms; Calcium-Binding Proteins; Cell Line, Tumor; Cell Proliferation; Cyclin D1; DNA-Binding Proteins; Humans; Mice; Mice, Nude; Microfilament Proteins; NF-kappa B; Signal Transduction; Transcription, Genetic; Transfection; Up-Regulation

We analyzed the expression of critical cell cycle regulators cyclin E and cyclin D1 in familial breast cancer, focusing on BRCA mutation-negative tumors. Cyclin E expression in tumors of BRCA1 or BRCA2 carriers is higher, and cyclin D1 expression lower, than in sporadic tumors. In familial non-BRCA1/2 tumors, cyclin E and cyclin D1 expression has not been studied.. Cyclin E and cyclin D1 immunohistochemical expression was studied in tissue microarrays consisting of 53 BRCA1, 58 BRCA2, 798 familial non-BRCA1/2, and 439 sporadic breast tumors.. In univariate analysis, BRCA1 tumors had significantly more frequently high cyclin E (88%) and low cyclin D1 (84%) expression than sporadic (54% and 49%, respectively) or familial non-BRCA1/2 (38% and 45%, respectively) tumors. BRCA2 tumors had significantly more frequently low cyclin D1 expression (68%) than sporadic or familial non-BRCA1/2 tumors and significantly more frequently high cyclin E expression than familial non-BRCA1/2 tumors. In a logistic regression model, cyclin expression, early age of onset, and estrogen receptor (ER) and human epidermal growth factor receptor-2 (HER2) status were the independent factors most clearly distinguishing tumors of BRCA1 mutation carriers from other familial breast cancers. High cyclin E and low cyclin D1 expression were also independent predictors of BRCA2 mutation when compared with familial non-BRCA1/2 tumors. Most interestingly, lower frequency of high cyclin E expression independently distinguished familial non-BRCA1/2 tumors also from sporadic ones.. Cyclin E and cyclin D1 expression distinguishes non-BRCA1/2 tumors from both sporadic and BRCA1- and BRCA2-associated tumors and may reflect different predisposition and pathogenesis in these groups.

Topics: Age of Onset; Biomarkers, Tumor; Breast Neoplasms; Cyclin D1; Cyclin E; Female; Genes, BRCA1; Genes, BRCA2; Genetic Predisposition to Disease; Humans; Immunohistochemistry; Middle Aged; Mutation

Insulin-like growth factor (IGF)-II is a required intermediate for prolactin-induced up-regulation of cyclin D1 and proliferation in normal murine mammary epithelial cells in vivo and in vitro. However, we have recently shown that prolactin can rapidly induce cyclin D1 protein expression and subsequent proliferation in the MCF-7 human breast cancer cell line, suggesting that prolactin actions can be independent of IGFs in breast disease. Here, we investigate the relationship between these factors and show that prolactin up-regulated transcript levels of both IGF-I and IGF-II, but only after increases in cyclin D1 protein were observed. Moreover, prolactin increased cyclin D1 in the presence of the IGF-I receptor neutralizing antibody alphaIR3. However, on cotreatment, IGF-I and prolactin elicited cooperative phosphorylation of extracellular signal-regulated kinases 1 and 2 and protein kinase B/AKT, but not signal transducer and activator of transcription 5. This interaction extended to increased activation of activating protein-1 enhancer elements, phosphorylation of glycogen synthase kinase 3beta, induction of cyclin D1, and ultimately, increased cell number. It also increased invasive behavior, which correlated with elevated matrix metalloproteinase-2 transcript levels. Interestingly, prolactin augmented phosphorylation at Tyr(1135) and Tyr(1136) of IGF-I receptor on cotreatment with IGF-I, although prolactin alone had no effect. Together, these data indicate that strong cooperative cross talk between prolactin and IGF-I augments biological processes associated with neoplastic progression, with implications for therapeutic strategies.

Topics: Breast Neoplasms; Cell Line, Tumor; Cyclin D1; Disease Progression; Enhancer Elements, Genetic; Female; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Insulin-Like Growth Factor I; Matrix Metalloproteinase 2; Phosphorylation; Prolactin; Receptor, IGF Type 1; Signal Transduction; Transcription Factor AP-1

Overexpression of the helix-loop-helix (HLH) protein Id1 has been associated with metastasis in breast cancer, but its role in models of early breast tumorigenesis is not well characterized. We show that the down-regulation of endogenous Id1 via proteosomal degradation and relocalization from the nucleus to the cytoplasm is an early event in the formation of mammary epithelial acini. Overexpression of Id1 in both human MCF-10A and primary mouse mammary epithelial cells disrupted normal acinar development by increasing acinar volume. This occurred in an HLH domain-dependent fashion via an increase in S phase. Id1 overexpression also increased apoptosis leading to accelerated luminal clearance, and this was reversed by coexpression of the proto-oncogene Bcl2, leading to large, disorganized structures with filled lumina. Id1 overexpression was unable to increase the volume of cyclin D1(-/-) acini, indicating that Id1 is dependent on cyclin D1 for its proliferative effects. In summary, Id1 may contribute to early breast cancer by promoting excessive proliferation through cyclin D1.

Topics: Animals; Breast Neoplasms; Cell Division; Cyclin D1; Epithelial Cells; Helix-Loop-Helix Motifs; Humans; Infant; Inhibitor of Differentiation Protein 1; Mammary Neoplasms, Experimental; Mice; Proto-Oncogene Mas

Pure invasive micropapillary carcinoma (MPC) is a special histological type that accounts for 0.7-3% of all breast cancers. MPC has a distinctive growth pattern and a more aggressive clinical behaviour than invasive ductal carcinomas of no special type (IDC-NSTs). To define the molecular characteristics of MPCs, we profiled a series of 12 MPCs and 24 grade and oestrogen receptor (ER)-matched IDC-NSTs using high-resolution microarray comparative genomic hybridization (aCGH). In addition, we generated a tissue microarray containing a series of 24 MPCs and performed immunohistochemical analysis with ER, PR, Ki-67, HER2, CK5/6, CK14, CK17, EGFR, topoisomerase-IIalpha, cyclin D1, caveolin-1, E-cadherin, and beta-catenin antibodies. In situ hybridization probes were employed to evaluate the prevalence of amplification of HER2, TOP2A, EGFR, CCND1, MYC, ESR1, and FGFR1 genes. aCGH analysis demonstrated that MPCs significantly differed from IDC-NSTs at the genomic level. Gains of 1q, 2q, 4p, 6p, 6q23.2-q27, 7p, 7q, 8p, 8q, 9p, 10p, 11q, 12p, 12q, 16p, 17p, 17q, 19p, 20p, 20q, and 21q, and losses of 1p, 2p, 6q11.1-q16.3, 6q21-q22.1, 9p, 11p, 15q, and 19q were more prevalent in MPCs. High-level gains/amplifications of 8p12-p11, 8q12, 8q13, 8q21, 8q23, 8q24, 17q21, 17q23, and 20q13 were significantly associated with MPCs. A comparison between 24 MPCs and a series of 48 grade and ER-matched IDC-NSTs revealed that high cyclin D1 expression, high proliferation rates, and MYC (8q24) amplification were significantly associated with MPCs. Our results demonstrate that MPCs have distinct histological features and molecular genetic profiles supporting the contention that they constitute a distinct pathological entity.

Topics: Breast Neoplasms; Carcinoma, Ductal, Breast; Cyclin D1; Disease Progression; Female; Gene Amplification; Gene Expression Profiling; Genetic Markers; Humans; Immunohistochemistry; Immunophenotyping; In Situ Hybridization, Fluorescence; Oligonucleotide Array Sequence Analysis; Oncogenes

Bisphosphonates may induce direct anti-tumor effects in breast cancer cells in vitro. In this study, six bisphosphonates were administered to three breast cancer cell lines. Cell proliferation was measured by quantification of the expression of Cyclin D1 mRNA. Apoptosis was determined by flow cytometry of a DNA fragmentation assay. We demonstrated that bisphosphonates have direct effects on cell proliferation and apoptosis in different breast cancer cell lines. However, not all bisphosphonates act equally on breast cancer cells in vitro. Zoledronate seems to be the most potent of the six bisphosphonates. This in vitro study showed that bisphosphonates possess promising anti-tumor potential.

Topics: Alendronate; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Clodronic Acid; Cyclin D1; Diphosphonates; DNA Fragmentation; Etidronic Acid; Female; Flow Cytometry; Humans; Ibandronic Acid; Imidazoles; Pamidronate; Risedronic Acid; RNA, Messenger; Zoledronic Acid

Biomarkers, commonly expressed in breast cancer cells, may be correlated with their expression in breast skin of the same subjects.. The expression of biomarkers in specimens from 33 breast tumours and breast skin from the same subject and from 32 normal controls was studied using immunohistochemical techniques.. (1) In normal women, there are significant correlations between the levels of expression of cyclin D1, bcl-2 and p53 in normal breast epithelial cells and breast skin epithelial cells. (2) These patterns of biomarker expression in normal women are similar in breast cancer and breast skin epithelial cells of women with invasive ductal carcinoma (IDC) and ductal carcinoma in situ (DCIS), but are at significantly higher levels in both breast cancer cells and skin from the same subjects. (3) In normal women, human epidermal growth factor receptor 2 (HER-2) is not expressed in either breast epithelial cells or skin epithelial cells. (4) HER-2 is expressed in the breast skin of some subjects with HER-2-positive breast cancer. (5) Positive oestrogen receptor alpha expression occurs significantly more frequently in the breast skin of women with IDC and DCIS than in normal controls.. The influence of localised breast cancer seems to be systemic, and leads to changes in skin and hair.

Topics: Biomarkers, Tumor; Breast; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Intraductal, Noninfiltrating; Cyclin D1; Estrogen Receptor alpha; Female; Humans; Neoplasm Proteins; Proto-Oncogene Proteins c-bcl-2; Receptor, ErbB-2; Skin; Tumor Suppressor Protein p53

Amplification of HER2, C-MYC and CCND1 oncogenes is a hallmark of breast cancer (BC); however, its involvement in the bilateral form of this disease has not been investigated yet. In this study, 50 bilateral BC (biBC) pairs (100 tumors) and 72 control unilateral BC were examined using real-time PCR analysis of microdissected archival tissues. In biBC, the frequency of >3-fold oncogene amplification was 6/100 (6%) for HER2, 6/100 (6%) for C-MYC and 7/100 (7%) for CCND1. Altogether, 18/100 (18%) biBC tumors had increased gene dosage of at least one oncogene. Tumors forming synchronous biBC pairs had amplification in 11/46 cases (24%). In 3 of 8 patients with amplification-positive carcinomas, the amplification was detected in both neoplasms: 2 biBC had concordant activation of the same oncogene (HER2 and CCND1, respectively), and in the remaining case distinct oncogenes were affected (HER2 and C-MYC). In contrast, amplifications in metachronous biBC were strongly discordant: none of 27 first carcinomas carried this abnormality, while the frequency of amplification in second tumors (7/27; 26%) was similar to the one observed in unilateral BC (20/72; 28%). The trend toward concordance of oncogene amplification status in synchronous but not in metachronous biBC pairs can be explained by the nearly identical natural history of the disease in simultaneously arising tumors. The skewed pattern of amplifications in metachronous biBC might be attributed to their association with adverse BC prognosis; it appears that only patients with amplification-negative first BC have sufficient chances to survive until the development of the contralateral carcinoma.

Topics: Adult; Aged; Aged, 80 and over; Breast Neoplasms; Carcinoma; Cyclin D1; DNA, Neoplasm; Female; Gene Amplification; Genes, erbB-2; Genes, myc; Humans; Middle Aged; Oncogenes; Polymerase Chain Reaction

Inhibition of protein kinase A (PKA) promotes estrogen-dependent growth of MCF7 breast cancer cells, although the mechanisms by which PKA regulates estrogen receptor (ER) function remain unclear. In this study elevation of cAMP by forskolin/3-isobutyl-1-methylxanthine (F/I) suppressed estradiol-dependent MCF7 and T47D breast cancer cell growth but not tamoxifen-resistant MCF7-LCC2 cells. Although F/I induced ligand independent activation of ERalpha, F/I also decreased estradiol-dependent reporter gene transcription. Overexpression of PKA or PKA inhibitor (PKI) demonstrated that F/I effects on repression of estradiol action occurred through the PKA pathway. 8CPT-2Me-cAMP, a selective inducer of non-PKA signaling, did not alter ER-dependent transcription. In contrast to F/I effects on reporter genes, F/I exhibited gene-specific effects on endogenous, ER-regulated genes. F/I enhanced estradiol induction of pS2 and cMyc but repressed estradiol induction of cyclin D1 mRNA and protein in MCF7 cells. To explore likely mechanisms by which F/I regulated ER, experiments examined estradiol binding, Hsp90 interaction, promoter recruitment, and ERalpha phosphorylation. F/I decreased estradiol binding and increased Hsp90 association with ERalpha. Chromatin immunoprecipitation revealed that F/I recruited ERalpha to both pS2 and cMyc promoters at earlier times than estradiol, and F/I shifted estradiol recruitment of ERalpha to earlier time points. F/I induced a unique ERalpha phosphorylation profile (increase in serine 305 and decrease in serine 118 phosphorylation) that was distinct from estradiol and estradiol + F/I. Taken together, F/I signaling through PKA selectively regulates estradiol-dependent genes in breast cancer, which is associated with reduced ligand binding and changes in promoter interaction and ERalpha phosphorylation.

Topics: 1-Methyl-3-isobutylxanthine; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclin D1; Estradiol; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; HSP90 Heat-Shock Proteins; Humans; Intracellular Signaling Peptides and Proteins; Ligands; Phosphorylation; Promoter Regions, Genetic; Signal Transduction; Tamoxifen; Transcription, Genetic

Cross-talk between insulin-like growth factor 1 (IGF-1) and estrogen receptor alpha (ER) regulates gene expression in breast cancer cells, but the underlying mechanisms remain unclear. Here, we studied how 17-beta-estradiol (E2) and IGF-1 affect ER transcriptional machinery in MCF-7 cells. E2 treatment stimulated ER loading on the estrogen response element (ERE) in the pS2 promoter and on the AP-1 motif in the cyclin D1 promoter. On ERE, similar amounts of liganded ER were found at 1-24-h time points, whereas on AP-1, ER binding fluctuated over time. At 1 h, liganded ER was recruited to ERE together with histone acetyltransferases SRC-1 and p300, ubiquitin ligase E6-AP, histone methyltransferase Carm1 (Carm), and polymerase (pol) II. This coincided with increased histone H3 acetylation and up-regulation of pS2 mRNA levels. At the same time, E2 moderately increased cyclin D1 expression, which was associated with the recruitment of liganded ER, SRC-1, p300, ubiquitin ligase E6-AP (E6L), Mdm2, and pol II, but not other regulatory proteins, to AP-1. In contrast, at 1 h, IGF-1 increased the recruitment of the ER.SRC-1.p300.E6L.Mdm2.Carm.pol II complex on AP-1, but not on ERE, and induced cyclin D1, but not pS2, mRNA expression. Notably, ER knockdown reduced the association of ER, E6L, Mdm2, Carm, and pol II with AP-1 and resulted in down-regulation of cyclin D1 expression. IGF-1 potentiated the effects of E2 on ERE but not to AP-1 and increased E2-dependent pS2, but not cyclin D1, mRNA expression. In conclusion, E2 and IGF-1 differentially regulate ER transcription at ERE and AP-1 sites.

Topics: Active Transport, Cell Nucleus; Breast Neoplasms; Cell Line, Tumor; Cyclin D1; Estradiol; Estrogen Receptor alpha; Humans; Insulin-Like Growth Factor I; Ligands; Response Elements; RNA, Messenger; Transcription Factor AP-1; Transcription, Genetic; Trefoil Factor-1; Tumor Suppressor Proteins; Up-Regulation

2-Methoxyestradiol (2ME) is an estradiol metabolite with anti-tumor and anti-angiogenic properties. We studied the effect of 2ME on apoptosis of MCF-7 breast cancer cells and explored a combination therapy using 2ME and a polyamine analogue, bis(ethyl)norspermine (BE-3-3-3). Determination of viable cells on day 4 of treatment with 2ME/BE-3-3-3 combinations showed synergistic effects by Chou-Talalay analysis. APO-BRDU analysis showed that there was only 1.5+/-0.5% apoptosis at 200 nM 2ME and 3.7+/-1.7% in the presence of 2.5 microM BE-3-3-3. Combination of 200 nM 2ME and 2.5 microM BE-3-3-3 resulted in 52.2+/-2.6% apoptosis. Up to 90% of the cells underwent apoptosis in the presence of 1000 nM 2ME and 2.5 microM BE-3-3-3. Combination treatments resulted in total disruption of microtubules and depletion of putrescine, spermidine and spermine. In addition, phosphorylation of Akt and nuclear localization of cyclin D1 were altered by 2ME/BE-3-3-3 combination. Our results suggest an important strategy to induce apoptosis of breast cancer cells, with potential applications in therapy.

Topics: 2-Methoxyestradiol; Apoptosis; Blotting, Western; Breast Neoplasms; Bromodeoxyuridine; Cell Cycle; Cell Line, Tumor; Cell Nucleus; Cyclin D1; Drug Synergism; Estradiol; Humans; Microscopy, Confocal; Phosphorylation; Proto-Oncogene Proteins c-akt; Spermine

The development of novel therapeutic strategies for breast cancer requires the identification of molecular targets involved in malignancy. Human Pygopus (Pygo)-1 and -2 are recently discovered components of the Wnt signaling pathway required for beta-Catenin/Tcf dependent transcription in embryos and colorectal cancer cells, but the role of these proteins in malignant cell growth and survival has not yet been determined. We report the expression and requirement for proliferation of hPygo2 in breast cancer cells. hPygo2 protein was overexpressed in malignant breast tumors and in the nuclei of five breast cancer cell lines, but was not expressed in the nuclei of non-malignant breast cells. Phosphorothioated antisense oligonucleotides were used to specifically knockdown expression hPygo2 in Mcf-7 and MDA-MB-468 cell lines. hPygo2 was required for the growth, in tissue culture and anchorage-independent assays, of both cell lines and for the expression of the Wnt target gene Cyclin D1. We conclude that hPygo2 is highly expressed in, and required for the growth of breast carcinoma cells.

Topics: Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Cyclin D1; Cytoskeletal Proteins; Gene Expression Regulation, Neoplastic; Humans; Intracellular Signaling Peptides and Proteins; Oligonucleotides, Antisense; RNA; RNA, Messenger; Signal Transduction; Transfection

We aimed at developing a more detailed understanding of cyclin D1 in early stage human breast cancer and defining the biologic profiles with different prognostic value correlating cyclin D1 gene amplification and chromosome 11 aneusomy with bio-pathologic variables of known clinical importance. Cyclin D1 gene amplification and chromosome 11 aneusomy were investigated using fluorescence in situ hybridization whereas cyclin D1, PgR, HER-2, Bcl2, p53, and Ki-67 expressions were analyzed by immunohistochemistry in 121 stage I or II breast cancer patients uniformly treated with cyclophosphamide/metotrexate/5-fluorouracil-based chemotherapy. Cyclin D1 was amplified in 6.6% and overexpressed in 32.2% of cases. Amplification was not associated with any selected bio-pathologic variables, whereas the chromosome 11 aneusomy level significantly increased in tumors with higher histologic grade (P < 0.01), higher tumor size (P < 0.003), p53 nuclear accumulation (P < 0.04), and ERalpha negativity (P < 0.049). Multiple correspondence analysis showed 4 different biologic tumor profiles. The first, characterized by high Ki-67 score, p53+, cyclin D1+, HER-2+, aneusomy level > 30%, ratio (cyclin D1 gene/CEP11) > 2, was associated with tumor relapse defining the most unfavorable biologic profile. Kaplan-Meier's method showed significantly shorter disease-free survival in patients with at least 3 variables positive out of the 6 detected by multiple correspondence analysis. In multivariate analysis, the identified biologic profile emerged as the only significant prognostic indicator. Our findings are of particular clinical interest for early stage breast cancer patients, because the assessment of biologic factors predictive of tumor aggressiveness may influence postoperative therapeutic strategies.

Topics: Adenocarcinoma; Adult; Aged; Aneuploidy; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Breast Neoplasms; Chemotherapy, Adjuvant; Chromosomes, Human, Pair 11; Cyclin D1; Cyclophosphamide; Female; Fluorouracil; Humans; Immunohistochemistry; In Situ Hybridization, Fluorescence; Mastectomy, Segmental; Methotrexate; Middle Aged; Neoplasm Staging; Survival Analysis; Survival Rate

The aim of the present study was to evaluate the association between different types of hormone replacement therapy (HRT) and risk of specific breast cancer subgroups. A population-based prospective cohort study including 12,583 peri- or postmenopausal women were followed using record-linkage with national cancer registries. During an average follow-up of 4.5 years, 332 cases of invasive breast cancer were diagnosed. Tumour samples were available from 283 cases. These tumours were re-evaluated according to histological type, grade, and mitotic index. Evaluation of tumours included estrogen and progesterone receptor status (ERalpha, ERbeta and PgR), as well as expression of Ki67, HER2, cyclin D1 and p27. The incidence of breast cancer in current users of combined HRT (CHRT) was significantly higher than in non-users. The difference corresponded to an adjusted relative risk (95% confidence interval) of 3.01 (2.35-3.84) as obtained using a Cox's proportional hazards analysis. CHRT was associated with lobular tumours (3.48:1.99-6.10), grade 1 tumours (4.46:2.79-7.13) and tumours with a low mitotic index (4.35:2.99-6.34). CHRT was not related to any specific subgroup in terms of ERalpha-, ERbeta- or PgR-expression. CHRT was associated with low proliferating tumours, defined by the Ki67 index (3.58:2.60-4.93), HER2 amplified tumours (4.40:1.93-10.06), low expression of the oncogene cyclin D1 (3.14:2.32-4.23) and high expression of the tumour suppressor gene p27 (3.47:2.40-5.01). Use of estrogen-alone HRT (ERT) was not associated with any statistically significant risk of breast cancer. We conclude that the use of CHRT is associated with an increased incidence of breast tumours with comparatively favourable prognostic factors.

Topics: Age Factors; Breast Neoplasms; Cohort Studies; Cyclin D1; Estrogen Receptor alpha; Estrogen Receptor beta; Estrogen Replacement Therapy; Female; Genes, erbB-2; Hormone Replacement Therapy; Humans; Mitotic Index; Prognosis; Proliferating Cell Nuclear Antigen; Prospective Studies; Receptor, ErbB-2; Receptors, Progesterone

Cyclin D1 plays an important role in cell cycle progression. In breast cancer, Cyclin D1 expression is deregulated by several mechanisms. We previously showed that in breast cancer cells, overexpression of BRCA1-IRIS induces Cyclin D1 overexpression and increases cell proliferation. BRCA1-IRIS alone or in complex with steroid receptor co-activators was targeted to the cyclin D1 promoter pre-bound by the c-Jun/AP1 and activated its transcription, which could explain the co-overexpression of BRCA1-IRIS and Cyclin D1 in breast cancer cells coupled with their increased proliferation. We report here an alternate or a complementary pathway by which BRCA1-IRIS activates Cyclin D1 expression. BRCA1-IRIS overexpression decreases the expression of the dual specificity phosphatase, DUSP3/VHR, an endogenous inhibitor of several MAPKs, including c-Jun N-terminal kinase. Although, the mechanism by which BRCA1-IRIS overexpression accomplishes that is not yet known, it is sufficient to induce Cyclin D1 overexpression in a human mammary epithelial cell model. Cyclin D1 overexpression could be blocked by co-overexpression of VHR in those cells. Furthermore, in 2 breast cancer cell lines that overexpress both BRCA1-IRIS and Cyclin D1 (MCF-7 and SKBR3) depletion of BRCA1-IRIS by RNA interference attenuated the expression of Cyclin D1 by elevating the expression level of VHR. These data demonstrate a critical role for BRCA1-IRIS in human breast cancer cell-cycle control and suggest that deregulated expression of BRCA1-IRIS is likely to reduce dependence on normal physiological growth stimuli, thereby providing a growth advantage to tumor cells and a potential mechanism of resistance to endocrine therapy.

Topics: BRCA1 Protein; Breast Neoplasms; Cell Cycle; Cell Line; Cyclin D1; Down-Regulation; Dual Specificity Phosphatase 3; Epithelial Cells; ErbB Receptors; Estrogen Receptor alpha; Gene Expression Regulation, Neoplastic; Humans; JNK Mitogen-Activated Protein Kinases; Phosphoprotein Phosphatases; Protein Tyrosine Phosphatases; Receptor, ErbB-2; RNA, Small Interfering

The objective of this study was to assess the anti-tumor efficacy of rapamycin alone or in combination with herceptin in breast cancer. A total of 20 human breast cancer lines were examined for expression of various receptor tyrosine kinases and activation of their down stream signaling molecules, as well as for their invasion and colony forming ability. The ErbB2 and PI3 kinase pathway inhibitors were tested for the inhibition on breast cancer cell growth and tumor development. Seven of the 20 lines displayed an elevated level of ErbB2, others had varying level of EGF, IGF-1 or insulin receptor. Over 30% of the lines also had constitutive activation of Akt and MAP kinase. The lines displayed a wide range of colony forming and invasion ability. The PI3 kinase pathway inhibitors LY294002 and rapamycin inhibited the colony forming ability of all of the lines with the ErbB2 overexpressing lines having a higher sensitivity. A similar trend was observed for inhibition of invasion by LY294002. Rapamycin alone and additively together with herceptin inhibited the breast cancer cell growth especially in ErbB2 overexpressing cells. Rapamycin and herceptin synergistically inhibited tumor growth and endpoint tumor load in a xenograft model using a MCF-7 subline and in a MMTV-ErbB2 transgenic model. Rapamycin and herceptin significantly reduced the level of cyclin D1 and D3 and increased the cleavage of caspase 3 suggesting an increased apoptosis. Our results suggest that rapamycin together with herceptin has an enhanced anti-cancer effect and could be developed as an improved therapeutic regimen for breast cancer.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin D3; Cyclins; Female; Humans; Intercellular Signaling Peptides and Proteins; Mice; Mice, Inbred BALB C; Mitogen-Activated Protein Kinases; Neoplasm Invasiveness; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Receptor Protein-Tyrosine Kinases; Receptor, ErbB-2; Signal Transduction; Sirolimus; Xenograft Model Antitumor Assays

Using whole sections of formalin-fixed paraffin-embedded material the expression of p27(KIP-1), cyclin A and cyclin D1 was examined in 60 cases of ductal carcinoma in situ (DCIS) using routine immunohistochemistry with a median follow up of 95 months (range 10-139 months) to identify any association with disease recurrence. Fifty-six patients were treated by local excision and radiotherapy and four by mastectomy without radiotherapy. There was a highly significant positive association between p27(KIP-1) and estrogen receptor/progesterone receptor (ER/PR) status (P = 0.002, P = 0.02) and with p27(KIP-1) and cyclin D1 expression (P = 0.002). A trend between cyclin A and PR status (P = 0.08) was also identified. These findings mirror those described in invasive ductal carcinoma, but there were no associations of any biomarker with histological parameters such as nuclear grade or with local recurrence on univariate analysis, which was present in four of the 56 locally excised group (7.1%). Further examination of a larger cohort may be worthwhile to explore the possible role as adjunctive predictive markers to aid clinical decision making.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Cyclin A; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Female; Gene Expression Regulation, Neoplastic; Humans; Middle Aged; Neoplasm Recurrence, Local; Receptors, Estrogen; Receptors, Progesterone

The inheritance of one defective BRCA1 or BRCA2 allele predisposes an individual to developing breast and ovarian cancers. BRCA1 is a multifunctional tumor suppressor protein, which through interaction with a vast array of proteins has implications in processes such as cell cycle, transcription, DNA damage response and chromatin remodeling. Conversely, the oncogene, cyclin D1 is overexpressed in about 35% of all breast cancer cases. In this study, we provide detailed analyses on the phosphorylation state of BRCA1 by cyclin D1/cdk4 complexes. In particular, we have identified Ser 632 of BRCA1 as a cyclin D1/cdk4 phosphorylation site in vitro. Using chromatin immunoprecipitation assays, we observed that the inhibition of cyclin D1/cdk4 activity resulted in increased BRCA1 DNA binding at particular promoters in vivo. In addition, we identified multiple novel genes that are bound by BRCA1 in vivo. Collectively, these results indicate that cyclin D1/cdk4-mediated phosphorylation of BRCA1 inhibits the ability of BRCA1 to be recruited to particular promoters in vivo. Therefore, cyclin D1/Cdk4 phosphorylation of BRCA1 could provide a mechanism to interfere with the DNA-dependent activities of BRCA1.

Topics: Amino Acid Sequence; BRCA1 Protein; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Chromatin Immunoprecipitation; Cyclin D1; Cyclin-Dependent Kinase 4; DNA; G1 Phase; Gene Expression Regulation, Neoplastic; Humans; Molecular Sequence Data; Phosphorylation; Promoter Regions, Genetic; Resting Phase, Cell Cycle

Cyclin-dependent kinases (Cdk) and their associated pathways represent some of the most attractive targets for the development of anticancer therapeutics. Based on antitumor activity in animal models, a variety of Cdk inhibitors are undergoing clinical evaluation either as a single agent or in combination with other approved drugs. In our anticancer drug discovery program, a novel series of flavones have been synthesized for evaluation against the activity of Cdk4-D1. This enzyme catalyzes the phosphorylation of retinoblastoma protein, thus inhibiting its function. We have identified a series of potent Cdk4-D1 inhibitors with IC(50) below 250 nmol/L. In this report, we have described the properties of one of the best compound, P276-00 of the flavone's series. P276-00 shows 40-fold selectivity toward Cdk4-D1, compared with Cdk2-E. The specificity toward 14 other related and unrelated kinases was also determined. P276-00 was found to be more selective with IC(50)s <100 nmol/L for Cdk4-D1, Cdk1-B, and Cdk9-T1, as compared with other Cdks, and less selective for non-Cdk kinases. It showed potent antiproliferative effects against various human cancer cell lines, with an IC(50) ranging from 300 to 800 nmol/L and was further compared for its antiproliferative activity against cancer and normal fibroblast cell lines. P276-00 was found to be highly selective for cancer cells as compared with normal fibroblast cells. To delineate its mechanism of action, the effect of P276-00 on cell cycle proteins was studied in human breast cancer cell line (MCF-7) and human non-small cell lung carcinoma (H-460). A significant down-regulation of cyclin D1 and Cdk4 and a decrease in Cdk4-specific pRb Ser(780) phosphorylation was observed. P276-00 produced potent inhibition of Cdk4-D1 activity that was found to be competitive with ATP and not with retinoblastoma protein. The compound also induced apoptosis in human promyelocytic leukemia (HL-60) cells, as evidenced by the induction of caspase-3 and DNA ladder studies. These data suggest that P276-00 has the potential to be developed as an anti-Cdk chemotherapeutic agent.

Topics: Antineoplastic Agents; Breast Neoplasms; Carcinoma, Non-Small-Cell Lung; Caspase 3; Cell Cycle; Cell Proliferation; Cells, Cultured; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; Down-Regulation; Enzyme Inhibitors; Fibroblasts; Flavones; HL-60 Cells; Humans; In Vitro Techniques; Lung Neoplasms; Molecular Structure; Phosphorylation; Retinoblastoma Protein

Recent evidence indicates that growth hormone-releasing hormone (GHRH) functions as an autocrine/paracrine growth factor for various human cancers. A splice variant (SV) of the full-length receptor for GHRH (GHRHR) is widely expressed in various primary human cancers and established cancer cell lines and appears to mediate the proliferative effects of GHRH. To investigate in greater detail the role of SV1 in tumorigenesis, we have expressed the full-length GHRHR and its SV1 in MCF-7 human breast cancer cells that do not possess either GHRHR or SV1. In accordance with previous findings, the expression of both GHRHR and SV1 restored the sensitivity to GHRH-induced stimulation of cell proliferation, with SV1 being more potent than the GHRHR. Furthermore, MCF-7 cells transfected with SV1 proliferated more quickly than the controls, even in the absence of exogenously added GHRH, suggesting the existence of intrinsic, ligand-independent activity of SV1 after its transfection. In agreement with the stimulation of cell proliferation, the levels of proliferation markers cyclin D1, cyclin E, and proliferating cell nuclear antigen were elevated in MCF-7 cells treated with GHRH, cultured in both serum-free and serum-containing media. In addition, SV1 caused a considerable stimulation of the ability of MCF-7 cells to grow in semisolid medium, an assay considered diagnostic for cell transformation. Collectively, our findings show that the expression of SV1 confers oncogenic activity and provide further evidence that GHRH operates as a growth factor in breast cancer and probably other cancers as well.

Topics: Agar; Alternative Splicing; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Neoplasms; Receptors, Neuropeptide; Receptors, Pituitary Hormone-Regulating Hormone; Transfection

Antitumour effects of retinoids are attributed to their influence on cell proliferation, differentiation, apoptosis and angiogenesis. In our effort to develop useful agents for breast cancer therapy, we evaluated the effects of four representative retinoic acid metabolism blocking agents (RAMBAs, VN/14-1, VN/50-1, VN/66-1 and VN/69-1) on growth inhibition of oestrogen receptor positive (ER +ve, MCF-7 and T-47D) and oestrogen receptor negative (ER -ve, MDA-MB-231) human breast cancer cells. Additionally, we investigated the biological effects/molecular mechanism(s) underlying their growth inhibitory properties as well as their antitumour efficacies against MCF-7 and MCF-7Ca tumour xenografts in nude mice. We also assessed the effect of combining VN/14-1 and all-trans-retinoic acid (ATRA) on MCF-7 tumour xenografts. The ER +ve cell lines were more sensitive (IC(50) values between 3.0 and 609 nM) to the RAMBAs than the ER -ve MDA-MB-231 cell line (IC(50)=5.6-24.0 microM). Retinoic acid metabolism blocking agents induced cell differentiation as determined by increased expression of cytokeratin 8/18 and oestrogen receptor-alpha (ER-alpha). Similar to ATRA, they also induced apoptosis via activation of caspase 9. Cell cycle analysis indicated that RAMBAs arrested cells in the G1 and G2/M phases and caused significant downregulation (>80%) of cyclin D1 protein. In vivo, the growth of MCF-7 mammary tumours was dose-dependently and significantly inhibited (92.6%, P<0.0005) by VN/14-1. The combination of VN/14-1 and ATRA also inhibited MCF-7 breast tumour growth in vivo (up to 120%) as compared with single agents (P<0.025). VN/14-1 was also very effective in preventing the formation of MCF-7Ca tumours and it significantly inhibited the growth of established MCF-7Ca tumours, being as effective as the clinically used aromatase inhibitors, anastrozole and letrozole. Decrease in cyclin D1 and upregulation of cytokeratins, Bad and Bax with VN/14-1 may be responsible for the efficacy of this compound in inhibiting breast cancer cell growth in vitro and in vivo. Our results suggest that our RAMBAs, especially VN/14-1 may be useful novel therapy for breast cancer.

Topics: Animals; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Caspase 9; Cell Cycle; Cell Proliferation; Cyclin D1; Female; Fenretinide; Humans; Imidazoles; Mice; Neoplasm Transplantation; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Transplantation, Heterologous; Tretinoin

In previous studies we identified a novel gene Dipl, also designated CCNDBP1, which encodes a 42kDa helix-loop-helix (HLH) nuclear protein. Although this protein was originally identified by its ability to bind to cyclin D1 its precise biochemical functions are not known. In the present study we carried out mechanistic studies on Dip1 focusing on the human breast cancer cell line MCF-7. We found that overexpression of Dip1 in MCF-7 cells inhibited colony formation and cell proliferation. Reporter assays in MCF-7 cells indicated that Dip1 strongly inhibited the transcriptional activities of the cyclin D1, c-fos, NF-kappaB, SRE and p21cP1 promoters. Furthermore studies with truncated and mutant forms of the cyclin D1 promoter suggest that Dip1 does not act on specific transcriptional elements. Assays with mutant and truncated forms of Dip1 indicated that both the LXXLL motif and the HLH domain play important, but not exclusive, roles in these inhibitory effects. Dip1 co-immunoprecipitated with the histone deacetylase (HDAC) proteins HDAC1 and HDAC3. Nevertheless Dip1 markedly inhibited the stimulation of cyclin D1 promoter activity obtained with trichostatin A [1], an inhibitor of HDAC. Taken together these findings suggest that Dip1 functions as a general repressor of transcription. Although the precise mechanism by which Dip1 inhibits gene transcription and the growth of MCF-7 cells remain to be determined, the present results suggest that Dip1 is a candidate tumor suppressor gene.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Helix-Loop-Helix Motifs; Histone Deacetylases; Humans; Promoter Regions, Genetic; Repressor Proteins; Transcription Factors

To screen the target genes regulated by the transcriptional repressor Syk (L) so as to explore the mechanism of Syk (L) in suppressing breast cancer.. Adeno-X expression system from Clontech Company was used to construct the adenovirus-Syk. Syk negative breast cancer cells of the line MB231 were cultured and infected with adeno-Syk (L), adeno-Syk (S), and the control adeno-Lac Z respectively. cDNA microarray was used to screen the genes regulated by Syk (L) and Syk (S). Northern blotting was used to examine the cDNA microarray results.. The transcriptional repressor Syk (L) was able to down-regulate the expression of the oncogenes ID1, cyclin D1, Fra1, and B-myb in the breast cancer cells. Northern blotting confirmed this regulation.. Syk (L) suppresses the progression of breast cancer by down regulating the expression of the oncogenes ID1, cyclin D1, Fra1, and B-myb.

Topics: Adenoviridae; Blotting, Northern; Breast Neoplasms; Cell Cycle Proteins; Cell Line, Tumor; Cyclin D1; Down-Regulation; Female; Gene Expression Profiling; Genetic Vectors; Humans; Inhibitor of Differentiation Protein 1; Intracellular Signaling Peptides and Proteins; Oligonucleotide Array Sequence Analysis; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-fos; Syk Kinase; Trans-Activators; Transfection

Vascular tumors comprise a minor subgroup of tumors arising in the breast and represent variants of hemangiomas and angiosarcomas. Diagnostic challenges may arise when differentiating hemangiomas from types I and II angiosarcomas. Ki-67 expression has been used as an adjunct to distinguish between benign and malignant lesions exhibiting histologic overlap at various anatomic sites.. To investigate the utility of Ki-67 and other cell cycle regulatory proteins (S-phase kinase-associated protein 2 [Skp2], p27, and cyclin D1) in the differential diagnosis of mammary vascular lesions.. Thirty-four vascular tumors (21 hemangiomas and 13 angiosarcomas) of the breast were studied. The Ki-67 index and immunoreactivity for Skp2, p27, and cyclin D1 were determined in each case. Appropriate statistical methods were used.. The mean value of Ki-67 index was statistically different when comparing hemangiomas and angiosarcomas (P < .001). Angiosarcomas were typically positive for Skp2, whereas hemangiomas were negative (P < .001). Sensitivity and specificity cutoffs for Ki-67 index to distinguish hemangiomas from angiosarcomas showed a candidate cutoff point of 175. The mean values of Ki-67 of low-grade angiosarcomas were significantly different from all hemangiomas (P < .001) and also different from the subset of atypical hemangiomas (P = .02). Sensitivity and specificity cutoffs for Ki-67 index to distinguish all hemangiomas from low-grade angiosarcomas showed a candidate cutoff point between 150 and 175. Among angiosarcomas, positivity for Ki-67 was inversely related to that of p27 but not to Skp2 or cyclin D1. This was also true among hemangiomas.. Ki-67 index can be used as a diagnostic tool to distinguish between benign and malignant vascular lesions of the breast. This can be particularly helpful in cases of histologic overlap such as low-grade angiosarcoma and hemangioma.

Topics: Biomarkers, Tumor; Breast Neoplasms; Breast Neoplasms, Male; Cell Cycle; Cyclin D1; Diagnosis, Differential; Female; Hemangioma; Hemangiosarcoma; Humans; Immunohistochemistry; Ki-67 Antigen; Male; Middle Aged; Proliferating Cell Nuclear Antigen; S-Phase Kinase-Associated Proteins; Sensitivity and Specificity

The incidence of ductal carcinoma in situ (DCIS) has risen dramatically with the introduction of screening mammography. The aim was to evaluate differences in pathological and biological characteristics between patients with screen-detected and interval DCIS.. From January 1992 to December 2001, 128 consecutive patients had been treated for pure DCIS at our institute. From these, 102 had been attending the Dutch breast cancer screening program. Sufficient paraffin-embedded tissue was available in 74 out of the 102 cases to evaluate biological marker expression (Her2/neu, ER, PR, p53 and cyclin D1) on tissue microarrays (TMA group). Differences in clinicopathological characteristics and marker expression between screen-detected and interval patients were evaluated. Screen-detected DCIS was classified as DCIS detected by screening mammography, when the two-year earlier examination failed to reveal an abnormality. Interval patients were classified as patients with DCIS detected within the two-year interval between two subsequent screening rounds.. Screen-detected DCIS was related with linear branching and coarse granular microcalcifications on mammography (p < .001) and with high-grade DCIS according to the Van Nuys classification (p = .025). In univariate analysis, screen-detected DCIS was related with Her2/neu overexpression (odds ratio [OR] = 6.5; 95%CI 1.3-31.0; p = .020), and interval DCIS was associated with low-grade (Van Nuys, OR = 7.3; 95% CI 1.6-33.3; p = .010) and PR positivity (OR = 0.3; 95%CI 0.1-1.0; p = .042). The multivariate analysis displayed an independent relation of Her2/neu overexpression with screen-detected DCIS (OR = 12.8; 95%CI 1.6-104.0; p = .018).. These findings suggest that screen-detected DCIS is biologically more aggressive than interval DCIS and should not be regarded as overdiagnosis.

Topics: Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Cyclin D1; Female; Genes, erbB-2; Genes, p53; Humans; Immunohistochemistry; Mammography; Mass Screening; Receptors, Estrogen; Receptors, Progesterone; Time Factors

Total tyrosine kinase activity is often elevated in both cytosolic and membrane fractions of malignant breast tissue and correlates with a decrease in disease-free survival. Breast tumor kinase (Brk; protein tyrosine kinase 6) is a soluble tyrosine kinase that was cloned from a metastatic breast tumor and found to be overexpressed in a majority of breast tumors. Herein, we show that Brk is overexpressed in 86% of invasive ductal breast tumors and coexpressed with ErbB family members in breast cancer cell lines. Additionally, the ErbB ligand, heregulin, activates Brk kinase activity. Knockdown of Brk by stable expression of short hairpin RNA (shRNA) in T47D breast cancer cells decreases proliferation and blocks epidermal growth factor (EGF)- and heregulin-induced activation of Rac GTPase, extracellular signal-regulated kinase (ERK) 5, and p38 mitogen-activated protein kinase (MAPK) but not Akt, ERK1/2, or c-Jun NH(2)-terminal kinase. Furthermore, EGF- and heregulin-induced cyclin D1 expression is dependent on p38 signaling and inhibited by Brk shRNA knockdown. The myocyte enhancer factor 2 transcription factor target of p38 MAPK and ERK5 signaling is also sensitive to altered Brk expression. Finally, heregulin-induced migration of T47D cells requires p38 MAPK activity and is blocked by Brk knockdown. These results place Brk in a novel signaling pathway downstream of ErbB receptors and upstream of Rac, p38 MAPK, and ERK5 and establish the ErbB-Brk-Rac-p38 MAPK pathway as a critical mediator of breast cancer cell migration.

Topics: Breast Neoplasms; Cell Growth Processes; Cell Movement; Cyclin D1; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Humans; MADS Domain Proteins; MEF2 Transcription Factors; Mitogen-Activated Protein Kinase 7; Myogenic Regulatory Factors; Neoplasm Proteins; Neuregulin-1; p38 Mitogen-Activated Protein Kinases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-akt; Receptor, ErbB-3; Receptor, ErbB-4; RNA, Small Interfering; Signal Transduction

Essential for embryonic development, the polycomb group protein enhancer of zeste homolog 2 (EZH2) is overexpressed in breast and prostate cancers and is implicated in the growth and aggression of the tumors. The tumorigenic mechanism underlying EZH2 overexpression is largely unknown. It is believed that EZH2 exerts its biological activity as a transcription repressor. However, we report here that EZH2 functions in gene transcriptional activation in breast cancer cells. We show that EZH2 transactivates genes that are commonly targeted by estrogen and Wnt signaling pathways. We demonstrated that EZH2 physically interacts directly with estrogen receptor alpha and beta-catenin, thus connecting the estrogen and Wnt signaling circuitries, functionally enhances gene transactivation by estrogen and Wnt pathways, and phenotypically promotes cell cycle progression. In addition, we identified the transactivation activity of EZH2 in its two N-terminal domains and demonstrated that these structures serve as platforms to connect transcription factors and the Mediator complex. Our experiments indicated that EZH2 is a dual function transcription regulator with a dynamic activity, and we provide a mechanism for EZH2 in tumorigenesis.

Topics: beta Catenin; Breast Neoplasms; Cell Cycle; Cyclin D1; DNA-Binding Proteins; Enhancer of Zeste Homolog 2 Protein; Estrogen Receptor alpha; Estrogens; Female; Gene Expression Regulation, Neoplastic; Genes, Neoplasm; Humans; Polycomb Repressive Complex 2; Promoter Regions, Genetic; Protein Binding; Protein Structure, Tertiary; Proto-Oncogene Proteins c-myc; Signal Transduction; Transcription Factors; Transcriptional Activation; Wnt Proteins

In order to better understand the mechanism of delivery of siRNAs by lipid-based vectors, we investigated the subcellular distribution of siRNAs directed against cyclin D1 delivered by the DLS system in the breast cancer cell line MCF-7. Cells were treated with cyclopentenone or 17beta-estradiol to modulate the level of expression of cyclin D1 mRNA. We qualitatively observed that siRNA localized to specific cytoplasmic compartments in the periphery of the nucleus in granular-like structures that do not correspond to early endosomal vesicles. In cells treated with either cyclopentenone or 17beta-estradiol cellular distribution of siRNAs was not affected but variations in the amount of siRNAs present in cells were found. We suggest these variations might be associated with the effects of cyclopentenone and 17beta-estradiol in cyclin D1 gene expression. Low cytotoxicity and highly cellular uptake of lipoplexes was observed in the presence of serum indicating that the DLS system could be a useful tool for siRNA vectorization in vitro and in vivo.

Topics: Breast Neoplasms; Cell Line, Tumor; Cyclin D1; Cyclopentanes; Estradiol; Humans; Immunohistochemistry; Intracellular Space; Liposomes; Membrane Proteins; Microscopy, Fluorescence; RNA, Small Interfering; Transfection; Vesicular Transport Proteins

BP1, a novel transcriptional factor, belongs to DLX family of homeobox genes. Recent researches showed that BP1 gene is correlated to genesis of breast cancer, but its correlation to cell cycle control factor has not been reported yet. This study was to observe the expression of BP1 in breast cancer, and to make clear its correlation to Cyclin D1.. The expression of BP1 and Cyclin D1 in 86 specimens of human breast cancer and 20 specimens of normal breast tissue (3 cm away from primary tumor) was detected by reverse transcription-polymerase chain reaction (RT-PCR). BP1 poly antibody was made and was certificated by Western blot. The expression of BP1 and Cyclin D1 in 86 specimens of human breast cancer were detected by immunohistochemistry; their correlation was analyzed.. The positive rate of BP1 mRNA was significanlty higher in breast cancer than in normal breast tissues (69.8% vs. 0, P < 0.001). The positive rate of Cyclin D1 mRNA was 64.0% in breast cancer. BP1 mRNA and Cyclin D1 mRNA were co-expressed in 52 specimens of breast cancer, and simultaneously negative in 23 specimens (P = 0.227); BP1 protein and Cyclin D1 protein were co-expressed in 43 specimens, and simultaneously negative in 31 specimens (P = 0.146).. BP1 gene is highly expressed in breast cancer. There is co-expression of Cyclin D1 and BP1 in breast cancer. BP1 gene may promote the genesis of breast cancer through regulating the expression of Cyclin D1.

Topics: Adult; Aged; Blotting, Western; Breast; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Medullary; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Genes, bcl-1; Homeodomain Proteins; Humans; Immunohistochemistry; Middle Aged; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transcription Factors

In transgenic mice overexpressing Stat5 or a constitutively activated Stat5 variant (STAT5ca), we show for the first time that parity is required for the development of tumors in postestropausal females. Tumors were detected in glands of multiparous transgenic female mice after latency period of 14 months, but rarely in their age-matched virgin (AMV) counterparts. This period was not affected by distinguishable tumor pathologies and was not dependent upon transgenic Stat5 variant. To associate Stat5 deregulation, parity and the postestropausal tumor occurrence with mammary cancer formation, the activities of endogenous and transgenic Stat5 were measured in the glands of aged multiparous and AMV females. No differences in phosphorylated Stat5 (pStat5) levels were found between the 2 cohorts. However, promoter sequences comprising the Stat5 binding sites from the cyclin D1 or the bcl-x genes associate differentially with acetylated histone H4 in aged multiparous and AMV STAT5ca transgenic females. Individual epithelial cells varied greatly with respect to the presence of nuclear pStat5. A small subset of epithelial cells, in which pStat5 and cyclin D1 were co-expressed, was exclusively present in the multiparous glands. Changes in chromatin structure might persist past the reproductive life time of the multiparous mice and contribute to the transcription of the cyclin D1 gene by activated Stat5. This may cause the detectable expression of cyclin D1 and add to the process of tumorigenesis.

Topics: Aging; Animals; Binding Sites; Breast Neoplasms; Cell Transformation, Neoplastic; Cyclin D1; Epithelial Cells; Female; Gene Expression Regulation, Neoplastic; Histones; Immunohistochemistry; Mammary Glands, Animal; Mice; Mice, Transgenic; Phenotype; Postmenopause; Protein Binding; STAT5 Transcription Factor

To clarify MUC1 patterns in invasive ductal breast carcinoma and to relate them to clinicopathological parameters, coexpression of other biological markers and prognosis.. Samples from 243 consecutive patients with primary ductal carcinoma were incorporated into tissue microarrays (TMAs). Slides were stained for MUC1, oestrogen receptor (ER), progesterone receptor (PR), Her2/neu, p53 and cyclin D1. Apical membrane MUC1 expression was associated with smaller tumours (P = 0.001), lower tumour grades (P < 0.001), PR positivity (P = 0.003) and increased overall survival (OS; P = 0.030). Diffuse cytoplasmic MUC1 expression was associated with cyclin D1 positivity (P = 0.009) and increased relapse-free survival (RFS; P = 0.034). Negativity for MUC1 was associated with ER negativity (P = 0.004), PR negativity (P = 0.001) and cyclin D1 negativity (P = 0.009). In stepwise multivariate analysis MUC1 negativity was an independent predictor of both RFS [hazard ratio (HR) 3.5, 95% confidence interval (CI) 1.5, 8.5; P = 0.005] and OS (HR 14.7, 95% CI 4.9, 44.1; P < 0.001).. The expression pattern of MUC1 in invasive ductal breast carcinoma is related to tumour characteristics and clinical outcome. In addition, negative MUC1 expression is an independent risk factor for poor RFS and OS, besides 'classical' prognostic indicators.

Topics: Adult; Aged; Aged, 80 and over; Breast Neoplasms; Carcinoma, Ductal, Breast; Cyclin D1; Female; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Middle Aged; Mucin-1; Multivariate Analysis; Prognosis; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone; Tissue Array Analysis; Treatment Outcome; Tumor Suppressor Protein p53

Cross-talk between receptor tyrosine kinases and estrogen receptor is at least partly responsible for the development of acquired resistance to endocrine therapies. Hence, targeting receptor tyrosine kinases and their downstream partners with inhibitors/antagonists may reverse this resistance. Although ras mutations are rare in breast cancer (2%), aberrant function of Ras signal transduction pathways is common. We therefore investigated the efficacy of the farnesyltransferase inhibitor (FTI) R115777 (tipifarnib) in combination with tamoxifen in MCF-7 human breast cancer models both in vitro and in vivo. There was a synergistic antiproliferative interaction between R115777 and 4-hydroxy-tamoxifen in vitro as calculated by median effect analysis. The combination resulted in a significantly greater G(1) arrest than either drug alone and this was associated with marked inhibition of cyclin D1 and induction of the cell cycle inhibitor p27(kip1). Combining R115777 with either tamoxifen or estrogen withdrawal in vivo produced a significantly greater inhibition of tumor growth and lower xenograft cell proliferation than either therapy alone. These results suggest that the combination of this FTI with endocrine therapy may be of therapeutic benefit in the treatment of breast cancer. Enhanced G1 arrest due to modulation of cell cycle regulatory proteins may be the underlying mechanism for the positive interaction between FTIs and tamoxifen.

Topics: Animals; Antineoplastic Agents; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Drug Therapy, Combination; Farnesyltranstransferase; Female; Flow Cytometry; Fluorescent Antibody Technique; G1 Phase; Humans; In Situ Nick-End Labeling; Mice; Mice, Nude; Quinolones; Receptors, Estrogen; Tamoxifen; Transcription, Genetic; Transplantation, Heterologous

For the female population in Asia, systematic investigation on alterations of cyclin D1 in breast carcinoma is rare, and correlation between cyclin D1 expression with clinicopathological parameters, survival rate, and other prognostic marker associated with cell cycle is unclear.. Expression of cyclin D1 protein, Ki-67, pRb, and p53 was determined by immunohistochemistry in 18 cases of early breast carcinomas and 80 cases of invasive ductal carcinomas. Genetic alteration of cyclin D1 gene and overexpression of cyclin D1 mRNA were detected by Southern blot and RT-PCR, respectively.. Expression of cyclin D1 is negative in usual ductal hyperplasia (UDH) and atypical ductal hyperplasia (ADH). However, in 52.0% (51/98) of all breast carcinomas, positive expression of cyclin D1 was observed. Five-year survival rate of the patients with positive expression of cyclin D1 (52.7%) is significantly lower than the cases with negative expression of cyclin D1 (72.1%). Positive rate of cyclin D1 protein in invasive ductal carcinoma (52.5%) is slightly higher than overexpression rate (40.8%) of cyclin D1 mRNA but significantly higher than amplification rate of cyclin D1 gene (18.4%). Expression of cyclin D1 is correlated with Ki-67 expression, but not correlated with pRb and p53 expression.. Positive expression of cyclin D1 could serve as a poor prognostic marker for Chinese patients with breast carcinoma independent of nodal metastasis and clinical stage. Expression of cyclin D1 protein is affected more directly by overexpression of cyclin D1 mRNA rather than cyclin D1 gene amplification. The cooperation between pRb and p53 with cyclin D1 protein in the carcinogenesis of breast carcinoma is not supported by the results.

Topics: Adult; Age Distribution; Blotting, Southern; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Intraductal, Noninfiltrating; China; Cyclin D1; Female; Follow-Up Studies; Gene Amplification; Humans; Immunohistochemistry; Ki-67 Antigen; Lymphatic Metastasis; Middle Aged; Neoplasm Staging; Retinoblastoma Protein; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Survival Rate; Time Factors; Tumor Suppressor Protein p53

The p53 protein has been a subject of intense research interest since its discovery as about 50% of human cancers carry p53 mutations. Mutations in the p53 gene are the most frequent genetic lesions in breast cancers suggesting a critical role of p53 in breast cancer development, growth and chemosensitivity. This report describes the derivation and characterization of MCF-7As53, an isogenic cell line derived from MCF-7 breast carcinoma cells in which p53 was abrogated by antisense p53 cDNA. Similar to MCF-7 and simultaneously selected hygromycin resistant MCF-7H cells, MCF-7As53 cells have consistent basal epithelial phenotype, morphology, and estrogen receptor expression levels at normal growth conditions. Present work documents investigation of molecular variations, growth kinetics, and cell cycle related studies in relation to absence of wild-type p53 protein and its transactivation potential as well. Even though wild-type tumor suppressor p53 is an activator of cell growth arrest and apoptosis-mediator genes such as p21, Bax, and GADD45 in MCF-7As53 cells, no alterations in expression levels of these genes were detected. The doubling time of these cells decreased due to depletion of G0/G1 cell phase because of constitutive activation of Akt and increase in cyclin D1 protein levels. This proliferative property was abrogated by wortmannin, an inhibitor of PI3-K/Akt signaling pathway. Therefore this p53 null cell line indicates that p53 is an indispensable component of cellular signaling system which is regulated by caveolin-1 expression, involving Akt activation and increase in cyclin D1, thereby promoting proliferation of breast cancer cells.

Topics: Apoptosis Regulatory Proteins; Breast Neoplasms; Caveolin 1; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; DNA, Antisense; Female; Humans; Proto-Oncogene Proteins c-akt; Transcriptional Activation; Tumor Suppressor Protein p53

C-erbB2 and estrogen receptors (ER) are well known for their cell proliferative capacity. Cyclin D1 is a major downstream target of both c-erbB2 and ER. This study was designed to analyze the expression of c-erbB2, cyclin D1 and ER and their prognostic implications in invasive ductal carcinoma of the breast.. The c-erbB2 status was evaluated by fluorescence in situ hybridization and immunohistochemistry (IHC) and cyclin D1 and ER were evaluated by IHC in 333 invasive breast cancer specimens.. The results of FISH and IHC for c-erbB2 showed 86.7% concordance. The overexpression of c-erbB2 was associated with the high expression of cyclin D1 and the negative expression of ER (P < 0.01 for both). The high expression of cyclin D1 was associated with the positive expression of ER (P < 0.01). When the group of patients who overexpressed c-erbB2 were analyzed, the patients with the low expression of cyclin D1 showed a significantly higher mortality than those with the high expression of cyclin D1 (RR = 3.2; 95% CI, 1.6-6.6). When the group of the high cyclin D1 expression was analyzed, the patients with negative expression of ER showed a significantly higher mortality than those with the positive expression of ER (RR = 2.1; 95% CI, 1.1-3.8).. Higher expression of cyclin D1 was associated with better prognosis in patients with c-erbB2 overexpression, and positive expression of ER was associated with better prognosis in patients with high cyclin D1 expression.

Topics: Adult; Aged; Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Ductal, Breast; Cyclin D1; Down-Regulation; Female; Follow-Up Studies; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; In Situ Hybridization, Fluorescence; Middle Aged; Multivariate Analysis; Predictive Value of Tests; Prognosis; Proportional Hazards Models; Receptor, ErbB-2; Receptors, Estrogen; Survival Analysis; Time Factors; Up-Regulation

Estrogen and estrogen receptor (ER)-mediated signaling are crucial for the etiology and progression of human breast cancer. Attenuating ER activities by natural products is a promising strategy to decrease breast cancer risk. We recently discovered that the pyranocoumarin compound decursin and its isomer decursinol angelate (DA) have potent novel antiandrogen receptor signaling activities. Because the ER and the androgen receptor belong to the steroid receptor superfamily, we examined whether these compounds affected ER expression and signaling in breast cancer cells.. We treated estrogen-dependent MCF-7 and estrogen-independent MDA MB-231 human breast cancer cells with decursin and DA, and examined cell growth, apoptosis, and ERalpha and ERbeta expression in both cell lines - and, in particular, estrogen-stimulated signaling in the MCF-7 cells. We compared these compounds with decursinol to determine their structure-activity relationship.. Decursin and DA exerted growth inhibitory effects on MCF-7 cells through G1 arrest and caspase-mediated apoptosis. These compounds decreased ERalpha in MCF-7 cells at both mRNA and protein levels, and suppressed estrogen-stimulated genes. Decursin and the pure antiestrogen Faslodex exerted an additive growth inhibitory effect on MCF-7 cells. In MDA MB-231 cells, these compounds induced cell-cycle arrests in the G1 and G2 phases as well as inducing apoptosis, accompanied by an increased expression of ERbeta. In contrast, decursinol, which lacks the side chain of decursin and DA, did not have these cellular and molecular activities at comparable concentrations.. The side chain of decursin and DA is crucial for their anti-ER signaling and breast cancer growth inhibitory activities. These data provide mechanistic rationales for validating the chemopreventive and therapeutic efficacy of decursin and its derivatives in preclinical animal models of breast cancer.

Topics: Antineoplastic Agents; Apoptosis; Benzopyrans; Breast Neoplasms; Butyrates; Caspases; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cell Nucleus; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cytosol; Estrogen Receptor alpha; Estrogen Receptor beta; Estrogens; Female; Gene Expression Regulation, Neoplastic; Humans; Immunoblotting; Intracellular Signaling Peptides and Proteins; Protein Kinase C; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Up-Regulation

Overexpression of cyclin D1 is associated with many cancers, and its overexpression is especially associated with a poor prognosis in breast cancer. Paradoxically, cyclin D1 is known to enhance radiation sensitivity, a finding that has not yet been therapeutically exploited. Proposed cyclin D1 functions that could be involved in this effect include cyclin-dependent kinase (CDK)-dependent phosphorylation of retinoblastoma gene product (pRb), titration of p21/p27 complexes, and less well-characterized effects on gene expression. In this report, we sought to clarify the functions of cyclin D1 that might contribute to enhanced radiation sensitivity. Breast cancer cells stably overexpressing a cyclin D1 mutant (KE) that cannot interact with its CDK partners to phosphorylate pRb were as radiation sensitive as those expressing wild-type D1. Although cyclin D1 has been proposed to affect radiation sensitivity through interactions with p21, a cyclin D1 mutant defective for p21 interactions also increased radiation sensitivity. Cyclin D1 overexpression is generally confined to hormone receptor-positive breast cancers, wherein standard therapies include both radiation and hormonal therapies. Among several proposed CDK-independent cyclin D1 targets, we have identified heat shock protein B8 (HSPB8) as a target particularly associated with cyclin D1 and ER-positive tumors. We therefore evaluated its potential contribution to radiation sensitivity. Overexpression of HSPB8 markedly increased radiation sensitivity, and HSPB8 small interfering RNA blocked cyclin D1's enhancement of radiation sensitivity. Taken together, our results show that some of cyclin D1's effects on radiation sensitivity are CDK and p21 independent and identify HSPB8 as a candidate CDK-independent cyclin D1 target that can mediate its effects.

Topics: Breast Neoplasms; Cell Line, Tumor; Cyclin D1; Gene Expression Regulation, Neoplastic; Heat-Shock Proteins; Humans; Molecular Chaperones; Mutation; Neoplasm Transplantation; Phosphorylation; Protein Serine-Threonine Kinases; Radiation Tolerance; Reverse Transcriptase Polymerase Chain Reaction; RNA

The Wnt/beta-catenin signaling cascade is an important signal transduction pathway in human cancers. Overexpression of beta-catenin and its downstream effector, cyclin D1, is implicated in malignant transformation and acquisition of an invasive tumor phenotype. This study aimed to determine the clinical significance of Wnt/beta-catenin canonical pathway components in breast cancer.. Expression of beta-catenin, dishevelled (Dvl) and cyclin D1 was examined in invasive ductal carcinomas (IDCs) of the breast by immunohistochemical analysis.. Of the 98 IDCs analyzed, 30% of tumors displayed both nuclear and cytoplasmic staining of Dvl protein, while 52% showed nuclear localization. Loss of cell surface beta-catenin was observed in 66% of breast carcinomas, whereas nuclear expression was observed in 48% IDCs. Cyclin D1 overexpression was observed in 60% IDCs; 31/59 (53%) of these tumors showed nuclear expression of beta-catenin, suggesting upregulation of the canonical Wnt/beta-catenin pathway. Our study demonstrates a significant association between nuclear localization of Dvl and beta-catenin (p < 0.01, OR = 15.8).. To our knowledge, this is the first study showing an association between nuclear localization of Dvl and beta-catenin in IDCs and suggests the upregulation of Wnt/beta-catenin pathway components, beta-catenin, Dvl and cyclin D1 in IDCs of the breast.

Topics: Adaptor Proteins, Signal Transducing; Adult; Aged; Aged, 80 and over; beta Catenin; Breast Neoplasms; Carcinoma, Ductal, Breast; Cyclin D1; Dishevelled Proteins; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Lymphatic Metastasis; Middle Aged; Neoplasm Staging; Phosphoproteins; Signal Transduction; Up-Regulation; Wnt Proteins

1,1-Bis(3'-indolyl)methane (DIM) and the 5,5'-dibromo ring substituted DIM (5,5'-diBrDIM) inhibited growth of MCF-7 and MDA-MB-231 breast cancer cells, and IC50 values were 10-20 and 1-5 microM, respectively, in both cell lines. DIM and 5,5'-diBrDIM did not induce p21 or p27 protein levels or alter expression of Sp1 or Sp3 proteins in either cell line. In contrast, 10 microM 5,5'-diBrDIM downregulated cyclin D1 protein in MCF-7 and MDA-MB-231 cells 12 and 24 h after treatment. DIM (20 microM) also decreased cyclin D1 in MCF-7 (24 h) and MDA-MB-231 (12 h), and the DIM/5,5'-diBrDIM-induced degradation of cyclin D1 was blocked by the proteasome inhibitor MG132. Both DIM and 5,5'-diBrDIM induced apoptosis in MCF-7 cells and this was accompanied by decreased Bcl-2, release of mitochondrial cytochrome c, and decreased mitochondrial membrane potential as determined by the red/green fluorescence of JC-1. DIM and 5,5'-diBrDIM induced extensive necrosis in MDA-MB-231 cells; however, this was accompanied by decreased mitochondrial membrane potential primarily in cells treated with 5,5'-diBrDIM but not DIM. Thus, DIM and 5,5'-diBrDIM induce cell death in MCF-7 and MDA-MB-231 cells by overlapping and different pathways, and the ring-substituted DIM represents a novel class of uncharged mitochondrial poisons that inhibit breast cancer cell and tumor growth.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cysteine Proteinase Inhibitors; Cytochromes c; Female; Humans; Indoles; Inhibitory Concentration 50; Leupeptins; Mitochondria; Necrosis; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Proto-Oncogene Proteins c-bcl-2; Time Factors

We have examined the role of cyclin D1 and cyclin-dependent kinase-4 (CDK4) in the cell cycle progression and proliferation of MCF-7 breast cancer cells. Forced expression of cyclin D1 using a tetracycline-regulated expression system, and suppression of endogenous cyclin D1 and CDK4 using small interfering RNA (siRNA) were used to validate this protein complex as a drug target in cancer drug discovery. Overexpression of cyclin D1 increased both phosphorylation of the retinoblastoma gene product (RB) and passage through the G1-S phase transition, resulting in increased proliferation of cells. When cyclin D1 expression was shut off, growth rates fell below those seen in control cell lines transfected with the vector, indicating an increased dependence on this protein for proliferation. Inhibition of endogenous cyclin D1 or CDK4 expression by RNA interference resulted in hypophosphorylation of RB and accumulation of cells in G1. These results support the prevailing view that pharmacological inhibition of cyclin D1/CDK4 complexes is a useful strategy to inhibit the growth of tumors. Furthermore, since MCF-7 cells appear to be dependent on this pathway for their continued proliferation, it is a suitable cell line to test novel cyclin D1/CDK4 inhibitors.

Topics: Breast Neoplasms; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Female; G1 Phase; Gene Silencing; Humans; Immunoprecipitation; Phosphorylation; Retinoblastoma Protein; RNA, Small Interfering; Tetracycline; Tumor Cells, Cultured

Breast tumors are usually classified according to their response to estrogens as hormone-dependent or -independent. In this work, we investigated the role of the proinflammatory cytokine TNF-alpha on the estrogen-receptor-positive T47D breast ductal tumor cells. We have found that TNF-alpha exerts a mitogenic effect, inducing cyclin D1 expression and activation of the transcription factor NF-kappaB. Importantly, activation of NF-kappaB was required for estrogen-induced proliferation and cyclin D1 expression. TNF-alpha enhanced the estrogen response by increasing the levels and availability of NF-kappaB. Chromatin immunoprecipitation analysis suggested that the action of estrogens is mediated by a protein complex that contains the activated estrogen receptor, the nuclear receptor coactivator RAC3 and a member of the NF-kappaB family. Finally, our results demonstrate that activation of this transcription factor could be one of the key signals for estrogen-mediated response.

Topics: Animals; Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Proliferation; Chromatin Immunoprecipitation; Cyclin D1; Estrogens; Female; Humans; Mice; NF-kappa B; Receptors, Estrogen; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

The Akt pathway, an important regulator of cell proliferation and survival, is deregulated in many cancers. The pathway has achieved considerable importance due to the development of kinase inhibitors that are able to successfully reduce tumor growth. This study was conducted to determine the status of the Akt pathway in human breast cancers and to study the relationship between the different component proteins. Expression levels of PTEN, phosphorylated forms of the constituent proteins (Akt, FKHR, mTOR, and S6) and cyclin D1 were evaluated by immunohistochemistry, on consecutive sections from a tissue microarray containing 145 invasive breast cancers and 140 pure ductal carcinomas in-situ. Aberrant expression was correlated statistically with tumor characteristics and disease outcome. The Akt pathway was found to be activated early in breast cancer, in the in-situ stage. In all, 33, 15, 32, and 60% of ductal carcinoma in-situ showed overexpression of Akt, FKHR, mTOR, and cyclin D1. PTEN loss did not correlate statistically with expression of AKT or any of the other proteins with the exception of S6, indicating that Akt activation was not a result of PTEN loss. Expression levels of PTEN and S6 were significantly different in in-situ and invasive cancers, indicating association with disease progression. Loss of PTEN was noted in 11% of in-situ as compared to 26% of invasive cancers, while S6 overexpression was seen in 47% in-situ and in 72% invasive cancers. High-grade carcinomas were associated with PTEN loss, while low-grade carcinomas with good prognostic features showed cyclin D1 overexpression and were associated with longer disease free survival. Additionally, cancers with mTOR overexpression showed a three times greater risk for disease recurrence. Overall, a large proportion of in-situ and invasive breast cancers overexpressed cyclinD1 and S6. Our results may have significant implications in the development and application of targeted therapy.

Topics: Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Cyclin D1; Disease Progression; Female; Humans; Immunohistochemistry; Logistic Models; Multivariate Analysis; Neoplasm Invasiveness; Protein Kinases; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Ribosomal Protein S6 Kinases; Signal Transduction; Survival Analysis; Tissue Array Analysis; TOR Serine-Threonine Kinases

The insulin-like growth factor receptor (IGF-IR) and insulin receptor are either overactivated and/or overexpressed in a wide range of tumor types and contribute to tumorigenicity, proliferation, metastasis, and drug resistance. Here, we show that BMS-554417, a novel small molecule developed as an inhibitor of IGF-IR, inhibits IGF-IR and insulin receptor kinase activity and proliferation in vitro, and reduces tumor xenograft size in vivo. In a series of carcinoma cell lines, the IC50 for proliferation ranged from 120 nmol/L (Colo205) to >8.5 micromol/L (OV202). The addition of stimulatory ligands was unnecessary for the antiproliferative effect in MCF-7 and OV202 cells. BMS-554417 treatment inhibited IGF-IR and insulin receptor signaling through extracellular signal-related kinase as well as the phosphoinositide 3-kinase/Akt pathway, as evidenced by decreased Akt phosphorylation at Ser473. At doses that inhibited proliferation, the compound also caused a G0-G1 arrest and prevented nuclear accumulation of cyclin D1 in response to LR3 IGF-I. In Jurkat T-cell leukemia cells, this agent triggered apoptotic cell death via the mitochondrial pathway. BMS-554417 was orally bioavailable and significantly inhibited the growth of IGF1R-Sal tumor xenografts in vivo. BMS-554417 is a member of a novel class of IGF-IR/insulin receptor inhibitors that have potential clinical applications because of their antiproliferative and proapoptotic activity in vitro and in vivo.

Topics: Animals; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Caspases; Cell Growth Processes; Cell Line, Tumor; Cell Nucleus; Cyclin D1; Dose-Response Relationship, Drug; Enzyme Activation; Female; G1 Phase; Humans; Insulin-Like Growth Factor I; Mice; Mice, Nude; Mitochondria; Mitogen-Activated Protein Kinase Kinases; Ovarian Neoplasms; Phosphorylation; Piperazines; Proto-Oncogene Proteins c-akt; Pyridones; Receptor, IGF Type 1; Receptor, Insulin; S Phase

Cyclin D1 is overexpressed in the majority of human breast cancers. We previously found that mice lacking cyclin D1 are resistant to mammary carcinomas triggered by the ErbB-2 oncogene. In this study, we investigated which function of cyclin D1 is required for ErbB-2-driven mammary oncogenesis. We report that the ability of cyclin D1 to activate cyclin-dependent kinase CDK4 underlies the critical role for cyclin D1 in breast cancer formation. We also found that the continued presence of CDK4-associated kinase activity is required to maintain breast tumorigenesis. We analyzed primary human breast cancers and found high cyclin D1 levels in a subset (approximately 25%) of ErbB-2-overexpressing tumors. We propose that this subset of breast cancer patients might benefit from inhibiting CDK4 kinase.

Topics: Animals; Breast Neoplasms; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Enzyme Activation; Female; Genes, erbB-2; Humans; Mammary Neoplasms, Experimental; Mice; Mice, Knockout; Protein Binding

Because of the reported immune-enhancing and anti-tumor activities of some mushroom polysaccharides, their applications as biological response modifiers have attracted significant attention. We have purified a water-soluble beta-glucan PCM3-II, comprising mainly 1right curved arrow 3 and 1right curved arrow 4 linkages, from the mycelia of Poria cocos (Schw.) Wolf (Fu-ling). In this study, the growth-inhibitory effect of PCM3-II was further explored on the human breast carcinoma MCF-7 cells in vitro. The dose effect of PCM3-II was studied by incubating the breast cancer cells with 12.5-400 microg/ml of the glucan for 72 h. The MTT study showed that PCM3-II reduced proliferation and viability of the MCF-7 cells dose-dependently, so that the cancer-cell growth was decreased by 50% of the control level at 400 microg/ml of the glucan. The time effect of PCM3-II was then investigated by treating the breast cancer cells with 400 microg/ml of the glucan for 24, 48 and 72 h, respectively. Results from the flow cytometry study demonstrated that PCM3-II induced cell-cycle G1 arrest time-dependently and about 90% of the cells in cell cycle were accumulated at G1 phase after 72 h of treatment. The G1 arrest was associated with downregulations of the unscheduled cyclin D1 and cyclin E expressions in the breast cancer cells. Apoptosis was also induced by PCM3-II in the MCF-7 cells, so that the subG1 cells in DNA histogram of the flow cytometry were elevated by 5-fold of the control level at 48 h and by 24-fold at 72 h of treatment. The immunoblot study also showed that the glucan induced depletion of the antiapoptotic Bcl-2 protein, but not the proapoptotic Bax protein, so that the Bax/Bcl-2 ratio was elevated in the breast cancer cells at the time when the most prominent apoptosis was also observed. In conclusion, although the detailed mechanism for the anti-tumor activity of the P. cocos beta-glucan still needs further investigation, this study provides preliminary insights into its mode of action and perspectives of its development as a water-soluble anti-tumor agent.

Topics: Apoptosis; bcl-2-Associated X Protein; beta-Glucans; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Cyclin E; Dose-Response Relationship, Drug; Down-Regulation; Female; Flow Cytometry; Growth Inhibitors; Humans; Immunoblotting; Mycelium; Polyporales; Proto-Oncogene Proteins c-bcl-2; Time Factors

Human breast cancer displays nuclear accumulation of beta-catenin and induction of cyclin D1 expression, which suggests that canonical Wnt/beta-catenin signaling is activated. In other cancers, the activation of canonical wnt/beta-catenin signaling is associated with APC, CTNNB1 or AXIN1 mutations. However, these mutations are rare or absent in breast cancer. In search of alternative mechanisms, we performed comprehensive expression analysis of Wnt signaling molecules, including 19 Wnt ligands, ten Frizzled receptors, two co-receptors and four Lef/TCF transcription factors in immortalized normal human mammary epithelial cells (HMEC) and six breast cancer cell lines. HMEC expressed all Frizzled receptors except FZD9 and FZD10. They also expressed LRP5 and LRP6 co-receptors, as well as four Lef/TCF transcription factors. HMEC cells also expressed many Wnt ligands, including WNT1, WNT2B, WNT3, WNT5A, WNT5B, WNT7B, WNT9A, WNT10B and WNT16. Redundant expression of Wnt ligands, Frizzled receptors, co-receptors and Lef/TCF transcription factors was maintained in breast cancer cell lines with some exceptions. The most important changes in cancer cell lines concerned Wnt ligand expression. We noticed that most breast cancer cell lines overexpressed WNT3A, WNT4, WNT6, WNT8B, WNT9A and WNT10B. In contrast, the expression of WNT5A, WNT5B and WNT16 was usually down-regulated. It is noteworthy that all six Wnt ligands that are overexpressed in malignant cell lines are known to signal through the canonical Wnt/beta-catenin signaling pathway, whereas down-regulated WNT5A and WNT5B ligands signal via the non-canonical pathway. The expression of both canonical Wnt ligands and most Frizzled receptors in breast cancer cell lines suggests that canonical Wnt/beta-catenin signaling is activated in these cell lines by an autocrine/paracrine mechanism. In support of this prediction, we observed nuclear beta-catenin accumulation and cyclin D1 induction in breast cancer cell lines, but not in HMEC. These results imply that ligand-dependent canonical Wnt/beta-catenin signaling is active in human breast cancer.

Topics: beta Catenin; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cell Nucleus; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Ligands; Receptors, Cell Surface; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; TCF Transcription Factors; Up-Regulation; Wnt Proteins

3-Methylcholanthrene (3MC) is an aryl hydrocarbon receptor (AhR) agonist, and it has been reported that 3MC induces estrogenic activity through AhR-estrogen receptor alpha (ER alpha) interactions. In this study, we used 3MC and 3,3',4,4',5-pentachlorobiphenyl (PCB) as prototypical AhR ligands, and both compounds activated estrogen-responsive reporter genes/gene products (cathepsin D) in MCF-7 breast cancer cells. The estrogenic responses induced by these AhR ligands were inhibited by the antiestrogen ICI 182780 and by the transfection of a small inhibitory RNA for ER alpha but were not affected by the small inhibitory RNA for AhR. These results suggest that 3MC and PCB directly activate ER alpha, and this was confirmed in a competitive ER alpha binding assay and in a fluorescence resonance energy transfer experiment in which PCB and 3MC induced CFP-ER alpha/YFP-ER alpha interactions. In a chromatin immunoprecipitation assay, PCB and 3MC enhanced ER alpha (but not AhR) association with the estrogen-responsive region of the pS2 gene promoter. Moreover, in AhR knockout mice, 3MC increased uterine weights and induced expression of cyclin D1 mRNA levels. These results show that PCB and 3MC directly activate ER alpha-dependent transactivation and extend the number of ligands that activate both AhR and ER alpha.

Topics: Animals; Binding, Competitive; Breast Neoplasms; Cell Line, Tumor; Chromatin Immunoprecipitation; Cyclin D1; Dimerization; Estradiol; Estrogen Receptor alpha; Female; Fluorescence Resonance Energy Transfer; Humans; Ligands; Methylcholanthrene; Mice; Mice, Inbred C57BL; Mice, Knockout; Organ Size; Polychlorinated Biphenyls; Receptors, Aryl Hydrocarbon; RNA, Messenger; Transfection; Uterus

This study was designed to produce a model to predict outcome in tamoxifen-treated breast cancer patients based on clinicopathologic features and multiple molecular markers.. This was a retrospective study of 324 stage I to III female breast cancer patients treated with tamoxifen for whom standard clinicopathologic data and tumor tissue microarrays were available. Nine molecular markers were studied by semiquantitative immunohistochemistry and/or fluorescence in situ hybridization. Cox proportional hazards analysis was used to determine the contributions of each variable to disease-specific and overall survival, and machine learning was used to produce a model to predict patient outcome.. On a univariate basis, the following features were significantly associated with worse survival: high pathologic tumor or nodal class, histologic grade, epidermal growth factor receptor, ERBB2, MYC, or TP53; absent estrogen receptor (ER) or progesterone receptor; and low BCL2. CCND1 and CDKN1B did not reach statistical significance. On a multivariate basis, nodal class, ER, and MYC were statistically significant as independent factors for survival. However, the benefit of ER-positive status was moderated by BCL2, ERBB2, and progesterone receptor. BCL2 and TP53 also interacted as an independent risk factor. A kernel partial least squares polynomial model was developed with an area under the receiver operating characteristic curve of 0.90.. Our data show the predictive value of BCL2, ERBB2, MYC, and TP53 in addition to the standard hormone receptors and clinicopathologic features, and they show the importance of conditional interpretation of certain molecular markers. Our multimarker predictive model performed significantly better than standard guidelines.

Topics: Aged; Aged, 80 and over; Biomarkers, Tumor; Breast Neoplasms; Cyclin D1; Estrogen Antagonists; Female; Humans; Immunohistochemistry; In Situ Hybridization, Fluorescence; Middle Aged; Multivariate Analysis; Predictive Value of Tests; Prognosis; Proportional Hazards Models; Proto-Oncogene Proteins c-bcl-2; Receptor, ErbB-2; Receptors, Estrogen; Retrospective Studies; Survival Analysis; Tamoxifen; Treatment Outcome; Tumor Suppressor Protein p53

Cyclin D1 is an important regulator of G1-S phase cell cycle transition and has been shown to be important for breast cancer development. GSK3beta phosphorylates cyclin D1 on Thr-286, resulting in enhanced ubiquitylation, nuclear export and degradation of the cyclin in the cytoplasm. Recent findings suggest that the development of small-molecule cyclin D1 ablative agents is of clinical relevance. We have previously shown that the histone deacetylase inhibitor trichostatin A (TSA) induces the rapid ubiquitin-dependent degradation of cyclin D1 in MCF-7 breast cancer cells prior to repression of cyclin D1 gene (CCND1) transcription. TSA treatment also resulted in accumulation of polyubiquitylated GFP-cyclin D1 species and reduced levels of the recombinant protein within the nucleus.. Here we provide further evidence for TSA-induced ubiquitin-dependent degradation of cyclin D1 and demonstrate that GSK3beta-mediated nuclear export facilitates this activity. Our observations suggest that TSA treatment results in enhanced cyclin D1 degradation via the GSK3beta/CRM1-dependent nuclear export/26S proteasomal degradation pathway in MCF-7 cells.. We have demonstrated that rapid TSA-induced cyclin D1 degradation in MCF-7 cells requires GSK3beta-mediated Thr-286 phosphorylation and the ubiquitin-dependent 26S proteasome pathway. Drug induced cyclin D1 repression contributes to the inhibition of breast cancer cell proliferation and can sensitize cells to CDK and Akt inhibitors. In addition, anti-cyclin D1 therapy may be highly specific for treating human breast cancer. The development of potent and effective cyclin D1 ablative agents is therefore of clinical relevance. Our findings suggest that HDAC inhibitors may have therapeutic potential as small-molecule cyclin D1 ablative agents.

Topics: Acetylcysteine; Breast Neoplasms; Cell Line, Tumor; Cell Nucleus; Cyclin D1; Cytoplasm; Enzyme Inhibitors; Exportin 1 Protein; Fatty Acids, Unsaturated; Female; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Karyopherins; Leupeptins; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Receptors, Cytoplasmic and Nuclear; Recombinant Fusion Proteins; RNA Interference; Transfection; Ubiquitin

HER2 overexpression is one of the most recognizable molecular alterations in breast tumors known to be associated with a poor prognosis. In the study described here, we explored the effect of HER2 overexpression on the sensitivity of breast cancer cells to the growth-inhibitory effects of 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO), a synthetic triterpenoid, both in vitro and in vivo in a xenograft model of breast cancer. Both cell growth and colony formation in the soft agar assay, a hallmark of the transformation phenotype, were preferentially suppressed in HER2-overexpressing cell lines at low concentrations of CDDO, whereas growth-inhibitory effects at high concentrations did not correlate with the expression level of HER2. CDDO dose-dependently inhibited phosphorylation of HER2 in HER2-overexpressing cells and diminished HER2 kinase activity in vitro. CDDO induced the transactivation of the nuclear receptor peroxisome proliferator-activated receptor-gamma in both vector control and HER2-transfected MCF7 cells. Dose-response studies showed that the growth inhibition seen at lower concentrations of CDDO correlated with induction of the tumor suppressor gene caveolin-1, which is known to inhibit breast cancer cell growth. CDDO also reduced cyclin D1 mRNA and protein expression. In vivo studies with liposomally encapsulated CDDO showed complete abrogation of the growth of the highly tumorigenic MCF7/HER2 cells in a xenograft model of breast cancer. These findings provide the first in vitro and in vivo evidence that CDDO effectively inhibits HER2 tyrosine kinase activity and potently suppresses the growth of HER2-overexpressing breast cancer cells and suggest that CDDO has a therapeutic potential in advanced breast cancer.

Topics: Animals; Breast Neoplasms; Caveolin 1; Cell Proliferation; Cyclin D1; Female; Humans; Mice; Mice, Mutant Strains; Oleanolic Acid; Phosphorylation; PPAR gamma; Receptor Protein-Tyrosine Kinases; Receptor, ErbB-2; RNA, Messenger; Transcriptional Activation; Xenograft Model Antitumor Assays

Signal transducers and activators of transcription 3 (STAT3) is a transcription factor that is aberrantly activated in many cancer cells. Constitutively activated STAT3 is oncogenic, presumably as a consequence of the genes that it differentially regulates. Activated STAT3 correlated with elevated cyclin D1 protein in primary breast tumors and breast cancer-derived cell lines. Cyclin D1 mRNA levels were increased in primary rat-, mouse-, and human-derived cell lines expressing either the oncogenic variant of STAT3 (STAT3-C) or vSrc, which constitutively phosphorylates STAT3. Mutagenesis of STAT3 binding sites within the cyclin D1 promoter and chromatin immunoprecipitation studies showed an association between STAT3 and the transcriptional regulation of the human cyclin D1 gene. Introduction of STAT3-C and vSrc into immortalized cyclin D1(-/-) and cyclin D1(-/+) fibroblasts led to anchorage-independent growth of only cyclin D1(-/+) cells. Furthermore, knockdown of cyclin D1 in breast carcinoma cells led to a reduction in anchorage-independent growth. Phosphorylation of the retinoblastoma (Rb) protein [a target of the cyclin D1/cyclin-dependent kinase 4/6 (cdk4/6) holoenzyme] was delayed in the cyclin D1(-/-) cells relative to cyclin D1(-/+) cells. The E7 oncogene, whose activity includes degradation of Rb and dissociation of Rb from E2F, did not confer anchorage-independent growth to the cyclin D1(-/-) cells but, in conjunction with vSrc, resulted in robust growth in soft agar. These results suggest both a cdk-dependent and cdk-independent role for cyclin D1 in modulating transformation by different oncogenes.

Topics: Animals; Binding Sites; Breast Neoplasms; Cell Adhesion; Cell Growth Processes; Cell Line, Tumor; Cell Transformation, Neoplastic; Cyclin D1; G1 Phase; Humans; Luciferases; Mice; Mutagenesis, Site-Directed; NIH 3T3 Cells; Papillomavirus E7 Proteins; Promoter Regions, Genetic; Rats; RNA, Messenger; RNA, Small Interfering; STAT3 Transcription Factor; Transcriptional Activation

The quaternary isoquinoline alkaloid sanguinarine is receiving increasing attention as a potential chemotherapeutic agent in the treatment of cancer. Previous studies have shown that this DNA-binding phytochemical can arrest a number of different types of transformed cells in G0/G1, and upregulate the CKIs p21 and p27 while downregulating multiple cyclins and CDKs. To more closely examine the responses of some of these cell cycle regulatory molecules to sanguinarine, we used immunocytochemical methods to visualize cyclin D1 and topoisomerase II behavior in MCF-7 breast cancer cells.. 5-10 microM sanguinarine effectively inhibits MCF-7 proliferation after a single application of drug. This growth inhibition is accompanied by a striking relocalization of cyclin D1 and topoisomerase II from the nucleus to the cytoplasm, and this effect persists for at least three days after drug addition. DNA synthesis is transiently inhibited by sanguinarine, but cells recover their ability to synthesize DNA within 24 hours. Taking advantage of the fluorescence characteristics of sanguinarine to follow its uptake and distribution suggests that these effects arise from a window of activity of a few hours immediately after drug addition, when sanguinarine is concentrated in the nucleus. These effects occur in morphologically healthy-looking cells, and thus do not simply represent part of an apoptotic response.. It appears that sub-apoptotic concentrations of sanguinarine can suppress breast cancer cell proliferation for extended lengths of time, and that this effect results from a relatively brief period of activity when the drug is concentrated in the nucleus. Sanguinarine transiently inhibits DNA synthesis, but a novel mechanism of action appears to involve disrupting the trafficking of a number of molecules involved in cell cycle regulation and progression. The ability of sub-apoptotic concentrations of sanguinarine to inhibit cell growth may be a useful feature for potential chemotherapeutic applications; however, a narrow effective range for these effects may exist.

Topics: Active Transport, Cell Nucleus; Alkaloids; Antineoplastic Agents, Phytogenic; Apoptosis; Benzophenanthridines; Breast Neoplasms; Cell Line, Tumor; Cell Nucleus; Cyclin D1; Cytoplasm; DNA Topoisomerases, Type II; DNA, Neoplasm; Female; Humans; Isoquinolines

In this study, tumour tissue samples of 85 primary breast cancer patients were evaluated for phosphatase and tensin homolog deleted on chromosome ten (PTEN), cyclin D1 and P27/Kip1 expression patterns. The results were correlated with clinicopathological parameters. Loss of PTEN protein expression was present in 32.5% of the cases. Cyclin D1 was overexpressed in 54.2% and P27/Kip1 in 89.3% of the cases. Statistically significant associations were found between PTEN and cyclin D1 expression patterns, and cyclin D1 expression and tumour size.

Topics: Breast Neoplasms; Carcinoma, Ductal, Breast; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Female; Gene Expression Regulation, Neoplastic; Humans; PTEN Phosphohydrolase

The G1 regulators of the cell cycle, cyclin D(1) and D(3), have been implicated in the regulation of Schwann cell proliferation and differentiation. The purpose of this study is to evaluate cyclin D(1) and D(3) protein expression and the corresponding clinical characteristics of vestibular schwannomas.. Tissue sections of 15 sporadic vestibular schwannomas were prepared. Immunohistochemical analysis of the vestibular schwannomas was performed with anticyclin D(1) and anticyclin D(3) antibodies. The immunoreactivity was evaluated in comparison with adjacent vestibular nerves. Tissue sections of breast carcinoma and prostate carcinoma were used as positive controls for cyclin D(1) and D(3) staining, respectively. Patient demographics, tumor characteristics, and cyclin D expression were reviewed, and statistical analysis was performed.. While the breast carcinoma control expressed abundant cyclin D(1) protein, none of the 15 vestibular schwannomas showed detectable cyclin D(1) staining. In contrast, seven of 15 vestibular schwannomas stained positive for the cyclin D(3) protein. Cyclin D(3) staining was taken up in the nucleus of schwannoma tumor cells in greater proportion than Schwann cells of adjacent vestibular nerve. Although sample size was small, no significant difference in the average age of presentation, tumor size, and male to female ratios for the cyclin D(3)(+) or cyclin D(3)(-) groups was found.. The Cyclin D(1) protein does not appear to play a prominent role in promoting cell cycle progression in vestibular schwannomas. In contrast, cyclin D(3) expression was seen in nearly half of the tumors examined, suggesting that it may have a growth-promoting role in some schwannomas. Further studies are needed to define its cellular mechanism.

Topics: Antibodies, Neoplasm; Biomarkers, Tumor; Breast Neoplasms; Carcinoma; Cyclin D1; Cyclin D3; Cyclins; Diagnosis, Differential; Female; Humans; Immunohistochemistry; Male; Neuroma, Acoustic; Prostatic Neoplasms

These studies were designed to determine whether ritonavir inhibits breast cancer in vitro and in vivo and, if so, how.. Ritonavir effects on breast cancer cell growth were studied in the estrogen receptor (ER)-positive lines MCF7 and T47D and in the ER-negative lines MDA-MB-436 and MDA-MB-231. Effects of ritonavir on Rb-regulated and Akt-mediated cell proliferation were studied. Ritonavir was tested for inhibition of a mammary carcinoma xenograft.. ER-positive estradiol-dependent lines (IC50, 12-24 micromol/L) and ER-negative (IC50, 45 micromol/L) lines exhibit ritonavir sensitivity. Ritonavir depletes ER-alpha levels notably in ER-positive lines. Ritonavir causes G1 arrest, depletes cyclin-dependent kinases 2, 4, and 6 and cyclin D1 but not cyclin E, and depletes phosphorylated Rb and Ser473 Akt. Ritonavir induces apoptosis independent of G1 arrest, inhibiting growth of cells that have passed the G1 checkpoint. Myristoyl-Akt, but not activated K-Ras, rescues ritonavir inhibition. Ritonavir inhibited a MDA-MB-231 xenograft and intratumoral Akt activity at a clinically attainable serum Cmax of 22 +/- 8 micromol/L. Because heat shock protein 90 (Hsp90) substrates are depleted by ritonavir, ritonavir effects on Hsp90 were tested. Ritonavir binds Hsp90 (K(D), 7.8 micromol/L) and partially inhibits its chaperone function. Ritonavir blocks association of Hsp90 with Akt and, with sustained exposure, notably depletes Hsp90. Stably expressed Hsp90alpha short hairpin RNA also depletes Hsp90, inhibiting proliferation and sensitizing breast cancer cells to low ritonavir concentrations.. Ritonavir inhibits breast cancer growth in part by inhibiting Hsp90 substrates, including Akt. Ritonavir may be of interest for breast cancer therapeutics and its efficacy may be increased by sustained exposure or Hsp90 RNA interference.

Topics: Animals; Blotting, Western; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinases; Dose-Response Relationship, Drug; Female; HIV Protease Inhibitors; HSP90 Heat-Shock Proteins; Humans; Immunohistochemistry; Immunoprecipitation; Mammary Neoplasms, Experimental; Mice; Mice, Nude; Mutant Proteins; Proto-Oncogene Proteins c-akt; Ritonavir; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

Most breast cancers are "lipogenic", defined by high fatty acid synthase (FAS) content and dependence on fatty acid synthesis for growth and survival. S14 (Spot 14; THRSP) is a nuclear protein that activates genes required for fatty acid synthesis. The S14 gene is amplified in approximately 15% of breast cancers, but clinical correlates of its expression were unknown. We analyzed 131 breast cancers by immunohistochemistry for S14 and FAS. Staining was graded 0, 1, or 2+, and scores were correlated with traditional tumor markers, histological features, and outcome. S14 and FAS staining were related to tumor size (p=0.05 for S14, p=0.038 for FAS), but not to stage. S14 but not FAS scores correlated with tumor grade in both DCIS (p=0.003) and invasive cases (p<0.001). Invasive cases (pooled node - and +) with weak S14 staining (n=21) showed no recurrence over 3000 d follow-up, including 10 cases with lymph node involvement, whereas 32% of 67 strongly-staining tumors recurred (log rank p<0.0001). S14 scores did not cosegregate with sex steroid receptors, Her2/neu, or cyclin D1. Low level S14 expression is associated with prolonged disease-free survival in invasive cases, including those with nodal metastasis. High-level expression of S14 identifies a subset of high-risk breast cancers that is not specified by analysis of sex steroid receptors, Her2/neu, or cyclin D1, and provides a molecular correlate to histologic features that predict recurrence.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Breast Neoplasms; Cyclin D1; Dietary Fats; Female; Humans; Immunohistochemistry; Middle Aged; Nuclear Proteins; Transcription Factors

Insulin-like growth factors (IGFs) play an important role in normal and cancerous cell proliferation. Moreover, in recent studies IGF-I has been implicated as a major cancer risk factor. The tomato carotenoid lycopene and all-trans retinoic acid (atRA) have been shown to inhibit growth factor-induced proliferation of different types of cancer cells. This action is associated with inhibition of cell cycle progression in G0/G1 phase. Cyclin D1 acts as a growth factor sensor in G1 phase and is overexpressed in many breast cancer tumors. We have previously demonstrated that slowdown of serum-stimulated cell cycle progression from G1 to S phase by lycopene correlates with reduction in cyclin D1 levels, suggesting that the expression of this protein is a main target for lycopene's action.. To determine whether the reported reduction in cyclin D1 level is the key mechanism for lycopene and atRA inhibitory action on IGF-I-induced cell cycle progression.. Human breast (MCF-7) and endometrial (ECC-1) cancer cells were synchronized in G0/G1 phase by serum deprivation followed by stimulation with IGF-I. Cell treatment with lycopene and atRA inhibited IGF-I-stimulated cell cycle progression from G1 to S phase and decreased retinoblastoma protein (pRb) phosphorylation. These events were associated with a reduction in cyclin D1 and p21(CIP1/WAF1) level, but not that of p27(KIP1). To test the hypothesis that the decrease in cyclin D1 has a major role in the inhibitory effects of lycopene and atRA, we examined the ability of these two agents to suppress cell cycle progression in MCF-7.7D1.13 cells which are capable of expressing cyclin D1 under the control of the Zn-inducible metallothionein promoter. Our results showed that ectopic expression of cyclin D1 can overcome cell cycle inhibition caused by lycopene and atRA.. Our findings suggest that attenuation of cyclin Dl levels by lycopene and atRA is an important mechanism for the reduction of the mitogenic action of IGF-I.

Topics: Antineoplastic Agents; Blotting, Western; Breast Neoplasms; Carotenoids; Cell Cycle; Cell Division; Culture Media, Serum-Free; Cyclin D1; Endometrial Neoplasms; Female; Humans; Insulin-Like Growth Factor I; Lycopene; Phosphorylation; Time Factors; Tretinoin; Tumor Cells, Cultured

Lauryl gallate is an antioxidant food additive showing low toxicity to normal cells. Here, its antiproliferative effect has been studied on three human breast cancer cell lines: estrogen-dependent, wild-type p53, MCF7; estrogen-independent, non-functional p53, MDA-MB-231 and MCF7 ADR, which overexpresses P-glycoprotein (P-gp) and displays a multidrug-resistant phenotype. Lauryl gallate inhibited proliferation and induced cell cycle alterations in all three cell lines without altering P-gp functionality in the drug-resistant cells. A stable arrest in G(1) phase was observed in MCF7, while a slow-down of cell cycle progression was induced in the other two cell lines. Lauryl gallate increased p53 expression only in MCF7, and upregulated p21(Cip1) and reduced cyclin D1 levels in all three cell lines. The induction of apoptosis, demonstrated by annexin V-FITC labeling, PARP cleavage and mitochondrial membrane depolarization and morphological alterations, were clearly detected in MCF7 ADR and MDA-MB-231 and to a minor extent in MCF7. Overexpression of Bcl-2 in MCF7 ADR cells demonstrated its protective role against morphological alterations and apoptosis. Lauryl gallate induction of p21(Cip1) and apoptosis observed in all three cell lines was regulated by Erk1/2 activation. These findings suggest a potential use of lauryl gallate against tumors harboring p53 mutations and drug-resistant phenotypes.

Topics: Apoptosis; ATP Binding Cassette Transporter, Subfamily B, Member 1; Breast Neoplasms; Cell Proliferation; Cyclin D1; Drug Resistance, Neoplasm; G1 Phase; Gallic Acid; Humans; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Estrogens and estrogen receptors (ER) play important roles in estrogen-dependent and ER-positive breast cancer development. Inhibitors against estrogen biosynthesis or anti-estrogens have been used in breast cancer treatment for many years. The aim of this study was to determine whether pentagalloylglucose (5GG) has inhibitory effects on ER function. In the present study, we found that 5GG significantly reduced the growth of estrogen-responsive human breast cancer MCF-7 cells, and suppressed the phosphorylation and protein level of estrogen receptor alpha (ERalpha). Interestingly, 5GG decreased ERalpha protein levels by promoting the degradation of ERalpha protein in the lysosome. The ERalpha can be activated through a ligand-dependent and/or a ligand-independent pathway. The activated Akt kinase was shown to directly phosphorylate ERalpha at its serine residues and cause ligand independent activation. Our results showed that 5GG might inhibit the phosphatidylinositol 3-kinase (PI3K)/Akt pathway either through directly inhibiting Akt kinase activity or through inhibiting phosphorylation of the upstream receptor tyrosine kinases. The depletion of ErbB family receptors, including epidermal growth factor receptor (EGFR), ErbB2, and ErbB3, was also observed. 5GG treatment also led to a dose-dependent decrease in the expression of the estrogen-activated cyclin D1 expression. These findings suggested that 5GG might be a useful chemopreventive or therapeutic agent for hormone-dependent breast cancer through suppressing the functions of ERalpha by lysosome-dependent depletion and modulating the ErbB/PI3K/Akt pathway.

Topics: Breast Neoplasms; Cell Line, Tumor; Cyclin D1; Estrogen Receptor alpha; Female; Humans; Hydrolyzable Tannins; Lysosomes; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proto-Oncogene Proteins c-akt; Receptor, ErbB-2; Receptor, ErbB-3

Conflicting results on the prevalence of cyclin D1 ovexpression and its correlation with CCND1 amplification and outcome of breast cancer patients have been reported. Owing to limited sensitivity and specificity of most antibodies against cyclin D1, evaluation of cyclin D1 immunoexpression is reported to be problematic. The aims of this study were to assess the prevalence of cyclin D1 expression in breast carcinomas using the SP4 rabbit monoclonal antibody; to correlate cyclin D1 expression with amplification, assessed using chromogenic in situ hybridisation (CISH); and to analyse the relationship between CCND1 amplification and overexpression with clinicopathological parameters and outcome in a tissue microarray containing replicate tumour samples from 245 breast cancer patients. Immunohistochemistry for cyclin D1 was performed using the SP4 and the results were scored according to the Allred scoring system. CISH was carried out using the Zymed CCND1 SpotLight probe. CISH signals were counted in 60 morphologically unequivocal neoplastic cells. Amplification was defined as >5 signals per nucleus in more than 50% of cancer cells, or when large gene copy clusters were seen. Strong cyclin D1 expression and CCND1 amplification were found in 67.4 and 14.5% of the cases, respectively. A strong correlation between cyclin D1 overexpression and CCND1 amplification was demonstrated (P<0.0001). Cyclin D1 expression showed a positive correlation with hormone receptor expression (both ER and PgR, P<0.0001). An inverse correlation was observed between an immunohistochemical panel of 'basal-like' markers and both cyclin D1 overexpression (P<0.0001) and CCND1 amplification (P<0.0001). On univariate analysis cyclin D1 expression showed a correlation with longer overall survival (OS). Neither cyclin D1 nor CCND1 were independent prognostic factors for disease-free survival or OS. The results of this study confirm the association between cyclin D1 overexpression and positivity for hormone receptors and the lack of CCND1 amplification in basal-like breast carcinomas.

Topics: Antibodies, Monoclonal; Breast Neoplasms; Cyclin D1; Female; Gene Amplification; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; In Situ Hybridization; Receptors, Estrogen; Receptors, Progesterone; Reproducibility of Results; Survival Analysis; Tissue Array Analysis

Zanthoxyli Fructus belongs to the family of oranges and is used as a seasoning in Asian countries including Japan. This study found that a water extract of Zanthoxyli Fructus possessed anti-tumor activity against a wide variety of cancer cells including those from prostate (LNCaP, DU145, PC-3), breast (MCF-7, T47D, MDA-MB231), lung (NCI-H460, -H520), as well as leukemia (HL-60, NB4, Jurkat) in vitro, as measured by the trypan blue exclusion test. Importantly, Zanthoxyli Fructus slowed the proliferation of LNCaP, DU145, and MDA-MB231 cells present as xenografts in BALB/c nude mice without adverse effects. Further studies explored the molecular mechanism by which Zanthoxyli Fructus inhibited the proliferation of androgen-dependent human prostate cancer LNCaP cells because Zanthoxyli Fructus possessed the strongest anti-tumor activity against these cells. Zanthoxyli Fructus blocked androgen receptor (AR) signaling in conjunction with down-regulation of nuclear levels of AR and induced apoptosis of these cells, as measured by the reporter assay, Western blot analysis, and TUNEL assay, respectively. As expected, Zanthoxyli Fructus also decreased the level of the AR-target molecule, prostate-specific antigen in these cells. Furthermore, Zanthoxyli Fructus inhibited AKT kinase and down-regulated levels of cyclin D1 protein, as measured by the AKT kinase assay with GSK-3alpha/beta as a substrate and Western blot analysis, respectively. Taken together, Zanthoxyli Fructus might be useful as an adjunctive therapeutic agent for the treatment of individuals with a variety of cancer types.

Topics: Androgen Receptor Antagonists; Animals; Apoptosis; Breast Neoplasms; Cell Growth Processes; Cyclin D1; Down-Regulation; HL-60 Cells; Humans; Jurkat Cells; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasms, Hormone-Dependent; Phosphorylation; Plant Extracts; Promoter Regions, Genetic; Prostate-Specific Antigen; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Receptors, Androgen; Signal Transduction; Transfection; Zanthoxylum

A Japanese study reported that up to 16% of breast cancer samples harbor a sporadic mutation within the human Cav-1 gene, namely P132L. To date, however, no studies have examined the United States' population. Here, we developed a novel allele-specific real-time PCR assay to detect the Cav-1 P132L mutation in mammary tumor cells isolated by laser capture microdissection from formalin-fixed paraffin-embedded breast cancer samples. We report that the Cav-1 P132L mutation is present in approximately 19% of estrogen receptor alpha (ERalpha)-positive breast cancers but not in ERalpha-negative breast cancers. This is the first demonstration that the P132L mutation is exclusively associated with ERalpha-positive mammary tumors. We also identified six novel Cav-1 mutations associated with ERalpha-positive breast cancers (W128Stop, Y118H, S136R, I141T, Y148H, and Y148S). Thus, the overall incidence of Cav-1 mutations in ERalpha-positive breast cancers approaches 35% (greater than one-third). To mechanistically dissect the functional relationship between Cav-1 gene inactivation and ERalpha expression, we isolated primary mammary epithelial cells from wild-type and Cav-1-/- mice and cultured them in a three-dimensional system, allowing them to form mammary acinar-like structures. Under conditions of growth factor deprivation, Cav-1-deficient mammary acini displayed increased ERalpha levels and enhanced sensitivity toward estrogen-stimulated growth, with specific up-regulation of cyclin D1. Finally, we discuss the possibility that sporadic Cav-1 mutations may act as an initiating event in human breast cancer pathogenesis.

Topics: Amino Acid Sequence; Animals; Breast Neoplasms; Caveolin 1; Cyclin D1; Estrogen Receptor alpha; Female; Humans; Mammary Neoplasms, Animal; Mice; Mice, Transgenic; Molecular Sequence Data; Mutation; Polymorphism, Single Nucleotide

Obesity has been recognized as a risk factor for breast cancer. Adipocyte-derived leptin may play as a paracrine regulator on the growth of breast cancer cells. Expression of both leptin and its OB-Rb receptor was detected in human breast cancer ZR-75-1 cells and further induced by leptin, suggesting that both expression and message mediation of leptin were autoregulated by itself. With cell counting and MTT assay, we had observed leptin stimulated ZR-75-1 growth in dose- and time-dependent manners. To study what steps of cell cycle progression leptin may involve in, we analyzed cell-cycle profile with flow cytometric analysis, mRNA and protein expressions of four cell-cycle regulators with RT-PCR and Western blotting analysis. Under the treatment of leptin, the G1 arrest of cells was reduced accompanied with up-regulation of G1 phase-specific cyclin D1 and proto-oncogene c-Myc, but down-regulation of cyclin-dependent kinase inhibitor p21(WAF1/CIP1) and tumor suppressor p53. Furthermore, JAK2 inhibitor AG490, PI3K/Akt inhibitor Wortmannin, and MEK/ERK1/2 inhibitor PD98059 were efficiently prevented leptin-promoted cell growth. Effect of cooperation between leptin and estrogen on ZR-75-1 growth had been observed. Collectively, the results showed that the proliferative effect of leptin on ZR-75-1 was associated with the up-regulation of cyclin D1 and c-Myc and down-regulation of tumor suppressor p53 and p21(WAF1/CIP1) plausibly through a hypothesized JAK2-PI3K/Akt-MEK/ERK pathway. The leptin- and OB-Rb-expressing capability of ZR-75-1 created a possible autocrine control of leptin, in which signal could be effectively amplified by itself, on cell growth.

Topics: Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Down-Regulation; Estrogens; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression Regulation; Genes, myc; Humans; Leptin; MAP Kinase Kinase Kinases; Phosphatidylinositol 3-Kinases; Proto-Oncogene Mas; Proto-Oncogene Proteins c-akt; Tumor Suppressor Protein p53; Up-Regulation

Rice protein isolate (RPI) has been reported to reduce the incidence of 7,12-dimethylbenz[a]anthracene-induced mammary tumors in rats. To determine the potential role of phytochemicals associated with the RPI, we studied in vitro antitumor activities of an ether fraction from RPI using human tumor cell lines, including two human breast carcinoma cell lines (MDA-MB-453 and MCF-7) and two myeloma cell lines (RPMI-8226 and IM-9). Concentration-dependent antiproliferative effects of the ether fraction were observed in all cell lines using the standard 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. Fraction-induced apoptosis (P < 0.05) was detected in all cell lines, and this was associated with the induction of proapoptotic bax protein and cdk inhibitors (p21) and the suppression of cdk4 and cyclin D1 activity. Liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) with both positive and negative modes was used to analyze the phytochemicals in the ether fraction from RPI. Fifty-seven phytochemicals were identified or characterized by their diagnostic fragmentation patterns and direct comparison with the authentic standards on the basis of electrospray ionization-MS/MS data. The major components bound to RPI were lysoglycerophospholipids, fatty acids, and fatty acid 3-[2-(2,3-dihydroxy-propoxycarbonyl)-2-hydroxy-ethoxy]-2-hydroxy-propyl esters.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Cell Division; Cell Line, Tumor; Chromatography, Liquid; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p21; Humans; Mass Spectrometry; Multiple Myeloma; Oryza; Plant Proteins

The B-cell translocation gene-2 (BTG2) is present in the nuclei of epithelial cells in many tissues, including the mammary gland where its expression is regulated during glandular proliferation and differentiation in pregnancy. In immortalized mammary epithelial cells and breast cancer cells, BTG2 protein localized predominantly to the nucleus and cytoplasm, respectively. The highly conserved domains (BTG boxes A, B, and C) were required for regulating localization, suppression of cyclin D1 and growth inhibitory function of BTG2. Expression analysis of BTG2 protein in human breast carcinoma (n = 148) revealed the loss of nuclear expression in 46% of tumors, whereas it was readily detectable in the nuclei of adjacent normal glands. Loss of nuclear BTG2 expression in estrogen receptor-alpha (ERalpha)-positive breast tumors correlated significantly with increased histologic grade and tumor size. Consistent with its ability to suppress cyclin D1 transcription, loss of nuclear BTG2 expression in ER-positive breast carcinomas showed a significant correlation with cyclin D1 protein overexpression, suggesting that loss of BTG2 may be a factor involved in deregulating cyclin D1 expression in human breast cancer.

Topics: Adult; Aged; Aged, 80 and over; Breast Neoplasms; Cell Growth Processes; Cell Line, Tumor; Cell Nucleus; Cyclin D1; Estrogen Receptor alpha; Female; Genes, Tumor Suppressor; Humans; Immediate-Early Proteins; Middle Aged; Protein Structure, Tertiary; Tumor Suppressor Proteins

The regulator of cell cycle progression, cyclin D1, is up-regulated in breast cancer cells; its expression is, in part, dependent on ERalpha signaling. However, many ERalpha-negative tumors and tumor cell lines (e.g., SKBR3) also show over-expression of cyclin D1. This suggests that, in addition to ERalpha signaling, cyclin D1 expression is under the control of other signaling pathways; these pathways may even be over-expressed in the ERalpha-negative cells. We previously noticed that both ERalpha-positive and -negative cell lines over-express BRCA1-IRIS mRNA and protein. Furthermore, the level of over-expression of BRCA1-IRIS in ERalpha-negative cell lines even exceeded its over-expression level in ERalpha-positive cell lines. In this study, we show that: (1) BRCA1-IRIS forms complex with two of the nuclear receptor co-activators, namely, SRC1 and SRC3 (AIB1) in an ERalpha-independent manner. (2) BRCA1-IRIS alone, or in connection with co-activators, is recruited to the cyclin D1 promoter through its binding to c-Jun/AP1 complex; this binding activates the cyclin D1 expression. (3) Over-expression of BRCA1-IRIS in breast cells over-activates JNK/c-Jun; this leads to the induction of cyclin D1 expression and cellular proliferation. (4) BRCA1-IRIS activation of JNK/c-Jun/AP1 appears to account for this, because in cells that were depleted from BRCA1-IRIS, JNK remained inactive. However, depletion of SRC1 or SRC3 instead reduced c-Jun expression. Our data suggest that this novel signaling pathway links BRCA1-IRIS to cellular proliferation through c-Jun/AP1 nuclear pathway; finally, this culminates in the increased expression of the cyclin D1 gene.

Topics: BRCA1 Protein; Breast Neoplasms; Cell Proliferation; Chromosomal Proteins, Non-Histone; Cyclin D1; Enzyme Activation; Estrogen Receptor alpha; Exons; Gene Silencing; Humans; JNK Mitogen-Activated Protein Kinases; Promoter Regions, Genetic; Protein Binding; Proto-Oncogene Proteins c-jun; Proto-Oncogene Proteins c-myc; Trans-Activators; Transcription Factor AP-1; Transcription, Genetic; Transcriptional Activation; Tumor Cells, Cultured

The molecular mechanisms underlying the mitogenic effect of ferulic acid (FA), an active compound derived from Angelica sinensis, have never been elucidated. It was the aim of this study to investigate the proliferative effect of FA on human breast cancer cell lines and to elucidate its modulation mechanism on HER2 expression in MCF7 line.. By using MCF7 (oestrogen receptor-positive; ER+, HER2-low), BT474 (ER+, HER2-high), MDAMB231 (ER-, HER2-low) and SKBR3 (ER-, HER2-high) human breast cancer cell lines as in vitro models, the mitogenic effects of FA were assessed by trypan blue dye exclusion assay and DNA flow cytometry. Ferulic acid-modulated cell signalling and HER2 gene expression were evaluated in MCF7 line by Western blot and real-time RT-PCR analysis.. Ferulic acid ER-dependently stimulated cell proliferation on MCF7 cells in a concentration-dependent manner. The HER2 oncogene (one of the prognostic factors of breast cancer) and ESR1 gene (oestrogen receptor-alpha; ERalpha) transcription were markedly up-regulated by FA treatment. Besides, HER2 signalling and its downstream molecules such as AKT and ERK1/2 were involved in FA-modulated ERalpha and cyclin D1 synthesis. Addition of anti-HER2 antibody, trastuzumab, abrogated FA-enhanced proliferative effect on MCF7 cells, indicated a positive feedback control for the action of HER2 in this setting. The fact that the ER antagonist blocked most of the FA-up-regulated HER2 expression, and that trastuzumab down-regulated ERalpha gene expression, suggested a cross-talk between ERalpha and HER2 signalling on MCF7 cells.. The authors' conclude that FA causes human breast cancer cell proliferation by up-regulation of HER2 and ERalpha expression.

Topics: Antibodies, Neoplasm; Breast Neoplasms; Cell Line, Tumor; Coumaric Acids; Cyclin D1; Dose-Response Relationship, Drug; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Genes, erbB-2; Humans; Mitogen-Activated Protein Kinase 1; Mitogens; Mitosis; Proto-Oncogene Proteins c-akt; Signal Transduction; Up-Regulation

To study the inhibitory effect of siRNA on cyclin D1 expression and cell proliferation in breast cancer MCF-7 cell line. The siRNA targeting cyclin D1 was chemically synthesized and transfected into MCF-7 cells by oligofectamine. The expression of cyclin D1 was analyzed by quantitive PCR and Western blot, and the cell growth inhibition was measured with CCK-8 assay. Then, cell cycle of the transfected cells was examined by flow cytometry, and cell colony forming ability was measured by soft-agar colony formation assay. After MCF-7 cells were transfected with 10, 50, 100nmol/L siRNA,the expression of cyclin D1 mRNA was respectively suppressed with inhibition rates of 57.85%, 63.22% and 68.02%, and the protein expression was suppressed with inhibition rates of 51.13%, 62.09% and 77.68% respectively. The proliferation of MCF-7 cells was inhibited after transfection with siRNA-cyclin D1, which caused cell cycle arrest at G1 phase and showed less colony forming ability in the breast cancer cell line MCF-7. These results indicate that siRNA-cyclin D1 could be a powerful anti-proliferative tool in breast cancer gene therapy.

Topics: Blotting, Western; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Humans; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Small Interfering

Cyclin D1 is frequently overexpressed in breast cancer, and its overexpression is, surprisingly, associated with improved survival. One potential mechanism for this association involves signal transducer and activator of transcription 3 (STAT3).. Cyclin D1 and STAT3 expression were assessed in human tumors using microarray analysis and in breast cancer cell lines HBL100, T47D, MCF7, MDA-MB-453, and BT20 and in HBL100 and T47D cells stably overexpressing cyclin D1 using immunoblot analysis. Cyclin D1 protein was stabilized by treatment with the proteasome inhibitor bortezomib, and the effects on STAT3 expression in vitro was determined by using immunoblotting and on xenograft tumor growth and apoptosis in vivo was determined by using terminal deoxyuridine nick-end labeling assays. All statistical tests were two-sided.. Tumors with high cyclin D1 expression (n = 17) had low STAT3 expression (mean = 274 arbitrary units), and those with low cyclin D1 expression (n = 31) had high STAT3 expression (mean = 882 arbitrary units) (P<.001). In HBL100 and T47D parental and cyclin D1-overexpressing cells, cyclin D1 overexpression was also inversely associated with STAT3 expression, and cyclin D1 directly reduced the expression of STAT3. Stabilization of cyclin D1 protein by bortezomib treatment further amplified the cyclin D1-dependent repression of STAT3 in vitro and slowed tumor growth in vivo (week 7: untreated mean = 185.7 mm3 versus treated mean = 136.2 mm3, difference = 49.5 mm3, 95% confidence interval [CI] = 18 to 81 mm3, P = .007; week 8: untreated mean = 240.2 mm3 versus treated mean = 157.3 mm3, difference = 82.9 mm3, 95% CI = 9.1 to 156.7 mm3, P = .0014; and week 9: untreated mean = 256.4 mm3 versus treated mean = 170.2 mm3, difference = 86.2 mm3, 95% CI = 22.8 to 149.6 mm3, P = .006) and increased apoptosis (untreated mean = 19% versus treated mean = 54%, difference = 35%, 95% CI = 24.7% to 45.4%; P = .013) of xenograft tumors.. Cyclin D1 repression of STAT3 expression may explain the association between cyclin D1 overexpression and improved outcome in breast cancer. In addition, bortezomib can amplify the proapoptotic function of cyclin D1, raising the possibility that cyclin D1 levels may be a marker for predicting the response to this novel drug.

Topics: Animals; Antineoplastic Agents; Apoptosis; bcl-X Protein; Biomarkers, Tumor; Boronic Acids; Bortezomib; Breast Neoplasms; Calcium; Caspase 3; Caspases; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; Humans; Immunoblotting; In Situ Nick-End Labeling; Mice; Mice, Inbred BALB C; Microarray Analysis; Protease Inhibitors; Proteasome Endopeptidase Complex; Pyrazines; STAT3 Transcription Factor; Transplantation, Heterologous; Up-Regulation

Carbamazepine, which has been used as an anti-epileptic drug in clinic for many years, is currently recognized as a histone deacetylase inhibitor (HDI), most of which showed anti-tumor characteristics. This study was to investigate the inhibitory effect of carbamazepine on estrogen dependent breast cancer cell lines with estrogen receptor alpha (ERalpha) expression and further explore the underlying mechanisms.. Sulforhodamine B viability assay was used to evaluate the viability of various cells treated with different drugs. Western blot and reverse transcription-polymerase chain reaction (RT-PCR) were performed to detect the protein and mRNA expression of ERalpha and Cyclin D1. Immunofluorescence assay was employed to observe HER-2 expression in MCF-7RT cells, which were resistant to tamoxifen. Immunoprecipitation was performed to detect the chaperon function and acetylation level of Hsp90.. Carbamazepine treatment could inhibit the proliferation of MCF-7 and T47D cells stimulated by estradiol (P<0.01). Carbamazepine and 4-hydroxytamoxifen (4-OHT) demonstrated a synergic effect on the inhibition of proliferation of MCF-7 cells stimulated by estradiol (q=1.00). Cabamazepine reversed the proliferation of MCF-7RT cells stimulated by 4-hydroxytamoxifen (P<0.01). Carbamazepine treatment could decrease the expression of ERalpha and Cyclin D1 at protein and mRNA level in ERalpha-positive cells and could reduce HER-2 expression in MCF-7RT cells. The decrease of ERalpha and Cyclin D1 expression was inhibited by MG132, an inhibitor of 26S proteosome. Carbamazepine treatment elevated the acetylation level of Hsp90 and disrupted its chaperon function.. Carbamazepine shows significant anti-proliferation effect in ERalpha-positive breast cancer cell lines and this might be due to the enhancement of proteosome-mediated degradation of ERalpha and Cyclin D1 by carbamazepine. Furthermore, carbamazepine could reverse HER-2 dependent drug resistance to 4-OHT by reducing HER-2 expression.

Topics: Acetylation; Breast Neoplasms; Carbamazepine; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Drug Resistance, Neoplasm; Drug Synergism; Estradiol; Estrogen Antagonists; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; HSP90 Heat-Shock Proteins; Humans; Receptor, ErbB-2; RNA, Messenger; Tamoxifen

Estrogen stimulates the proliferation of the most common type of human breast cancer that expresses estrogen receptor alpha (ERalpha) through the activation of the cyclin D1 (CCND1) oncogene. However, our knowledge of ERalpha transcriptional mechanisms remains limited. Hence, it is still elusive why ERalpha ectopically expressed in ER-negative breast cancer cells (BCC) is functional on ectopic reporter constructs but lacks activity on many endogenous target genes, including CCND1. Here, we show that estradiol (E2) stimulation of CCND1 expression in BCC depends on a novel cell-type-specific enhancer downstream from the CCND1 coding region, which is the primary ERalpha recruitment site in estrogen-responsive cells. The pioneer factor FoxA1 is specifically required for the active chromatin state of this enhancer and as such is crucial for both CCND1 expression and subsequent cell cycle progression. Interestingly, even in BCC, CCND1 levels and proliferation are tightly controlled by E2 through the establishment of a negative feedforward loop involving the induction of NFIC, a putative tumor suppressor capable of directly repressing CCND1 transcription. Taken together, our results reveal an estrogen-regulated combinatorial network including cell-specific cis- and trans-regulators of CCND1 expression where ERalpha collaborates with other transcription factors associated with the ER-positive breast cancer phenotype, including FoxA1 and NFIC.

Topics: Adaptor Proteins, Signal Transducing; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cyclin D1; DNA Polymerase II; Enhancer Elements, Genetic; Estradiol; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Hepatocyte Nuclear Factor 3-alpha; Humans; LIM Domain Proteins; Microfilament Proteins; Models, Biological; Neoplasms, Hormone-Dependent; NFI Transcription Factors; Transcription, Genetic

Obstacles to the expansion of cells with proliferative potential include the induction of cell death, telomere-based senescence, and the pRb and p53 tumor suppressors. Not infrequently, the molecular pathways regulating oncogenesis recapitulate aberrations of processes governing embryogenesis. The genetic network, consisting of the dachshund (dac), eyes absent (eya), eyeless, and sine oculis (so) genes, regulates cell fate determination in metazoans, with dac serving as a cointegrator through a So DNA-binding factor. Here, DACH1 inhibited oncogene-mediated breast oncogenesis, blocking breast cancer epithelial cell DNA synthesis, colony formation, growth in Matrigel, and tumor growth in mice. Genetic deletion studies demonstrated a requirement for cyclin D1 in DACH1-mediated inhibition of DNA synthesis. DACH1 repressed cyclin D1 through a novel mechanism via a c-Jun DNA-binding partner, requiring the DACH1 alpha-helical DS domain which recruits corepressors to the local chromatin. Analysis of over 2,000 patients demonstrated increased nuclear DACH1 expression correlated inversely with cellular mitosis and predicted improved breast cancer patient survival. The cell fate determination factor, DACH1, arrests breast tumor proliferation and growth in vivo providing a new mechanistic and potential therapeutic insight into this common disease.

Topics: Animals; Binding Sites; Breast Neoplasms; Cells, Cultured; Cyclin D1; DNA; Epithelial Cells; Eye Proteins; Female; Humans; Mammary Glands, Animal; Mammary Glands, Human; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; Phenotype; Promoter Regions, Genetic; Protein Structure, Tertiary; Proto-Oncogene Proteins c-myc; ras Proteins; Transcription Factor AP-1; Transcription Factors; Tumor Stem Cell Assay

MCF-7 human breast cancer cells are relatively resistant to cisplatin treatment compared to other breast cancer cell lines. In order to identify possible targets for sensitizing the breast cancer cells to cisplatin treatment protein expression levels and the phosphorylation status of 27 different signaling proteins were examined. MCF-7 cells expressed high levels of anti-apoptotic Bcl-2 protein relative to more cisplatin sensitive breast cancer cells. After cisplatin treatment a decrease in cyclin D1 was seen in all the breast cancer cells studied. Therefore, Bcl-2 and cyclin D1 were chosen as putative targets for increasing cell death and growth arrest induced by cisplatin, thereby enhancing the drug sensitivity in MCF-7. RNA interference, using Bcl-2- and cyclin D1- siRNAs sensitized MCF-7 cells to cisplatin treatment and by simultaneous knockdown of both Bcl-2 and cyclin D1 further sensitization was seen. This shows the potential of targeting both apoptotic- and cell cycle-regulating pathways to enhance the effect of chemotherapy.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cisplatin; Cyclin D1; Down-Regulation; Drug Screening Assays, Antitumor; G1 Phase; Humans; Proto-Oncogene Proteins c-bcl-2; RNA, Small Interfering; Transfection

Although alterations in the expressions of protein kinase C (PKC) have been implicated in breast carcinogenesis, the roles of specific isoforms in this process remain elusive. In the present study, we examined the specific roles of PKCbeta1 and beta2 in growth control in human breast cancer cell lines. The PKCbeta-specific inhibitor LY379196 significantly inhibited growth of the breast cancer cell lines MCF-7, MDA-MB-231, and BT474, but not the normal mammary epithelial cell line MCF-10F. Treatment of MCF-7 cells with LY379196 caused an increase in the fraction of cells in the G(1) phase of the cell cycle. To explore the roles of PKCbeta1 and beta2, we used cDNA expression vectors that encode wild-type and constitutively activated or dominant negative mutants of these two proteins. When compared with vector controls, derivatives of MCF-7 cells that stably overexpress wild-type PKCbeta1 or PKCbeta2 displayed a slight increase in growth rate; derivatives that stably express the constitutively active mutants of PKCbeta1 or PKCbeta2 displayed a marked increase in growth rate; and derivatives that stably express a dominant negative mutant of PKCbeta1 or beta2 displayed inhibition of growth. The derivatives of MCF-7 cells that stably express the constitutively activated mutants of PKCbeta1 or beta2 were more resistant to growth inhibition by LY379196 than the vector control MCF-7 cells. Immunoblot analysis indicated that MCF-7 cells that stably overexpress wild-type or constitutively activated mutants of PKCbeta1 or beta2 had higher cellular levels of cyclin D1 than vector control cells, whereas cells that express a dominant negative mutant had decreased levels of cyclin D1. The derivatives that stably express the constitutively activated mutants of PKCbeta1 or beta2 also displayed increased cyclin D1 promoter activity in transient transfection luciferase reporter assays, and this induction of activity requires activator protein 1. Constitutively activated PKCbeta1 and beta2 also enhanced the transcription of c-fos in transient transfection luciferase reporter assays. Thus, PKCbeta1 and beta2 may play important positive roles in the growth of at least a subset of human breast cancers. Therefore, inhibitors of these isoforms may be useful in breast cancer chemoprevention or therapy.

Topics: Breast Neoplasms; Cell Division; Cell Line; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Immunoblotting; Isoenzymes; Luciferases; Mesylates; Mutation; Protein Kinase C; Protein Kinase C beta; Proto-Oncogene Proteins c-fos; Pyrroles; Recombinant Fusion Proteins; Response Elements; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transfection

Adiponectin is an adipokine that has pleiotropic beneficial roles in systemic insulin resistance and inflammation. Several recent clinical studies suggest that low serum levels of adiponectin are associated with increased risks of breast cancer. Here, we investigated the direct effects of adiponectin on breast cancer development in vitro and in vivo. Our results showed that adiponectin significantly attenuated the proliferations of two typical human breast cancer cells, MDA-MB-231 and T47D, in a cell type-specific manner. Further analysis revealed that adiponectin could induce apoptosis and arrest the cell cycle progression at G(0)-G(1) phase in MDA-MB-231 cells. Prolonged treatment with adiponectin in this cell line blocked serum-induced phosphorylation of Akt and glycogen synthase kinase-3beta (GSK-3beta), suppressed intracellular accumulation of beta-catenin and its nuclear activities, and consequently reduced expression of cyclin D1. Adiponectin-mediated suppression of cyclin D1 expression and attenuation of cell proliferation was abrogated by the GSK-3beta inhibitor lithium chloride. These results suggest that the inhibitory role of adiponectin on MDA-MB-231 cell growth might be attributed to its suppressive effects on the GSK-3beta/beta-catenin signaling pathway. Furthermore, our in vivo study showed that both supplementation of recombinant adiponectin and adenovirus-mediated overexpression of this adipokine substantially reduced the mammary tumorigenesis of MDA-MB-231 cells in female nude mice. Taken together, these data support the role of adiponectin as a negative regulator of breast cancer development and also suggest that adiponectin might represent a novel therapeutic target for this disease.

Topics: Active Transport, Cell Nucleus; Adiponectin; Animals; Apoptosis; beta Catenin; Breast Neoplasms; Cattle; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Nucleus; Cell Proliferation; Culture Media; Cyclin D1; Female; Fetal Blood; Genetic Therapy; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Mice; Mice, Nude; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction; Xenograft Model Antitumor Assays

Despite the success of tamoxifen in treating hormone-responsive breast cancer, its use is limited by the development of resistance to the drug. Understanding the pathways involved in the growth of tamoxifen-resistant cells may lead to new ways to treat tamoxifen-resistant breast cancer. Here, we investigate the role of cyclin D1, a mediator of estrogen-dependent proliferation, in growth of tamoxifen-resistant cells using a cell culture model of acquired resistance to tamoxifen. We show that tamoxifen and 4-hydroxytamoxifen (OHT) promoted cell cycle progression of tamoxifen-resistant cells after growth-arrest mediated by the estrogen receptor down-regulator ICI 182,780. Down-regulation of cyclin D1 with small interfering RNA blocked basal cell growth of tamoxifen-resistant cells and induction of cell proliferation by OHT. In addition, pharmacologic inhibition of phosphatidylinositol 3-kinase/Akt or mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 pathways decreased basal cyclin D1 expression and impaired OHT-mediated cyclin D1 induction and cell cycle progression. These findings indicate that cyclin D1 expression is necessary for proliferation of tamoxifen-resistant cells and for tamoxifen-induced cell cycle progression. These results suggest that therapeutic strategies to block cyclin D1 expression or function may inhibit development and growth of tamoxifen-resistant tumors.

Topics: Blotting, Western; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Chromones; Cyclin D1; Drug Resistance, Neoplasm; Estrogen Antagonists; Flavonoids; Humans; Mitogen-Activated Protein Kinases; Morpholines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proto-Oncogene Proteins c-akt; RNA Interference; RNA, Small Interfering; S Phase; Signal Transduction; Tamoxifen; Time Factors

ErbB2 overexpressing breast tumors have a poor prognosis and a high risk to develop chemoresistance to therapeutic treatment. "Chemoresistance" is a response of cells to toxic stress, and, although it is a common phenomenon, it is still poorly defined. However, a detailed understanding is required to target desensitized pathways and mechanisms for successful reactivation as part of a tailored therapy. To gain insight, which malfunctions contribute to chemoresistance, two mechanisms relevant for tissue homeostasis, the regulation of the cell cycle and of apoptosis, were investigated. Maternal MCF-7- and ErbB2-overexpressing MCF-7(erbB2) breast cancer cells were long term pretreated with 2'-deoxy-5-fluorodeoxyuridine (5-FdUrd) or 1-beta-d-arabinofuranosylcytosine (AraC) and the acquisition of drug-insensitivity was analyzed. A phosphate-conjugated heterodinucleoside consisting of one 5-FdUrd- and one AraC-moiety (5-fluoro-2'-desoxyuridylyl-(3'-->5')-Arabinocytidine) was utilized as a tool to assess the type of acquired resistances. ErbB2-overexpression disrupted proper cell cycle regulation and furthermore facilitated the development of an apoptosis-refractory phenotype upon exposure to 5-FdUrd. Experiments with dimer 5-FdUrd-araC in ErbB2-overexpressing MCF-7(erbB2) cells, and also with nucleoside 5-FdUrd in maternal MCF-7 cells, evidenced that the phenotypes of resistance to cell cycle inhibition and to apoptosis induction were differently affected. The expression profile of cyclin D1 (but not that of p53, p21, or p27) correlated with the proliferative phenotypes and nuclear accumulation of apoptosis inducing factor (but not activation of caspase 7) with apoptotic phenotypes. Dimer 5-FdUrd-araC overrode acquired chemoresistances, whereas combined application of 5-FdUrd and AraC exhibited significantly less activity. Dimer 5-FdUrd-araC remained active in MCF-7 clones most likely by circumventing the prerequisite of first-step phosphorylation. The acquisition of chemoresistance encompassed the affection of apoptosis- and cell-cycle regulation to, respectively, different extents. Thus, drug-induced cell cycle arrest and apoptosis induction are independent of each other.

Topics: Antimetabolites, Antineoplastic; Apoptosis; Breast Neoplasms; Caspase 7; Cell Cycle; Cell Proliferation; Cyclin D1; Cytarabine; Dimerization; Drug Resistance, Neoplasm; Female; Floxuridine; Humans; Receptor, ErbB-2; Tumor Cells, Cultured

Tumors can become lethal when they progress from preinvasive lesions to invasive carcinomas. Here, we identify candidate tumor progression genes using gene array analysis of preinvasive and invasive tumors from mice, which were then evaluated in human cancers. Immediate early response protein IEX-1, small stress protein 1 (HSPB8), and tumor necrosis factor-associated factor-interacting protein mRNAs displayed higher expression levels in invasive lesions than in preinvasive lesions using samples obtained by laser capture microdissection (LCM) from transgenic erbB2, ras, and cyclin D1 mice. LCM-isolated tissues from patient-matched normal, ductal carcinoma in situ, and invasive ductal carcinoma revealed similar increased expression in invasive human cancers compared with preinvasive and normal samples. These genes induced anchorage independence, increased cell proliferation, and protected against apoptosis, singly or in collaboration with erbB2. Surprisingly, they were all up-regulated by 17beta-estradiol and cyclin D1, and cyclin D1 overexpression increased p300/CBP binding to their promoters, supporting the model that cyclin D1-estrogen receptor (ER) coactivator interactions may be important to its role in ER-positive breast cancer. Additionally, an irreversible dual kinase inhibitor of ErbB signaling inhibited expression of the same genes. The up-regulation of genes contributing to increased invasiveness of ER-positive cancers offers a novel explanation for the contribution of cyclin D1 to a worse prognosis in ER-positive cancers. As targets of estrogen, cyclin D1, and erbB2 signaling, these candidates offer insights into the nature of the second events involved in breast cancer progression, regulatory events contributing to invasion, and potential targets of combined inhibition of hormone and growth factor signaling pathways.

Topics: 3T3 Cells; Animals; Breast; Breast Neoplasms; Cyclin D1; Disease Progression; Estrogens; Female; Gene Expression Regulation, Neoplastic; Humans; Hyperplasia; Mice; Mice, Transgenic; Reference Values; RNA, Messenger; RNA, Neoplasm

To study the value of immunocytochemical study for cyclin D1, c-erbB-2, Ki-67, p21(CIP1/WAF1) and 34betaE12 in fine needle aspiration cytology (FNAC) diagnosis of mammary lesions.. One hundred and thirty-five cases of breast diseases, all with FNAC performed and follow-up histologic correlation available, were enrolled into the study. These included 43 cases of benign non-proliferative diseases, 45 cases of benign proliferative diseases and 47 cases of mammary carcinoma. Immunostaining for cyclin D1, c-erbB-2, Ki-67, p21(CIP1/WAF1) and 34betaE12 was carried out on FNAC smears and paraffin sections of the corresponding biopsy specimens. The statistical significance was analyzed using SPSS11.5 software.. No statistically significant difference was observed in the expression of cyclin D1, c-erbB-2, Ki-67, p21(CIP1/WAF1) and 34betaE12 within the groups of benign non-proliferative and benign proliferative breast diseases. On the other hand, a significant difference in immunostaining results was found between benign breast lesions and mammary carcinoma (P < 0.001). A panel of cyclin D1, 34betaE12 and c-erbB-2 immunostaining is highly sensitive and specific in confirming the diagnosis of mammary carcinoma in FNAC samples. A positive reaction for cyclin D1 and c-erbB-2, when coupled with a negative reaction for 34betaE12, showed to be the most reliable supportive evidence for the malignant cytologic diagnosis. When taking the results of either cyclin D1 or 34betaE12 immunostaining into consideration, the sensitivity and specificity for diagnosing carcinoma was 95.7% and 94.3% respectively. On the other hand, when any of the three immunostains suggested carcinoma, the diagnostic sensitivity and specificity became 97.9% and 92.0% respectively. If the immunostaining results of any two of the three markers suggested carcinoma, the diagnostic sensitivity and specificity became 72.3% and 100% respectively. Within the carcinoma group, the degree of expression of cyclin D1, p21(CIP1/WAF1) and 34betaE12 showed little difference amongst different cytologic grades (according to Robinson cytologic grading system). There were however differences in expression of c-erbB-2 and Ki-67. Highest expression rate was observed in grade 3 carcinoma, while lowest expression rate was observed in grade 1 carcinoma (only in 40.0% and 33.3% of cases respectively). Whenever either cyclin D1 positivity or 34betaE12 negativity was demonstrated, the diagnostic accuracy for grade 1 and grade 2 carcinoma was 93.3% and 96.2 % respectively.. Immunocytochemical study using a panel of antibodies for cyclin D1, c-erbB-2, and 34betaE12 has significant diagnostic value in distinguishing between benign breast diseases and mammary carcinoma in FNAC samples. Cyclin D1 and 34betaE12 are especially useful in confirming the cytologic diagnosis of low-grade cancer.

Topics: Biopsy, Fine-Needle; Breast; Breast Diseases; Breast Neoplasms; Carcinoma; Cyclin D1; Diagnosis, Differential; Female; Humans; Immunohistochemistry; Keratins; Receptor, ErbB-2

Amplified in breast cancer 1 (AIB1, also known as ACTR, SRC-3, RAC-3, TRAM-1, p/CIP) is a member of the p160 nuclear receptor coactivator family involved in transcriptional regulation of genes activated through steroid receptors, such as estrogen receptor alpha (ER(alpha)). The AIB1 gene and a more active N-terminally deleted isoform (AIB1-Delta3) are overexpressed in breast cancer. To determine the role of AIB1-Delta3 in breast cancer pathogenesis, we generated transgenic mice with human cytomegalovirus immediate early gene 1 (hCMVIE1) promoter-driven over-expression of human AIB1/ACTR-Delta3 (CMVAIB1/ACTR-Delta3 mice). AIB1/ACTR-Delta3 transgene mRNA expression was confirmed in CMV-AIB1/ACTR-Delta3 mammary glands by in situ hybridization. These mice demonstrated significantly increased mammary epithelial cell proliferation (P < 0.003), cyclin D1 expression (P = 0.002), IGF-I receptor protein expression (P = 0.026), mammary gland mass (P < 0.05), and altered expression of CCAAT/enhancer binding protein isoforms (P = 0.029). At 13 months of age, mammary ductal ectasia was found in CMV-AIB1/ACTR-Delta3 mice, but secondary and tertiary branching patterns were normal. There were no changes in the expression patterns of either ER(alpha) or Stat5a, a downstream mediator of prolactin signaling. Serum IGF-I levels were not altered in the transgenic mice. These data indicate that overexpression of the AIB1/ACTR-Delta3 isoform resulted in altered mammary epithelial cell growth. The observed changes in cell proliferation and gene expression are consistent with alterations in growth factor signaling that are thought to contribute to either initiation or progression of breast cancer. These results are consistent with the hypothesis that the N-terminally deleted isoform of AIB1 can play a role in breast cancer development and/or progression.

Topics: Alternative Splicing; Animals; Antigens, Viral; Blotting, Southern; Blotting, Western; Breast Neoplasms; Cell Proliferation; Cyclin D1; DNA; DNA-Binding Proteins; Enzyme-Linked Immunosorbent Assay; Epithelial Cells; Estrogen Receptor alpha; Gene Expression Regulation; Genotype; Humans; Immediate-Early Proteins; Immunoblotting; Immunohistochemistry; In Situ Hybridization; Insulin-Like Growth Factor I; Mammary Glands, Animal; Mammary Glands, Human; Mammary Neoplasms, Animal; Mice; Mice, Transgenic; Milk Proteins; Models, Genetic; Nuclear Receptor Coactivator 3; Promoter Regions, Genetic; Protein Isoforms; Receptor, IGF Type 1; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; STAT5 Transcription Factor; Trans-Activators; Transcription Factors; Transgenes; Tumor Suppressor Proteins

Estrogen and insulin/insulin-like growth factor-I (IGF-I) are major mitogens for breast epithelial cells and when co-administered, synergistically induce G(1)-S phase cell cycle progression. We investigated this cooperativity by evaluating if the key cell cycle regulators, c-Myc and cyclin D1, represent points of convergence in the action of these mitogens in MCF-7 breast cancer cells. These studies demonstrated that estrogen significantly increased both c-Myc and cyclin D1 protein, while insulin predominantly increased cyclin D1 levels. This cumulative increase in c-Myc and cyclin D1 contributes to the cooperativity of these mitogens, since ectopic expression of c-Myc or cyclin D1 cooperates with either the estrogen or insulin signaling pathways to increase cell cycle progression. Inhibition of the MAPK or PI3-kinase pathways significantly reduced c-Myc and cyclin D1 protein levels and cell cycle progression. Ectopic expression of cyclin D1 partially overcame this inhibition, while ectopic expression of c-Myc partially overcame MAPK but not PI3-kinase inhibition. Therefore, estrogen and insulin/IGF-1 differentially regulate c-Myc and cyclin D1 to cooperatively stimulate breast cancer cell proliferation.

Topics: Breast Neoplasms; Cell Cycle; Cell Proliferation; Cyclin D1; Estrogens; Female; Gene Expression Regulation, Neoplastic; Humans; Hypoglycemic Agents; Insulin; Insulin-Like Growth Factor I; Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-myc; Signal Transduction; Tumor Cells, Cultured

We have shown that exposure of the HER2/neu-overexpressing breast cancer cells to apigenin resulted in induction of apoptosis by depleting HER2/neu protein and, in turn, suppressing the signaling of the HER2/HER3-PI3K/Akt pathway. Here, we examined whether inhibition of this pathway played a role in the anti-tumor effect. The results revealed that treatment with apigenin induced apoptosis through cytochrome c release and caused a rapid induction of caspase-3 activity and stimulated proteolytic cleavage of DFF-45. Furthermore, apigenin downregulated cyclin D1, D3 and Cdk4 and increased p27 protein levels. Colony formation in the soft agar assay, a hallmark of the transformation phenotype, was preferentially suppressed in HER2/neu-overexpressing breast cancer cells in the presence of apigenin. In addition, a structure-activity relationship study indicated that (1) the position of B ring; and (2) the existence of the 3', 4'-hydroxyl group on the 2-phenyl group were important for the depletion of HER2/neu protein by flavonoids. These results provided new insights into the structure-activity relationship of flavonoids.

Topics: Apigenin; Apoptosis; Apoptosis Regulatory Proteins; Breast Neoplasms; Caspase 3; Caspases; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cyclin D1; Cyclin D3; Cyclin-Dependent Kinase Inhibitor p27; Cyclins; Cytochromes c; Flavonoids; Humans; Phosphoinositide-3 Kinase Inhibitors; Proteins; Receptor, ErbB-2; Tumor Suppressor Proteins

In light of the clinical relevance of targeting cyclin D1 in breast cancer, we have investigated the mechanism underlying the effect of the peroxisome proliferator-activated receptor-gamma (PPARgamma) agonists troglitazone and ciglitazone on cyclin D1 repression. We obtain evidence that the ability of high doses of troglitazone and ciglitazone to repress cyclin D1 is independent of PPARgamma activation. PPARgamma-inactive troglitazone and ciglitazone analogs 5-[4-(6-hydroxy-2,5,7,8-tetramethyl-chroman-2-yl-methoxy)-benzylidene]-2,4-thiazolidinedione (Delta2-TG) and 5-[4-(1-methyl-cyclohexylmethoxy)-benzylidene]-thiazolidine-2,4-dione are able to facilitate cyclin D1 ablation with potency similar to that of troglitazone and ciglitazone in MCF-7 cells. Reverse transcription-polymerase chain reaction shows that the mRNA level of cyclin D1 remains unaltered in drug-treated cells, indicating the repression is mediated at the post-transcriptional level. Moreover, the ablative effect of these agents is specific to cyclin D1, in that the expression levels of many other cyclins and cyclin-dependent kinases examined remain unchanged after drug treatment. Our data indicate that troglitazone- and Delta2-TG-induced cyclin D1 repression is mediated via proteasome-facilitated proteolysis because it is inhibited by different proteasome inhibitors, including N-carbobenzoxy-l-leucinyl-l-leucinyl-l-norleucinal (MG132), lactacystin, and epoxomicin, and is preceded by increased ubiquitination. The dissociation of these two pharmacological activities (i.e., PPARgamma activation and cyclin D1 ablation) provides a molecular basis to use Delta2-TG as a scaffold to develop a novel class of cyclin D1-ablative agents. Therefore, a series of Delta2-TG derivatives have been synthesized. Among them, 5-[4-(6-allyoxy-2,5,7,8-tetramethyl-chroman-2-yl-methoxy)-benzylidene]-2,4-thiazolidinedione represents a structurally optimized agent with potency that is an order of magnitude higher than that of Delta2-TG in cyclin D1 repression and MCF-7 cell growth inhibition.

Topics: Breast Neoplasms; Cell Line, Tumor; Chromans; Cyclin D1; Down-Regulation; Female; Humans; PPAR gamma; Proteasome Endopeptidase Complex; Thiazolidinediones; Troglitazone

Besides its function as a cell cycle regulator, cyclin D1 interacts with transcription factors to regulate gene activation. In this study, we show that cyclin D1 is recruited to the p21waf1 promoter by a STAT3-NcoA complex. The association of cyclin D1 with DNA prevented the recruitment of the CBP histone acetylase and RNA polymerase II, leading to an inhibition of the p21waf1 gene. Confirming the transcriptional function of the protein, the expression of the p21waf1 gene was enhanced in cyclin D1-/- fibroblasts or upon siRNA-mediated down-regulation of the cyclin. Moreover, the STAT3-mediated activation of p21waf1 was also inhibited in breast cancer cells containing elevated levels of cyclin D1. Altogether, these results suggest that the transcriptional activities of cyclin D1 might play an important role in the regulation of cell-cycle regulatory genes and that these functions are probably involved in cell transformation.

Topics: Animals; Breast Neoplasms; Cell Cycle Proteins; Cell Line, Tumor; CREB-Binding Protein; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; DNA; DNA-Binding Proteins; Down-Regulation; Gene Expression Regulation; Humans; Mice; Mice, Knockout; Nuclear Proteins; Promoter Regions, Genetic; Protein Binding; RNA Polymerase II; STAT3 Transcription Factor; Trans-Activators; Transcription, Genetic; Transcriptional Activation

The human growth hormone (hGH) gene is expressed in the normal human mammary epithelial cell and its expression increases concomitant with the acquisition of proliferative lesions. Herein we demonstrate that autocrine production of hGH in human mammary carcinoma cells dramatically enhances anchorage-independent growth in a Janus kinase 2-dependent manner. Forced expression of the hGH gene in immortalized human mammary epithelial cells increased proliferation, decreased apoptosis, altered the cellular morphology and resulted in oncogenic transformation. Autocrine hGH was therefore sufficient to support anchorage-independent growth of immortalized human mammary epithelial cells and tumor formation in vivo. Moreover, autocrine hGH disrupted normal mammary acinar architecture with luminal filling and deregulated proliferation in three-dimensional epithelial cell culture. Autocrine hGH utilized homeobox A1 to govern the transcriptional program required for autocrine hGH-stimulated oncogenic transformation of human mammary epithelial cells, including transcriptional up-regulation of c-Myc, cyclin D1, and Bcl-2. Forced expression of a single orthotopically expressed wild-type gene is therefore sufficient for oncogenic transformation of the immortalized human mammary epithelial cell.

Topics: Breast; Breast Neoplasms; Cell Division; Cell Line; Cell Line, Tumor; Cell Survival; Cell Transformation, Neoplastic; Cyclin D1; DNA Primers; Epithelial Cells; Female; Genes, myc; Genetic Vectors; Human Growth Hormone; Humans; Reverse Transcriptase Polymerase Chain Reaction; Transcription, Genetic; Transfection

Epidermal growth factor receptor (EGFR) has been detected in the nucleus of cancer cells and primary tumors for decades. While localized in the nucleus, EGFR functions as a transcriptional regulator resulting in the activation of the cyclin D1 gene. Despite nuclear accumulation of EGFR is linked to increased DNA synthesis and proliferative potential, the pathological significance of nuclear EGFR, however, remains uninvestigated. Furthermore, expression of EGFR has not provided a consistent predictive value for survival of breast cancer patients. Here, we analyzed 130 breast carcinomas via immunohistochemical analyses for the levels of nuclear and non-nuclear EGFR. We found 37.7% of the cohort immunostained positively for nuclear EGFR and 6.9% with high levels of expression. Importantly, Kaplan-Meier survival analysis and log-rank test revealed a significant inverse correlation between high nuclear EGFR and overall survival (P = 0.009). Expression of nuclear EGFR correlated positively with increased levels of cyclin D1 and Ki-67, both are indicators for cell proliferation. In contrast, expression of non-nuclear EGFR did not significantly correlate with those of cyclin D1 and Ki-67 or the overall survival rate. In addition, we analyzed 37 oral squamous carcinomas for EGFR expression and found 24.3% of the cases to contain moderate/high levels of nuclear EGFR. Taken together, our findings indicate pathological significance of nuclear EGFR and may have important clinical implication.

Topics: Breast Neoplasms; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Nucleus; Cyclin D1; ErbB Receptors; Female; Humans; Ki-67 Antigen; Mouth Neoplasms; Survival Analysis

Cyclin D1 (CCND1) is a key cell cycle regulatory protein that governs cell cycle progression from the G(1) to S phase. A common polymorphism (A870G) in exon 4 of the CCND1 gene produces an alternate transcript (transcript-b) that preferentially encodes a protein with enhanced cell transformation activity and possible prolonged half-life. We evaluated the association of CCND1 A870G polymorphism with breast cancer risk and survival in 1,130 breast cancer cases and 1,196 controls who participated in the Shanghai Breast Cancer Study. Approximately 81% of cases and 79% of controls carried the A allele at A870G of the CCND1 gene [odds ratio, 1.1; 95% confidence interval (95% CI), 0.9-1.4]. As lightly stronger but nonsignificant association was found for the A allele among younger women (odds ratio, 1.3; 95% CI, 0.9-1.8). However, this polymorphism seems to modify the effect of hormonal exposures on postmenopausal breast cancer, as the positive associations of postmenopausal breast cancer with body mass index (Pfor interaction = 0.02) and waist-to-hip ratios (P for interaction < 0.03; all Ps are two sided) were only observed among women who carry the A allele at A870G of the CCND1 gene. Following up with this cohort of patients for an average of 4.84 years, we found that the CCND1 A870G polymorphism was inversely associated with overall and disease-free survival, particularly among women with late stage or estrogen/progesterone receptor-negative breast cancer. The adjusted hazard ratios for disease-free survival associated with GA and AA genotypes were 0.94 (95% CI, 0.49-1.82) and 0.41 (95% CI, 0.19-0.91) for tumor-node-metastasis stage III to IV breast cancer, and 0.35 (95% CI, 0.15-0.80) and 0.32 (95% CI, 0.13-0.79) for estrogen/progesterone receptor-negative breast cancer. This study suggests that CCND1 A870G polymorphism may modify the postmenopausal breast cancer risk associated with hormonal exposure and predict survival after breast cancer diagnosis.

Topics: Adult; Alleles; Body Mass Index; Breast Neoplasms; Case-Control Studies; Chi-Square Distribution; China; Cyclin D1; Data Collection; Female; Genetics, Population; Genotype; Humans; Middle Aged; Polymorphism, Genetic; Postmenopause; Prognosis; Proportional Hazards Models; Registries; Survival Analysis

We studied the effects of 2-methoxyestradiol (2-ME2) and 16alpha-hydroxyestrone (16alpha-OHE1), two metabolites of estradiol (E2), on DNA synthesis, cell cycle progression and cyclin D1 protein levels in estrogen receptor-positive MCF-7 cells. E2 and 16alpha-OHE1 stimulated DNA synthesis, and 2-ME2 inhibited the stimulatory effects of these agents. E2 and 16alpha-OHE1 stimulated the progression of cells from G1 to S phase and this effect was attenuated by 2-ME2. Western blot analysis showed that E2 and 16alpha-OHE1 increased cyclin D1 protein level by about fourfold compared with control. 2-ME2 had no significant effect on cyclin D1; however, it prevented the accumulation of cyclin D1 in the presence of E2 and 16alpha-OHE1. Cells transfected with a cyclin D1 reporter gene and treated with E2 or 16alpha-OHE1 showed 7- and 9.5-fold increase respectively in promoter activity compared with control. This activity was significantly inhibited by 2-ME2. Cyclin D1 transactivation was mediated by the cAMP response element (CRE) region, which binds activating transcription factor 2 (ATF-2). DNA affinity assay showed 2.5- and 3.5-fold increases in ATF-2 binding to CRE in the presence of E2 and 16alpha-OHE1 respectively. The binding of ATF-2 was inhibited by the presence of 2-ME2. These results show that 2-ME2 can downregulate cyclin D1 and thereby cell cycle progression by a mechanism involving the disruption of ATF-2 binding to cyclin D1 promoter.

Topics: 2-Methoxyestradiol; Activating Transcription Factor 2; Blotting, Western; Breast Neoplasms; Cyclic AMP Response Element-Binding Protein; Cyclin D1; DNA Replication; Enzyme Activation; Estradiol; Female; Gene Expression Regulation, Neoplastic; Humans; Hydroxyestrones; p38 Mitogen-Activated Protein Kinases; Transcription Factors

Mayven is a member of the kelch-related superfamily of proteins, characterized by a series of 'kelch' repeats at their carboxyl terminus and a BTB/POZ domain at their NH2-terminus. Little is known about the role of Mayven in cancer. Here, we report that Mayven expression was abundant and diffuse in primary human epithelial breast tumor cells as compared to normal breast epithelial cells, where Mayven was detected in the normal breast layer of the mammary ducts. Overexpression of Mayven resulted in an induction of c-Jun protein levels, as well as increased AP-1 (activating protein 1) transcriptional activity in MCF-7 and T47D breast cancer cells through its BTB/POZ domain. Furthermore, Mayven activated c-Jun N-terminal kinase in breast cancer cells. Mayven, through its BTB/POZ domain, induced cyclin D1 expression and cyclin D1 promoter activity and promoted cell cycle progression from the G1 to S phase. MCF-7 cells transduced with the recombinant retroviral sense Mayven (pMIG-W-Mayven) showed significant induction of c-Jun and cyclin D1 mRNA expression and activities as compared to the retroviral vector alone, while MCF-7 cells transduced by the recombinant retroviral antisense Mayven (pMIG-W-Mayven-AS) demonstrated a significant decrease in c-Jun and cyclin D1 expression and activities. Given the crucial functions of cyclin D1 and AP-1 signaling in oncogenesis, our results strongly suggest that overexpression of Mayven may promote tumor growth through c-Jun and cyclin D1.

Topics: Breast Neoplasms; Cell Line, Tumor; Cyclin D1; G1 Phase; Humans; Immunohistochemistry; Microfilament Proteins; Microscopy, Fluorescence; Nerve Tissue Proteins; Proto-Oncogene Proteins c-jun; S Phase

Curcumin (diferuloylmethane) is known to induce apoptosis in tumor cells. In asynchronous cultures, with time-lapse video-micrography in combination with quantitative fluorescence microscopy, we have demonstrated that curcumin induces apoptosis at G(2) phase of cell cycle in deregulated cyclin D1-expressed mammary epithelial carcinoma cells, leaving its normal counterpart unaffected. In our search toward delineating the molecular mechanisms behind such differential activities of curcumin, we found that it selectively increases p53 expression at G(2) phase of carcinoma cells and releases cytochrome c from mitochondria, which is an essential requirement for apoptosis. Further experiments using p53-null as well as dominant-negative and wild-type p53-transfected cells have established that curcumin induces apoptosis in carcinoma cells via a p53-dependent pathway. On the other hand, curcumin reversibly inhibits normal mammary epithelial cell cycle progression by down-regulating cyclin D1 expression and blocking its association with Cdk4/Cdk6 as well as by inhibiting phosphorylation and inactivation of retinoblastoma protein. In addition, curcumin significantly up-regulates cell cycle inhibitory protein (p21Waf-1) in normal cells and arrests them in G(0) phase of cell cycle. Therefore, these cells escape from curcumin-induced apoptosis at G(2) phase. Interestingly, these processes remain unaffected by curcumin in carcinoma cells where cyclin D1 expression is high. Similarly, in ectopically overexpressed system, curcumin cannot down-regulate cyclin D1 and thus block cell cycle progression. Hence, these cells progress into G(2) phase and undergo apoptosis. These observations together suggest that curcumin may have a possible therapeutic potential in breast cancer patients.

Topics: Animals; Antineoplastic Agents; Apoptosis; Breast; Breast Neoplasms; Cell Cycle Proteins; Cell Line; Cell Line, Tumor; Curcumin; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Epithelial Cells; Female; G2 Phase; Humans; Mice; Mice, Knockout; Microscopy, Fluorescence; Microscopy, Video; NIH 3T3 Cells; Tumor Suppressor Protein p53

Estrogen receptors (ER) are ligand-dependent transcription factors that regulate growth, differentiation, and maintenance of cellular functions in a wide variety of tissues. We report here that p21WAF1/CIP1, a cyclin-dependent kinase (Cdk) inhibitor, cooperates with CBP to regulate the ERalpha-mediated transcription of endogenous target genes in a promoter-specific manner. The estrogen-induced expression of the progesterone receptor and WISP-2 mRNA transcripts in MCF-7 cells was enhanced by p21WAF1/CIP1, whereas that of the cyclin D1 mRNA was reduced and the pS2 mRNA was not affected. Chromatin immunoprecipitation assays revealed that p21WAF1/CIP1 was recruited simultaneously with ERalpha and CBP to the endogenous progesterone receptor gene promoter in an estrogen-dependent manner. Experiments in which the p21WAF1/CIP1 protein was knocked down by RNA interference showed that the induction of the expression of the gene encoding the progesterone receptor required p21WAF1/CIP1, in contrast with that of the cyclin D1 and pS2 genes. p21WAF1/CIP1 induced not only cell cycle arrest in breast cancer cells but also milk fat globule protein and lipid droplets, indicators of the differentiated phenotype, as well as cell flattening and increase of the volume of the cytoplasm. These results indicate that p21WAF1/CIP1, in addition to its Cdk-regulatory role, behaves as a transcriptional coactivator in a gene-specific manner implicated in cell differentiation.

Topics: Breast Neoplasms; Cell Cycle; Cell Cycle Proteins; Cell Differentiation; Chromatin Immunoprecipitation; CREB-Binding Protein; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cytoplasm; Estradiol; Estrogen Receptor alpha; Female; Gene Expression Regulation; Humans; Nuclear Proteins; Promoter Regions, Genetic; Proteins; RNA Interference; RNA, Messenger; Trans-Activators; Transcription, Genetic; Transcriptional Activation; Trefoil Factor-1; Tumor Cells, Cultured; Tumor Suppressor Proteins

The mammalian target of rapamycin is a serine-threonine kinase that regulates cell cycle progression. Rapamycin and its analogues inhibit the mammalian target of rapamycin and are being actively investigated in clinical trials as novel targeted anticancer agents. Although cyclin D1 is down-regulated by rapamycin, the role of this down-regulation in rapamycin-mediated growth inhibition and the mechanism of cyclin D1 down-regulation are not well understood. Here, we show that overexpression of cyclin D1 partially overcomes rapamycin-induced cell cycle arrest and inhibition of anchorage-dependent growth in breast cancer cells. Rapamycin not only decreases endogenous cyclin D1 levels but also decreases the expression of transfected cyclin D1, suggesting that this is at least in part caused by accelerated proteolysis. Indeed, rapamycin decreases the half-life of cyclin D1 protein, and the rapamycin-induced decrease in cyclin D1 levels is partially abrogated by proteasome inhibitor N-acetyl-leucyl-leucyl-norleucinal. Rapamycin treatment leads to an increase in the kinase activity of glycogen synthase kinase 3beta (GSK3beta), a known regulator of cyclin D1 proteolysis. Rapamycin-induced down-regulation of cyclin D1 is inhibited by the GSK3beta inhibitors lithium chloride, SB216763, and SB415286. Rapamycin-induced G1 arrest is abrogated by nonspecific GSK3beta inhibitor lithium chloride but not by selective inhibitor SB216763, suggesting that GSK3beta is not essential for rapamycin-mediated G1 arrest. However, rapamycin inhibits cell growth significantly more in GSK3beta wild-type cells than in GSK3beta-null cells, suggesting that GSK3beta enhances rapamycin-mediated growth inhibition. In addition, rapamycin enhances paclitaxel-induced apoptosis through the mitochondrial death pathway; this is inhibited by selective GSK3beta inhibitors SB216763 and SB415286. Furthermore, rapamycin significantly enhances paclitaxel-induced cytotoxicity in GSK3beta wild-type but not in GSK3beta-null cells, suggesting a critical role for GSK3beta in rapamycin-mediated paclitaxel-sensitization. Taken together, these results show that GSK3beta plays an important role in rapamycin-mediated cell cycle regulation and chemosensitivity and thus significantly potentiates the antitumor effects of rapamycin.

Topics: Aminophenols; Antibiotics, Antineoplastic; Antimanic Agents; Apoptosis; Breast Neoplasms; Cell Cycle; Cyclin D1; Cysteine Proteinase Inhibitors; Down-Regulation; Drug Resistance, Neoplasm; Female; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Half-Life; Humans; Indoles; Leupeptins; Lithium Chloride; Maleimides; Mitochondria; NF-kappa B; Paclitaxel; Proteasome Inhibitors; Sirolimus

BCL-2 overexpression occurs in many cancer types and is associated with chemoresistance and radioresistance. The mechanisms responsible for its aberrant expression are thought to be transcriptionally mediated but remain unclear. We examined the cell type-specific mechanism of BCL-2 gene transcription in various solid organ malignancies.. Regulation of BCL-2 gene transcription was examined in seven different human cancer cell lines including two pancreatic (MIA-PaCa-2, PANC-1), two prostate (LNCaP, PC-3), two lung (Calu-1, A549) and one breast (MCF-7) cancer cell line. Cells were treated with inhibitors of phosphatidylinositol-3 kinase (PI3K), MEK/ERK, and p38MAPK. The effect of mutation of a NF-kappaB site in the BCL-2 promoter was determined, as was the effect of inhibition of NF-kappaB function using a 26S proteasome inhibitor (bortezomib) on both BCL-2 transcription and induction of apoptosis.. BCL-2 expression varied both between and within tumor types; four of seven cell lines demonstrated high BCL-2 levels (MIA-PaCa-2, PC-3, Calu-1 and MCF-7). No signaling pathway was uniformly responsible for overexpression of BCL-2; however, mutation of the NF-kappaB site decreased BCL-2 promoter activity in all cell lines. Inhibition of NF-kappaB activity decreased BCL-2 protein levels independently of the signaling pathway involved in transcriptional activation of the BCL-2 gene.. Diverse signaling pathways variably regulate BCL-2 gene expression in a cell type-specific fashion. Therapy to decrease BCL-2 levels in various human cancers would be more broadly applicable if targeted to transcriptional activation rather than signal transduction cascades. Finally, the apoptotic efficacy of proteasome inhibition with bortezomib paralleled the ability to inhibit NF-kappaB activity and decrease BCL-2 levels.

Topics: Boronic Acids; Bortezomib; Breast Neoplasms; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Male; NF-kappa B; Pancreatic Neoplasms; Prostatic Neoplasms; Protease Inhibitors; Pyrazines; Signal Transduction; Transcription, Genetic; Tumor Cells, Cultured

Recent data have suggested considerable molecular differences in cancers from various ethnical groups. As molecular features are increasingly used for predicting cancer prognosis and response to therapy, better knowledge of ethnic molecular features is important. To identify potential molecular differences between breast cancers in Europe and the Middle East, we analyzed consecutive breast cancer series from Switzerland (n=2197) and Saudi Arabia (n=204). Tissue microarrays were analyzed by fluorescence in situ hybridization for HER2, CCND1, MYC, and EGFR amplification. The data revealed marked differences between Saudi and Swiss patients. Saudi breast cancers had a markedly higher frequency of HER2 (31 vs 17%; P<0.0001) and MYC (16 vs 5%; P<0.0001) amplifications than Swiss breast cancers. Remarkably, this was partly due to a much higher incidence of grade 3 cancers in the Saudi than in the Swiss population (65 vs 32%; P<0.0001). However, differences in amplification frequency hold also true within grade 3 cancers (HER2: 40 vs 30%, P<0.05; MYC: 22 vs 11%, P=0.002). Interestingly, in combination with known age standardized incidence rates of breast cancer in Saudi Arabia (21.6/100 000) and Switzerland (70.1/100 000), these data suggest that the incidence of high-grade breast cancer is comparable for Saudi and Swiss women, while the incidence of low-grade breast cancers is about 14 times lower in Saudi than for Swiss women. These observations suggest that a difference in genetic susceptibility and/or lifestyle between Saudi and Swiss women has a substantial and much higher than expected impact on the risk of low-grade breast cancer.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Breast Neoplasms; Cyclin D1; ErbB Receptors; Female; Gene Amplification; Genetic Predisposition to Disease; Humans; In Situ Hybridization, Fluorescence; Middle Aged; Neoplasm Staging; Proto-Oncogene Proteins c-myc; Receptor, ErbB-2; Saudi Arabia; Switzerland; Tissue Array Analysis

To study the function and prognostic value of the recently identified tumor suppressor gene LKB1 in human breast cancer.. Estrogen receptor-positive human breast cancer cells of MCF-7 line and estrogen receptor-negative human breast cancer cells of the lines MDA-MB-435 and MDA-MB-231 were cultured and tested for the LKB1 expression. Plasmid pcDNA4 TOPO HisMAX/LKB1 expressing LKB1 was constructed and transfected into the MDA-MB-435 cells. PCR and Western blotting were used to detect the expressions of LKB1 mRNA and protein. Flow cytometry was used to determine the cell cycle. PCR and Western blotting were used to detect the expressions of LKB1 mRNA and protein in 121 human breast cancer samples. The association with relapse free survival (RFS) and overall survival (OS) was assessed by univariate analysis (log-rank test and Kaplan-Meier method).. Human breast cells of MCF-7 line expressed LKB1 and the cells of MDA-MB-435 and MDA-MB-231 lines did not. Flow cytometry showed that 40.9% of the MDA-MB-435 transfected with pcDNA4 TOPO HisMAX/LKB1 was at the G(1) stage, a proportion significantly higher than that of the control (18.3%). The expressions of cyclin D1 and cyclin E were significantly down-regulated and the expression of p21(WAF1/CIP1) was significantly up-regualed. LKB1 was lowly expressed in 43 out of the 121 cases of breast cancer (35.4%) and highly expressed in 78 cases (64.6%). Low expression of LKB1 was correlated with pathological grade (P = 0.013), cancer size (P = 0.001), status of estrogen receptor (P = 0.048), and status of lymph nodes metastasis (P = 0.003). Univariate analysis showed that low expression of LKB1 was significantly correlated with shorter RFS (P = 0.048) and low OS (P = 0.003).. Reintroducing LKB1 into breast cell of the lines lacking LKB1 expression restores LKB1 activity and induces growth suppression by G(1) cell cycle block. The LKB1 mediated G(1) cell cycle arrest is caused by up-regulation of the expression of p21(WAF1/CIP1) in MDA-MB-435 breast cancer cells.

Topics: AMP-Activated Protein Kinase Kinases; Biomarkers, Tumor; Breast Neoplasms; Cell Line, Tumor; Cyclin D1; Cyclin E; Female; Humans; Prognosis; Protein Serine-Threonine Kinases; RNA, Messenger; Transfection

The S14 (spot 14) gene encodes a protein that is predominantly expressed in lipogenic tissues, such as the liver, white and brown adipose tissues and the lactating mammary glands. Accumulated evidence suggests that S14 could play an important role in the induction of lipogenic enzymes. In humans, the S14 locus resides in the chromosome region 11q13, which is frequently amplified in breast tumours, and as a result, it has been suggested that this protein could play a role in the metabolism and growth of these kinds of tumours. In the present study, we have examined the effects of S14 overexpression in MCF-7 human breast cancer cells. We found that S14 causes (i) an inhibition of cell proliferation and of anchorage-independent growth, (ii) a marked reduction in the number of viable cells and (iii) the induction of differentiation and cell death of these cells. The inhibition of cell growth was associated with a decrease in the expression of cyclin D1 and a reduction of cyclin D1 promoter activity. Increased expression of S14 also caused the accumulation of cytochrome c in the cytosol and loss of mitochondrial membrane potential. These findings suggest that S14 may function as an important modulator of tumorigenesis in human breast by decreasing cell growth and inducing cell death and differentiation.

Topics: Breast Neoplasms; Cell Death; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cholecalciferol; Cyclin D1; Gene Expression; Humans; Nuclear Proteins; Promoter Regions, Genetic; Prostaglandin D2; Proteins; Time Factors; Transcription Factors; Tretinoin

Cyclin D1 (CCND1), an intracellular cell-cycle regulatory protein with checkpoint function, can promote cell proliferation or induce growth arrest and apoptosis depending on the cellular context. We hypothesized that the direction of the association between the (CCND1) G870A-polymorphism and breast cancer risk may be modified by dietary and genetic factors influencing the oxidant-antioxidant balance, such as a dietary pattern with a high intake of n-6 fatty acids and a low intake of n-3 fatty acids, or a genetic profile that is deficient in glutathione S-transferases. We tested our hypothesis in a case-control study nested into the Singapore Chinese Health Study, a prospective investigation of diet and cancer in 63,000 Chinese men and women. Genomic DNA collected from 258 incident cases of breast cancer and 670 female cohort controls was examined for CCND1, GSTM1, GSTT1 and GSTP1 genes using fluorogenic 5'-nuclease assay. Unconditional logistic regression models were used to assess the effects with adjustment for potential confounders. All statistical tests were two-sided. The heterozygous CCND1 GA genotype significantly reduced the breast cancer risk in all subjects (OR=0.67, 95% CI 0.45-0.99) when compared with the GG genotype. The association was restricted to women with a high (above median value) intake level of n-6 fatty acids (OR=0.51, 95% CI 0.30-0.87), a low (below median value) intake level of the antagonistic marine n-3 fatty acids (OR=0.54, 95% CI 0.32-0.93) or a total lack of the antioxidative GSTM1 (OR=0.44, 95% CI 0.25-0.80) or GSTT1 genes (OR=0.46, 95% CI 0.24-0.87). The effects were consistently stronger in cases with advanced disease. The AA genotype did not affect breast cancer risk. The results of this study are compatible with the hypothesis that the oxidant-antioxidant balance in cells is an important determinant of the direction of the cyclin D1 effect, leading either to cell proliferation or cell death.

Topics: Adult; Breast Neoplasms; Case-Control Studies; China; Cyclin D1; Female; Genotype; Glutathione Transferase; Humans; Menopause; Middle Aged; Oxidative Stress; Parity; Polymorphism, Single Nucleotide; Reference Values; Risk Factors; Singapore; Socioeconomic Factors

To explore the potential prognostic value of cyclin D1 in invasive breast cancer and its correlation with basic histopathological parameters, hormonal status (estrogen [ER] and progesterone receptor [PR]), and bcl-2.. Medical records of 48 patients, diagnosed in 1998, from the Central Database of the Institute of Oncology, Clinical Center University of Sarajevo, were analyzed. The mean follow-up was 61 months (range: 4-103 months). Routine histopathological evaluation was performed for 48 formalin-fixed and paraffin-embedded tissue samples. For immunohistochemical staining, we used monoclonal antibodies for ER, PR, bcl-2, and cyclin D1.. Cyclin D1 expression inversely correlated with tumor grade (P=0.010) and tumor size (P=0.023), whereas significant positive association was found with ER (P=0.001) and bcl-2 (P=0.001) expression. Patients with higher cyclin D1 expression had longer both overall survival (P=0.014) and relapse-free survival (P=0.037). Cox regression analysis for overall survival (OS) showed that lymph node status, ER expression, therapy, and cyclin D1 expression were independent prognostic factors. (P range from 0.003 to 0.04).. Expression of cyclin D1 is associated with better disease outcome in breast cancer.

Topics: Adult; Aged; Aged, 80 and over; Breast Neoplasms; Carcinoma, Ductal, Breast; Cyclin D1; Female; Follow-Up Studies; Humans; Immunohistochemistry; Middle Aged; Prognosis; Proto-Oncogene Proteins c-bcl-2; Receptors, Estrogen

beta2-Chimerin is a member of the "non-protein kinase C" intracellular receptors for the second messenger diacylglycerol and the phorbol esters that is yet poorly characterized, particularly in the context of signaling pathways involved in proliferation and cancer progression. beta2-Chimerin possesses a C-terminal Rac-GAP (GTPase-activating protein) domain that accelerates the hydrolysis of GTP from the Rac GTPase, leading to its inactivation. We found that beta2-chimerin messenger levels are significantly down-regulated in human breast cancer cell lines as well as in breast tumors. Adenoviral delivery of beta2-chimerin into MCF-7 breast cancer cells leads to inhibition of proliferation and G(1) cell cycle arrest. Mechanistic studies show that the effect involves the reduction in Rac-GTP levels, cyclin D1 expression, and retinoblastoma dephosphorylation. Studies using the mutated forms of beta2-chimerin revealed that these effects were entirely dependent on its C-terminal GAP domain and Rac-GAP activity. Moreover, MCF-7 cells stably expressing active Rac (V12Rac1) but not RhoA (V14RhoA) were insensitive to beta2-chimerin-induced inhibition of proliferation and cell cycle progression. The modulation of G(1)/S progression by beta2-chimerin not only implies an essential role for Rac in breast cancer cell proliferation but also raises the intriguing possibility that diacylglycerol-regulated non-protein kinase C pathways can negatively impact proliferation mechanisms controlled by Rho GTPases.

Topics: Adenoviridae; Blotting, Western; Breast Neoplasms; Cell Cycle; Cell Line; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Diglycerides; G1 Phase; Guanosine Triphosphate; Humans; Mutation; Neoplasm Proteins; Phosphorylation; Protein Binding; Protein Kinase C; Protein Structure, Tertiary; rac GTP-Binding Proteins; Retinoblastoma Protein; RNA, Messenger; Signal Transduction; Time Factors

Cyclin D1 plays an important role in the regulation of the G1 phase in the cell cycle. In mammary epithelial cells the expression of cyclin D1 is regulated through the oestrogen receptor and via ErbB2 signalling. Here we investigated the prognostic significance of cyclin D1 among 230 breast cancer patients randomised for tamoxifen, CMF chemotherapy and radiotherapy. The importance of combined cyclin D1 and ErbB2 overexpression was also analysed. Immunohistochemical analysis of the cyclin D1 expression resulted in 69 (29.8%) weakly positive, 107 (46.5%) moderately positive and 54 (23.7%) strongly positive cases. The prognostic importance of ErbB2 was significantly greater for patients whose tumours overexpressed cyclin D1 than for other patients (p = 0.026). In the former group, ErbB2 overexpression was strongly associated with increased risk of recurrence (RR = 4.7; 95% CI, 2.1-10.4) and breast cancer death (RR = 5.4; 95% CI, 2.3-12.6). This result is in accordance with experimental studies demonstrating a link between cyclin D1 and ErbB2 in oncogenesis. Among oestrogen receptor positive patients, those with moderate cyclin D1 expression significantly did benefit from tamoxifen treatment (RR = 0.42; 95% CI, 0.21-0.82) whereas those with weak or strong expression did not. Therefore cyclin D1 might be a predictive marker for tamoxifen resistance.

Topics: Antineoplastic Agents, Hormonal; Biomarkers; Breast Neoplasms; Cyclin D1; Disease-Free Survival; Drug Resistance, Neoplasm; Female; Genes, erbB-2; Humans; Neoplasm Recurrence, Local; Prognosis; Proportional Hazards Models; Survival Rate; Sweden; Tamoxifen

To investigate the expression of cyclin D1, ER, and PR gene proteins and to analyze their relevance to tumor biological characteristics, chemotherapy effects, diseases free survival (DFS) and overall survival (OS) of patients.. Immunohistochemical staining techniques was used to detect the expression of cyclin D1, ER and PR gene protein in 100 samples of breast infiltrating ductal carcinoma patients, all female, aged 49.49 +/- 10.81 (28 approximately 92).. The positive expression rate of gene protein was 60& for ER 58% for PR, and 55% for cyclin D1 ER, PR presented a negative correlation to SBR grading. Patients with cyclin D1 positive tumor had longer OS than those with cyclin D1 negative tumor (P = 0.0053). Coexpression of cyclin D1 and ER was significantly correlated with longer DFS and OS (P(dfs) = 0.0108, P(os) = 0.0030). The positivity of ER and PR was significantly correlated with longer DFS (P(ER) = 0.0322, P(PR) = 0.0129). For those patients receiving postoperative adjuvant chemotherapy, the expression of ER and PR were correlated with a better prognosis and longer DFS. The patients with cyclin D1 negative tumor, who received CAF, had a mean DFS of 51.6 months and a mean OS of 57 months in comparison with 24.8 months and 31.2 months for those patients who received other chemotherapy.. In breast infiltrating ductal carcinoma patients expression of ER and of PR are correlated with longer DFS, cyclin D1 expression is correlated with longer OS. For the patients receiving postoperative adjuvant chemotherapy, the expression of ER and PR correlated to a better prognosis and longer DFS. The patients with cyclin D1 negative tumor who receive CAF chemotherapy have longer DFS and OS than those receiving other chemotherapy.

Topics: Adult; Aged; Aged, 80 and over; Breast Neoplasms; Carcinoma, Ductal, Breast; Cyclin D1; Female; Humans; Middle Aged; Receptors, Estrogen; Receptors, Progesterone

The cyclin-dependent kinase (CDK) inhibitor p27(Kip1) (p27) is an important regulator of cell cycle progression controlling the transition from G to S-phase. Low p27 levels or accelerated p27 degradation correlate with excessive cell proliferation and poor prognosis in several forms of cancer. Phosphorylation of p27 at Thr187 by cyclin E-CDK2 is required to initiate the ubiquitination-proteasomal degradation of p27. Protecting p27 from ubiquitin-mediated proteasomal degradation may increase its potential in cancer gene therapy. Here we constructed a non-phosphorylatable, proteolysis-resistant p27 mutant containing a Thr187-to-Ala substitution (T187A) which is not degraded by ubiquitin-mediated proteasome pathway, and compared its effects on cell growth, cell-cycle control, and apoptosis with those of wild-type p27. In muristerone A-inducible cell lines overexpressing wild-type or mutant p27, the p27 mutant was more resistant to proteolysis in vivo and more potent in inducing cell-cycle arrest and other growth-inhibitory effects such as apoptosis. Transduction of p27(T187A) in breast cancer cells with a doxycycline-regulated adenovirus led to greater inhibition of proliferation, more extensive apoptosis, with a markedly reduced protein levels of cyclin E and increased accumulation of cyclin D1, compared with wild-type p27. These findings support the potential effectiveness of a degradation-resistant form of p27 in breast cancer gene therapy.

Topics: Adenoviridae; Alanine; Annexin A5; Apoptosis; Biomarkers, Tumor; Breast Neoplasms; CDC2-CDC28 Kinases; Cell Cycle; Cell Cycle Proteins; Cell Line; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p27; Doxycycline; Ecdysterone; Humans; Immunoblotting; Mutagenesis, Site-Directed; Mutation; Phosphorylation; Prognosis; Proteasome Endopeptidase Complex; Proteins; S Phase; Threonine; Time Factors; Tumor Suppressor Proteins; Ubiquitin

Topics: Adult; Aged; Aged, 80 and over; Breast Neoplasms; Cyclin D1; Female; Humans; Logistic Models; Middle Aged; Retrospective Studies

Estradiol (E2) and the naturally occurring polyamines (putrescine, spermidine, and spermine) play important roles in breast cancer cell growth and differentiation. We examined the effects of E2 and spermine on the phosphorylation and DNA binding of activating transcription factor-2 (ATF-2) in MCF-7 breast cancer cells. ATF-2 is a transcription factor involved in estrogenic regulation of cyclin D1 gene, and thereby cell cycle progression. DNA affinity immunoblot assays showed a six- to eightfold increase in the binding of ATF-2 to a 74-mer ATF/CRE oligonucleotide (ODN1) from cyclin D1 promoter in the presence of 4 nM E2 and 0.5 mM spermine, compared to untreated control. Individual treatments with E2 or spermine caused a twofold or lower increase in ATF-2 binding to ODN1. Immunoblotting with phospho-ATF-2 antibody showed that increased DNA binding of ATF-2 was associated with its phosphorylation. A p38 MAP kinase inhibitor, PD169316, inhibited ATF-2 phosphorylation. In contrast, the MEK-ERK1/2 inhibitor, PD98059, or the JNK inhibitor, SP600125, had no significant effect on DNA binding of ATF-2. Cyclin D1 promoter (-1745CD1) activity increased by approximately 12-fold (above control) in the presence of E2 and spermine, compared to a sixfold increase in the presence of E2 alone and a twofold increase with spermine. Cells transfected with a dominant negative mutant of ATF-2 showed decreased transactivation of cyclin D1 promoter in response to E2 and spermine. These results indicate that spermine can enhance E2-induced cell signaling and cyclin D1 transcription by activation of the p38 MAP kinase and phosphorylation of ATF-2, contributing to breast cancer cell proliferation.

Topics: Activating Transcription Factor 2; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Chromatography, High Pressure Liquid; Cyclic AMP Response Element-Binding Protein; Cyclin D1; Drug Synergism; Estradiol; Humans; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Promoter Regions, Genetic; Spermine; Transcription Factors; Transcriptional Activation

The cyclin D1 gene is frequently overexpressed in human breast cancer and is capable of inducing mammary tumorigenesis when overexpressed in transgenic mice. The BRCA1 breast tumor susceptibility gene product inhibits breast cancer cellular growth and the activity of several transcription factors. Herein, cyclin D1 antagonized BRCA1-mediated repression of estrogen receptor alpha (ERalpha)-dependent gene expression. Cyclin D1 repression of BRCA1 function was mediated independently of its cyclin-dependent kinase, retinoblastoma protein, or p160 (SRC-1) functions in human breast and prostate cancer cells. In vitro, cyclin D1 competed with BRCA1 for ERalpha binding. Cyclin D1 and BRCA1 were both capable of binding ERalpha in a common region of the ERalpha hinge domain. A novel domain of cyclin D1, predicted to form a helix-loop-helix structure, was required for binding to ERalpha and for rescue of BRCA1-mediated ERalpha transcriptional repression. In chromatin immunoprecipitation assays, 17beta-estradiol (E2) enhanced ERalpha and cyclin D1 recruitment to an estrogen response element (ERE). Cyclin D1 expression enhanced ERalpha recruitment to an ERE. E2 reduced BRCA1 recruitment and BRCA1 expression inhibited E2-induced ERalpha recruitment at 12 hours. Cyclin D1 expression antagonized BRCA1 inhibition of ERalpha recruitment to an ERE, providing a mechanism by which cyclin D1 antagonizes BRCA1 function at an ERE. As cyclin D1 abundance is regulated by oncogenic and mitogenic signals, the antagonism of the BRCA1-mediated ERalpha repression by cyclin D1 may contribute to the selective induction of BRCA1-regulated target genes.

Topics: Binding, Competitive; Breast Neoplasms; Carrier Proteins; Cyclin D1; Estradiol; Estrogen Receptor alpha; Female; Genes, BRCA1; Humans; Male; Membrane Proteins; Presenilin-2; Promoter Regions, Genetic; Prostatic Neoplasms; Protein Structure, Tertiary; Response Elements; Transcriptional Activation; Transfection; Ubiquitin-Protein Ligases

FIP200 is a novel protein inhibitor for focal adhesion kinase (FAK), which binds to FAK directly and inhibits its kinase activity and associated cellular functions, such as cell adhesion, spreading, and motility in fibroblasts. Here we show that FIP200 inhibits G1-S phase progression, proliferation, and clonogenic survival in human breast cancer cells. Consistent with the G1 arrest induced by FIP200, we found that FIP200 increased p21 and decreased cyclin D1 protein levels in breast cancer cells. In addition, FIP200 significantly induced p21 promoter activity in MCF-7 cells and this response was abolished upon deletion of p53 binding sites within p21 promoter. Furthermore, we found that FIP200 could interact with exogenous and endogenous p53 protein and significantly increase its half-life compared with the control cells. We also found that the NH2-terminal 154 residues of FIP200 were sufficient to mediate p53 interaction and G1 arrest in cells. The increase in p53 half-life correlated with the increased phosphorylation at Ser15 and decreased proteasomal degradation via ubiquitin and Hdm2-independent mechanism. Stabilization of p53 by FIP200 could be partially reversed by NQO1 inhibitor, dicoumarol. In contrast to p53, FIP200 decreased cyclin D1 protein half-life by promoting proteasome-dependent degradation of cyclin D1. In summary, our results suggest that FIP200 increases p21 protein levels via stabilization of its upstream regulator p53 and decreases cyclin D1 protein by promoting its degradation. Both effects are critical for FIP200-induced G1 arrest and may contribute to the putative antitumor activities of FIP200 in breast cancer.

Topics: Autophagy-Related Proteins; Breast Neoplasms; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; G1 Phase; Humans; Phosphorylation; Proteasome Endopeptidase Complex; Protein-Tyrosine Kinases; Transfection; Tumor Suppressor Protein p53; Up-Regulation

The mechanism of medroxyprogesterone acetate (MPA)-induced cell proliferation in human breast cancer cells remains elusive. We examined the mechanism by which MPA affects the cyclin D1 expression in progesterone receptor (PR)-positive T47D human breast cancer cells. MPA (10 nM) treatment for 48 h induced proliferation of the cells (1.6-fold induction). MPA induced cyclin D1 expression (3.3-fold induction), and RU486, a selective PR antagonist, blocked the MPA-induced cell proliferation and cyclin D1 expression (23% inhibition). MPA increased both the protein level (2.2-fold induction) and promoter activity (2.7-fold induction) of cyclin D1 in MCF-7 cells transfected with PRB but not with PRA. Although MPA transcriptionally activated cyclin D1 expression, cyclin D1 promoter does not have progesterone-responsive element-related sequence. We further examined the mechanism for the regulation of the cyclin D1 expression. Because the cyclin D1 promoter contains three putative nuclear factor-kappaB (NFkappaB)-binding motifs and NFkappaB is a substrate of Akt, we investigated the effect of the phosphatidylinositol 3-kinase (PI3K)/Akt/NFkappaB cascade on the responses of cyclin D1 to MPA. MPA induced the transient phosphorylation of Akt (2.7-fold induction at 5 min), and treatment with PI3K inhibitor (wortmannin) attenuated the MPA-induced up-regulation of cyclin D1 expression (40% inhibition) and cell proliferation (40% inhibition). MPA also induced phosphorylation of inhibitor of NFkappaBalpha (IkappaBalpha) (2.3-fold induction), and treatment with wortmannin attenuated the MPA-induced IkappaBalpha phosphorylation (60% inhibition). Treatment with an IkappaBalpha phosphorylation inhibitor (BAY 11-7085) or a specific NFkappaB nuclear translocation inhibitor (SN-50) attenuated the MPA-induced up-regulation of both cyclin D1 expression (80 and 50% inhibition, respectively) and cell proliferation (55 and 34% inhibition, respectively). Because MPA induced a transient phosphorylation of Akt and the cyclin D1 promoter contains no progesterone-responsive element-related sequence, the MPA-induced cell proliferation through PRB by up-regulation of cyclin D1 expression via the PI3K/Akt/NFkappaB cascade may be a nongenomic mechanism.

Topics: Antineoplastic Agents, Hormonal; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Humans; Medroxyprogesterone Acetate; NF-kappa B; Phosphatidylinositol 3-Kinases; Phosphorylation; Up-Regulation

Targeting the mitogen-activated protein kinases (MAPKs) has been suggested as a novel strategy to treat cancer. Chlorophyllin (CHL) is the sodium-copper salt of chlorophyll derivative and is a commonly used food dye for green coloration; CHL was found previously to retard growth of the human breast carcinoma MCF-7 cells. Extracellular signal-regulated kinases (ERKs) constitute a subfamily of MAPKs, participating in cell survival, proliferation and differentiation. We report here the first evidence that CHL deactivates ERKs to inhibit the breast cancer cell proliferation. The results from flow cytometry showed that 200 microg/ml CHL reduced the phosphorylated and activated ERK-positive cells in different cell cycle phases from the control of >96 to <38% at 24 h of incubation; the ERK deactivations occurred in both dose- and time-dependent manner, so that nearly all ERKs were de-activated by 400 microg/ml CHL at 72 h of treatment. Immunoblot studies, however, illustrated that the levels of total ERKs were not significantly affected by the CHL treatments, suggesting that the phytochemical retards the enzyme activation rather than its expression. Cyclin D1, but not its enzyme Cdk6, was also depleted after the CHL treatments; the depletions were associated with elevations of G0/G1 cells. Apoptosis occurred time-dependently with the ERK deactivations by 400 microg/ml CHL; the apoptotic cells elevated from 2.7-fold of the control level at 24 h, to 4.7-fold at 48 h and to 16.6-fold at 72 h of treatment. Bcl-2 was also depleted at 72 h when there was the most prominent elevation of the apoptotic cells, suggesting that it participates during the exacerbation rather than the initiation phases of the CHL-induced apoptosis. Results from this study support further research on CHL for preventing and treating those tumors with deregulated ERK activations.

Topics: Apoptosis; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Chlorophyllides; Cyclin D1; Dose-Response Relationship, Drug; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Flow Cytometry; Humans; Immunoblotting; Phosphorylation; Proto-Oncogene Proteins c-bcl-2; Time Factors

Hypoxia, a common consequence of solid tumor growth in breast cancer or other cancers, serves to propagate a cascade of molecular pathways which include angiogenesis, glycolysis, and various cell-cycle control proteins. As we have shown previously, hypoxia activates STAT5 (signal transducer and activator of transcription 5) and increases its binding activity to the GAS element in mammary epithelial cells. In this study we attempted to elucidate the mechanism by which cyclin D1 is regulated by the STAT5 protein under hypoxic conditions. Our data demonstrate that hypoxia (2% O(2)) or desferrioxamine (DFO) induces tyrosine and serine phosphorylation of STAT5 in human breast cancer cells (MCF-7) and mammary epithelial cells (HC11). Imunoprecipitation and subsequent Western analysis showed that Jak2 leads to the tyrosine phosphorylation and activation of STAT5a or STAT5b under hypoxic conditions. Using a transfected COS-7 cell model system, we demonstrate that the activity of a cyclin D1 promoter-luciferase construct increased under hypoxic conditions or DFO treatment. The activity of the STAT5b/cyclin D1 promoter increased significantly by 12 h of hypoxia, whereas the activity of the STAT5a/cyclin D1 promoter was unaffected under hypoxic conditions. These increases in promoter activity are predominantly mediated by the Jak2/STAT5b signaling pathway. We have shown by EMSA that hypoxia induces STAT5 to bind to the cyclin D1 promoter (GAS-1) in MCF-7 and HC11 cells. These data suggest that STAT5b may mediate the transcriptional activation of cyclin D1 after hypoxic stimulation.

Topics: Anaerobiosis; Animals; Breast Neoplasms; Cell Hypoxia; Chlorocebus aethiops; COS Cells; Cyclin D1; Deferoxamine; Female; Gene Expression Regulation, Neoplastic; Humans; Janus Kinase 2; Phosphorylation; Promoter Regions, Genetic; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Serine; Tumor Cells, Cultured; Tyrosine

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN), a tumor suppressor phosphatase that dephosphorylates both protein and lipid substrates, is found to be mutated in both heritable and sporadic breast cancer. Cellular PTEN has been shown to regulate Akt phosphorylation, mitogen-activated protein kinase (MAPK) phosphorylation, p27(kip1), and cyclin D1 protein levels. Additionally, we and others have shown that PTEN can regulate not only the cell cycle but also cellular apoptosis. Until recently, the functions of PTEN have been thought to occur through cytoplasmic PTEN. However, we have shown that PTEN localizes to the nucleus and that this localization coincides with the G0-G1 phases of the cell cycle. Furthermore, we have shown that PTEN has bipartite nuclear localization sequence (NLS)-like sequences that are required for major vault protein-mediated nuclear import. These findings suggest that subcellular localization of PTEN may regulate its function and that nuclear-localized PTEN may regulate unique cellular functions that have been attributed to cytoplasmic PTEN. To examine this possibility, we analyzed downstream PTEN readouts using MCF-7 Tet-Off breast cancer cell lines stably transfected with two different NLS mutant PTEN constructs, which do not localize to the nucleus, and compared these with cells transfected with wild-type PTEN and empty vector control cells. We found that cytoplasmic PTEN down-regulates phosphorylation of Akt and up-regulates p27(kip1), whereas nuclear PTEN down-regulates cyclin D1 and prevents the phosphorylation of MAPK. Additionally, whereas we observe that nuclear PTEN is required for cell cycle arrest, we found that cytoplasmic PTEN is required for apoptosis. Our observations show that nuclear-cytoplasmic partitioning differentially regulates the cell cycle and apoptosis and, in this manner, provide further evidence that nuclear import of PTEN should play a role in carcinogenesis.

Topics: Apoptosis; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Nucleus; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Cytoplasm; Disease Progression; Down-Regulation; G1 Phase; Humans; Intracellular Signaling Peptides and Proteins; Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Resting Phase, Cell Cycle

To investigate the way of nuclear factor kappa B (NF-kappaB) activation and the mechanism of NF-kappaB-promoted proliferation in estrogen receptor (ER)-negative breast cancer cells.. The protein of IkappaB kinase alpha (IKKalpha) was measured by Western blot and the influence on cell-cycle was assayed by flow cytometry (FCM).. The IKKalpha was tested higher in three ER-negative breast cancer cell lines than in MCF-7. The influence caused by epidermal growth factor (EGF), tumor necrosis factor (TNF)-alpha and E(2) to tumor cells' proliferation was tested. EGF could remarkably enhance cyclin D(1) expression about 83% more in EGF group than that in control group and proliferation index from 0.22 to 0.31 (P < 0.01). On the other hand, TNF-alpha inhibited cyclin D(1) expression. Protein kinase C inhibitor, Go6976, could peculiarly prevent NF-kappaB over-expression caused by EGF. The cell-cycle was assayed by FCM in phase G(0)/G(1) 69.36% and in phase S 22.77% when adding EGF and in phase G(0)/G(1) 91.54% and in phase S 7.81% when adding EGF and Go6976. The proliferation index decreased from 0.31 to 0.09 (P < 0.01).. EGF-EGFR pathway can provide ER-negative breast cancer cells the signal for the autonomous growth. This signal promoted tumor cells' proliferation is transmitted by activating NF-kappaB and expressing more cyclin D(1). In this pathway, NF-kappaB play an important role as signal transmitting. The strategy to NF-kappaB activating may provide new way to treat ER-negative breast cancers.

Topics: Breast Neoplasms; Carbazoles; Cell Proliferation; Cyclin D1; Epidermal Growth Factor; Estradiol; Female; Humans; I-kappa B Kinase; Indoles; NF-kappa B; Receptors, Estrogen; Signal Transduction; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Human Rad9 (hRad9), a structural homologue of yeast Schizosaccharomyces pombe rad9, is involved in cell cycle checkpoints and apoptosis. hRad9 can serve as a corepressor of androgen receptor in prostate cancer cells, but little is known about its role in the development of breast or other cancers. In the present study, semiquantitative reverse transcription-PCR showed that Rad9 mRNA levels were up-regulated in 52.1% (25 of 48) of breast tumors, and this up-regulation correlated with tumor size (P = 0.037) and local recurrence (P = 0.033). Overexpression of Rad9 mRNA was partly due to an increase in Rad9 gene number as measured by quantitative PCR. In other breast tumors with Rad9 mRNA overexpression but without increase in gene number, there was differential methylation of two putative Sp1/3 binding sites within the first and second introns of the Rad9 gene, which was similarly found in MCF-7 breast cancer cell line with increased Rad9 mRNA. Silencing Rad9 expression by RNA interference in MCF-7 cell line inhibited its proliferation in vitro. Promoter assays indicated that the Sp1/3 site in intron 2 may act as a silencer. In vivo binding of Sp3 to intron 2 was shown by chromatin immunoprecipitation assays. Treatment of MCF-7 cell line with 5'-aza-2'-deoxycytidine reduced Rad9 mRNA expression and also increased binding of Sp3 to the demethylated intron 2 region. Collectively, these findings suggest that Rad9 is a novel oncogene candidate activated by 11q13 amplification and DNA hypermethylation in breast cancer and may play a role in tumor proliferation and local invasion.

Topics: Azacitidine; Base Sequence; Breast Neoplasms; Breast Neoplasms, Male; Cell Cycle Proteins; Cell Growth Processes; Cell Line, Tumor; Chromosomes, Human, Pair 11; Cyclin D1; Decitabine; DNA Methylation; Female; Gene Amplification; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Introns; Male; Middle Aged; Molecular Sequence Data; RNA, Messenger

Classical NF-kappaB (p65/p50) transcription factors display dynamic induction in the mammary gland during pregnancy. To further elucidate the role of NF-kappaB factors in breast development, we generated a transgenic mouse expressing the IkappaB-alpha S32/36A superrepressor (SR) protein under control of the mouse mammary tumor virus (MMTV) long terminal repeat promoter. A transient delay in mammary ductal branching was observed in MMTV-SR-IkappaB-alpha mice early during pregnancy at day 5.5 (d5.5) and d7.5; however, development recovered by mid- to late pregnancy (d14.5). Recovery correlated with induction of nuclear cyclin D1 and RelB/p52 NF-kappaB complexes. RelB/p52 complexes induced cyclin D1 and c-myc promoter activities and failed in electrophoretic mobility shift assay to interact with IkappaB-alpha-glutathione S-transferase, indicating that their weak interaction with IkappaB-alpha can account for the observed recovery of mammary gland development. Activation of IKKalpha and NF-kappaB-inducing kinase was detected by d5.5, implicating the alternative NF-kappaB signaling pathway in RelB/p52 induction. Constitutively active IKKalpha induced p52, RelB, and cyclin D1 in untransformed mammary epithelial cells. Moreover, mouse mammary tumors induced by 7,12-dimethylbenz(a)anthracene treatment displayed increased RelB/p52 activity. Inhibition of RelB in breast cancer cells repressed cyclin D1 and c-Myc levels and growth in soft agar. These results implicate RelB/p52 complexes in mammary gland development and carcinogenesis.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Agar; Animals; Breast Neoplasms; Cell Line, Tumor; Cell Nucleus; Cyclin D1; Female; Glutathione Transferase; Humans; I-kappa B Kinase; I-kappa B Proteins; Immunoblotting; Mammary Glands, Animal; Mammary Neoplasms, Animal; Mice; Mice, Transgenic; NF-kappa B; NF-kappa B p52 Subunit; NF-KappaB Inhibitor alpha; Phenotype; Pregnancy; Pregnancy, Animal; Promoter Regions, Genetic; Protein Binding; Proto-Oncogene Proteins c-myc; RNA; Time Factors; Transcription Factor RelA; Transcription Factor RelB; Transfection; Transgenes

Although great progress has been made at identifying and characterizing individual genes involved in cancer, less is known about how the combination of such genes collaborate to form tumors in humans. To this end, we sought to genetically recreate tumorigenesis in normal human cells using genes altered in human cancer. We now show that expression of mammalian proteins that inactivate the tumor suppressors Rb and p53 in conjunction with the oncoproteins Ras and Myc and the telomerase subunit hTERT is sufficient to drive a number of normal human somatic cells to a tumorigenic fate. This provides a blueprint of the events that lead to human cancer, allowing different cancers to be genetically modeled from normal human cells.

Topics: Animals; Breast Neoplasms; Cell Transformation, Neoplastic; Cyclin D1; DNA-Binding Proteins; Female; Humans; Mammary Glands, Human; Mice; Muscle, Skeletal; Proto-Oncogene Proteins c-myc; ras Proteins; Retinoblastoma Protein; Telomerase; Tumor Suppressor Protein p53

Both cyclin D1 and c-myc are key molecules in breast cancer carcinogenesis, and their transcriptional level and stability are regulated through several signaling pathways, including the Wnt signaling pathway. We performed immunohistochemical and mutational analyses of Wnt signaling components to investigate the association of Wnt signaling alterations with breast cancer carcinogenesis using 49 surgically resected primary breast cancer samples. Positive staining of cyclin D1 and c-myc was observed in 55.1% and 30.6% of the 49 breast cancer samples, respectively. Aberrant cytoplasmic expression of beta-catenin, which indicates the existence of alterations in the Wnt signaling pathway, was observed in 38.8% of breast cancer samples, though no mutation was found in the beta-catenin and Axin 1 genes. Reduced expression of APC was observed in 34.7% of samples. Statistical analysis revealed strong correlations between overexpression of beta-catenin and that of cyclin D1 and c-myc (p=0.0001 and 0.0117, respectively). Furthermore, overexpression of beta-catenin was significantly correlated with reduced expression of APC (p=0.0127). Wnt signaling alterations were frequently observed in breast cancer from the results of beta-catenin immunohistochemistry, although no mutation in the components of the Wnt signaling pathway was found in the present study. Based on the statistical analyses, we speculated that reduced expression of APC leads to overexpression of beta-catenin, and aberrant expression of cyclin D1 and c-myc mainly depends on alterations in the Wnt signaling pathway in breast cancer.

Topics: Adenomatous Polyposis Coli Protein; Adult; Axin Protein; beta Catenin; Breast Neoplasms; Cyclin D1; DNA Mutational Analysis; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Middle Aged; Mutation; Proto-Oncogene Proteins c-myb; Repressor Proteins; Signal Transduction; Wnt1 Protein

The development and progression of epithelial cancers are the result of an imbalance in signals promoting and inhibiting cellular proliferation and apoptosis. The aim of this study is to evaluate the expression of cell-cycle and apoptosis regulators and correlate them with clinical outcome in the most frequent carcinomas, in order to establish common prognostic biomarkers independent of cancer origin. Using tissue microarrays (TMAs), we have analysed the immuno-expression of Ki-67, Bcl-2, Bax, cyclin D1, cyclin D3, CDK1, CDK2, CDK6, p16, p21, and p27 in a series of 205 carcinomas of the large bowel, breast, lung and prostate (80, 73, 37 and 15 cases, respectively). By univariate analysis, positivity for p27, p16 and Bcl-2 was associated with better overall survival (P<0.0135, P<0.0442 and P<0.0001, respectively). The risk of mortality was 2.3-fold greater in patients without Bcl-2 expression. TMA immunohistochemical analysis identified a subset of epithelial cancers with overlapping alterations in cell-cycle checkpoints, apoptosis regulators and tumour suppressor pathways. We found that in most common epithelial cancers, regardless of origin, Bcl-2 appears to be the key biological factor influencing clinical behaviour.

Topics: Adult; Analysis of Variance; bcl-2-Associated X Protein; Biomarkers, Tumor; Breast Neoplasms; Cell Cycle Proteins; Cyclin D1; Cyclin D3; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Cyclins; Female; Humans; Immunohistochemistry; Intestinal Neoplasms; Ki-67 Antigen; Lung Neoplasms; Male; Middle Aged; Neoplasms; Prognosis; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Survival Analysis; Tissue Array Analysis

Aberrant activation of nuclear factor-kappaB (NF-kappaB) transcription factors has been implicated in the pathogenesis of breast cancer. We previously showed elevated activity of IkappaB kinase alpha (IKKalpha), IKKbeta, and protein kinase CK2 in primary human breast cancer specimens and cultured cells. A novel inducible IKK protein termed IKK-i/IKKepsilon has been characterized as a potential NF-kappaB activator. Here, we provide evidence that implicates IKK-i/IKKepsilon in the pathogenesis of breast cancer. We show IKK-i/IKKepsilon expression in primary human breast cancer specimens and carcinogen-induced mouse mammary tumors. Multiple breast cancer cell lines showed higher levels of IKK-i/IKKepsilon and kinase activity compared with untransformed MCF-10F breast epithelial cells. Interestingly, IKK-i/IKKepsilon expression correlated with CK2alpha expression in mammary glands and breast tumors derived from MMTV-CK2alpha transgenic mice. Ectopic CK2 expression in untransformed cells led to increased IKK-i/IKKepsilon mRNA and protein levels. Inhibition of CK2alpha via the pharmacologic inhibitor apigenin or upon transfection of a CK2 kinase-inactive subunit reduced IKK-i/IKKepsilon levels. Expression of a kinase-inactive IKK-i/IKKepsilon mutant in breast cancer cells reduced NF-kappaB activity as judged by transfection assays of reporters driven either by NF-kappaB elements or the promoters of two NF-kappaB target genes, cyclin D1 and relB. Importantly, the kinase-inactive IKK-i/IKKepsilon mutant reduced the endogenous levels of these genes as well as the ability of breast cancer cells to grow in soft agar or form invasive colonies in Matrigel. Thus, CK2 induces functional IKK-i/IKKepsilon, which is an important mediator of the activation of NF-kappaB that plays a critical role in the pathogenesis of breast cancer.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Apigenin; Breast; Breast Neoplasms; Carcinogens; Casein Kinase II; Cell Movement; Collagen; Cyclin D1; Drug Combinations; Enzyme Activation; Epithelial Cells; Female; Gene Expression Regulation, Neoplastic; Humans; I-kappa B Kinase; Laminin; Mice; Mice, Transgenic; NF-kappa B; Proteoglycans; Proto-Oncogene Proteins c-rel; Transcription Factor RelB; Transfection; Tumor Cells, Cultured

A diagnosis of atypical ductal hyperplasia (ADH) after breast core biopsy usually is followed by an excisional biopsy to exclude the presence of a more significant lesion. To determine whether the immunohistochemical expression of cyclin D1 (CyD1) and Ki-67 can aid in case stratification for the likelihood of finding ductal carcinoma in situ (DCIS) on subsequent excision, we immunohistochemically stained 21 consecutive ADH cases diagnosed by core biopsy, and proliferation indices (PIs) were calculated for each case. Fluorescence in situ hybridization to detect CCND1 amplification was performed in 10 cases. In 5 cases, DCIS (with or without invasive carcinoma) was identified in the subsequent excision. The mean PICyD1 and PIKi-67 for these cases were significantly higher than in the remainder (P = .03 and P = .05, respectively). The sensitivities of PICyD1 and PIKi-67 for the presence of DCIS on subsequent excision were 100%, and the specificities were 75% and 69%, respectively. The specificity of the 2 markers combined was 88%. The number of cells with CCND1 amplification was higher in cases with DCIS or ADH on subsequent excision. Immunostaining for CyD1 and Ki-67 might help stratify cases of ADH on core biopsy and identify patients unlikely to have DCIS found on excision.

Topics: Aged; Biomarkers, Tumor; Biopsy, Needle; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Cyclin D1; Female; Humans; Hyperplasia; Immunohistochemistry; In Situ Hybridization, Fluorescence; Ki-67 Antigen; Middle Aged; Precancerous Conditions

p21-activated kinase 1 (Pak1) has been shown recently to induce hyperplasia in the mammary epithelium, a phenotype also manifested by overexpression of cyclin D1, a known indicator of the proliferative stage. Here we investigated the role of the Pak1 pathway in the expression of cyclin D1 using tissue culture models and transgenic mice expressing activated Pak1 in mammary glands. We found that hyperplastic mammary glands from catalytically active Pak1 transgenic mice exhibit a 5- to 7-fold increased expression of cyclin D1 as compared with stage-matched wild-type mice. In addition, Pak1 levels were elevated in human breast tumors and also correlated well with increased cyclin D1 expression. Increased expression of Pak1 in breast cancer cells stimulated cyclin D1 promoter activity, elevated levels of cyclin D1 mRNA, protein, and nuclear accumulation of cyclin D1. Conversely, Pak1 inhibition by an auto-inhibitory peptide (amino acids 83-149) or Pak1 knockdown by short interference RNA markedly reduced the expression of cyclin D1, suggesting a requirement of a functional Pak1 pathway for optimal expression of cyclin D1. Results from deletion and mutant analysis indicate that Pak1 regulates cyclin D1 transcription by means of an NF-kappaB-dependent pathway. Together, these findings suggest a model wherein Pak1 regulation of cyclin D1 expression might involve an NF-kappaB-dependent pathway and that hyperplasia in the mammary glands of Pak1-TG mice may be associated, at least in part, with the up-regulation of cyclin D1, and that Pak1 is up-regulated in human breast tumors.

Topics: Animals; Breast Neoplasms; Cell Division; Cell Line; Cell Line, Tumor; Cyclin D1; HeLa Cells; Humans; Immunohistochemistry; Mammary Glands, Animal; Mammary Neoplasms, Animal; Mice; Mice, Transgenic; NF-kappa B; p21-Activated Kinases; Peptides; Phosphorylation; Protein Serine-Threonine Kinases; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Time Factors; Transcription, Genetic; Up-Regulation

Estrogens are mitogenic for estrogen receptor (ER)-positive breast cancer cells. Current treatment of ER-positive breast tumors is directed towards interruption of estrogen activity. We report that treatment of ER-positive breast cancer cells with kaempferol resulted in a time- and dose-dependent decrease in cell number. The concentration required to produce 50% growth inhibition at 48 h was approximately 35.0 and 70.0 microM for ER-positive and ER-negative breast cancer cells, respectively. For MCF-7 cells, a reduction in the ER-alpha mRNA equivalent to 50, 12, 10% of controls was observed 24 h after treatment with 17.5, 35.0, and 70.0 microM of kaempferol, respectively. Concomitantly, these treatments led to a 58, 80, and 85% decrease in ER-alpha protein. The inhibitory effect of kaempferol on ER-alpha levels was seen as early as 6 h post-treatment. Kaempferol treatment also led in a dose-dependent decrease in the expression of progesterone receptor (PgR), cyclin D1, and insulin receptor substrate 1 (IRS-1). Immunocytochemical study revealed that ER-alpha protein in kaempferol-treated MCF-7 cells formed an aggregation in the nuclei. Kaempferol also induced degradation of ER-alpha by a different pathway than that were observed for the antiestrogen ICI 182,780 and estradiol. Estradiol-induced MCF-7 cell proliferation and expression of the estrogen-responsive-element-reporter gene activity were abolished in cells co-treated with kaempferol. These findings suggest that modulation of ER-alpha expression and function by kaempferol may be, in part, responsible for its anti-proliferative effects seen in in vitro.

Topics: Blotting, Northern; Blotting, Western; Breast Neoplasms; Cell Division; Cell Line, Tumor; Cyclin D1; Dose-Response Relationship, Drug; Estradiol; Estrogen Antagonists; Estrogen Receptor alpha; Female; Fulvestrant; Humans; Immunohistochemistry; Insulin Receptor Substrate Proteins; Kaempferols; Phosphoproteins; Receptors, Estrogen; Receptors, Progesterone; Time Factors; Transfection

Blockade of the estrogen receptor (ER) with antiestrogens and aromatase inhibitors is effective in the treatment of breast cancer. Why ER plays such a dominant role in breast cancer and represents such an excellent target remains to be defined. The ability of ER to respond to multiple inputs and to control expression of multiple downstream genes may be one of the reasons why ER is such a powerful target for breast cancer treatment. The recent modest performance of a number of targeted therapies in breast cancer has raised the question whether we will ever develop therapies that have such success as antiestrogens. Targeted therapies tend to inhibit a single pathway that is probably altered in only a subset of patients. Even within this subset, only a limited number of patients respond. The evidence that virtually all pathways can cross-talk and that they exhibit several layers of redundancy reveals a complexity of signaling networks that may defy the generation of targeted therapies with efficacy similar to antiestrogens. However, there are clearly regulatory nodes that can integrate multiple upstream inputs and elicit diverse downstream outputs. We provide evidence and rationales for integrins, insulin receptor substrates (IRSs), and cyclin D1 as potential therapeutic targets. These proteins, similar to ER, can integrate and coordinate multiple signals in breast cancer cells and thus mediate diverse aspects of breast cancer progression. New treatment targets will emerge in light of more global models of signal transduction that fully integrate all aspects of cell biology such as the role of the extracellular matrix and will hopefully result in the development of targeted therapies that show efficacy similar to antiestrogens.

Topics: Breast Neoplasms; Cell Nucleus; Cyclin D1; Cytoplasm; Disease Progression; Estrogens; Extracellular Matrix; Gene Expression Regulation, Neoplastic; Growth Substances; Humans; Neoplasms, Hormone-Dependent; Receptors, Estrogen; Signal Transduction

Rapamycin inhibits the serine-threonine kinase mammalian target of rapamycin (mTOR), blocking phosphorylation of p70 S6 kinase (S6K1) and 4E-binding protein 1 (4E-BP1) and inhibiting protein translation and cell cycle progression. Rapamycin and its analogues are currently being tested in clinical trials as novel-targeted anticancer agents. Although rapamycin analogues show activity in clinical trials, only some of the treated patients respond. The purpose of this study is to identify determinants of rapamycin sensitivity that may assist the selection of appropriate patients for therapy.. Breast cancer cell lines representing a spectrum of aberrations in the mTOR signaling pathway were tested for rapamycin sensitivity. The expression and phosphorylation state of multiple components of the pathway were tested by Western blot analysis, in the presence and absence of rapamycin.. Cell proliferation was significantly inhibited in response to rapamycin in 12 of 15 breast cancer cell lines. The ratio of total protein levels of 4E-BP1 to its binding partner eukaryotic initiation factor 4E did not predict rapamycin sensitivity. In contrast, overexpression of S6K1, and phosphorylated Akt independent of phosphatase and tensin homologue deleted from chromosome 10 status, were associated with rapamycin sensitivity. Targeting S6K1 and Akt with small interfering RNA and dominant-negative constructs, respectively, decreased rapamycin sensitivity. Rapamycin inhibited the phosphorylation of S6K1, ribosomal S6 protein, and 4E-BP1 in rapamycin-resistant as well as -sensitive cells, indicating that its ability to inhibit the mTOR pathway is not sufficient to confer sensitivity to rapamycin. In contrast, rapamycin treatment was associated with decreased cyclin D1 levels in the rapamycin-sensitive cells but not in rapamycin-resistant cells.. Overexpression of S6K1 and expression of phosphorylated Akt should be evaluated as predictors of rapamycin sensitivity in breast cancer patients. Furthermore, changes in cyclin D1 levels provide a potential pharmacodynamic marker of response to rapamycin.

Topics: Adaptor Proteins, Signal Transducing; Antibiotics, Antineoplastic; Blotting, Western; Breast Neoplasms; Carrier Proteins; Cell Cycle; Cell Cycle Proteins; Cell Division; Cell Line, Tumor; Cyclin D1; Dose-Response Relationship, Drug; Genes, Dominant; Humans; Phosphoproteins; Phosphorylation; Protein Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Ribosomal Protein S6 Kinases, 70-kDa; RNA, Small Interfering; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases

Intact cyclin D1 functions are essential for transformation by erbB2 in tissue culture and murine models. Because cyclin D1 may alter cell proliferation through a variety of mechanisms, we used transgenic models and human tumor samples to particularly address the role of cyclin D1-cyclin-dependent kinases in transformation by erbB2. The p16 tumor suppressor specifically blocks cyclin-dependent kinase 4 and 6 activity. Here we show that an MMTV-p16 transgene blocked tumorigenesis by erbB2, demonstrating that deregulation of the cyclin-dependent kinase partner of cyclin D1 is an essential target of erbB2. ErbB2 overexpression was a determining factor in deregulation of cyclin D1-cdk4/6 interactions because neither transgenic cyclin D1 nor loss of p16 accelerated tumorigenesis in MMTV-erbB2-transgenic mice. ErbB2 was also a deciding factor in deregulation of cyclin D1-cdk4/6 in human tumors because no loss of pRb or p16 was found in tumors overexpressing erbB2, although erbB2-negative invasive breast adenocarcinomas frequently lacked expression of p16 or pRb. We conclude that deregulation of cyclin D1-Cdk4/6 interactions is a critical target of erbB2 function in human and mouse breast tumors, and erbB2's overexpression may be sufficient to deregulate cyclin D1-cdk4/6 activity in breast cancer.

Topics: Animals; Breast Neoplasms; CDC2 Protein Kinase; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinases; DNA Primers; Genes, erbB-2; Genes, p16; Humans; Immunohistochemistry; Membrane Proteins; Mice; Mice, Transgenic; Proto-Oncogene Proteins; Receptors, Virus; Reverse Transcriptase Polymerase Chain Reaction

Previous studies have suggested that cells may differ in their response to metal stress. This study was undertaken to investigate the role of PI3K/Akt signaling pathway in metal resistance in human breast cancer epithelial cells with different p53 and estrogen receptor status. Exposure to copper and zinc increased Akt phosphorylation with its nuclear localization only in MDA-MB-231 cells with no estrogen receptor and mutated p53. Cyclin D1 expression and cell-cycle progression followed the metal-induced Akt phosphorylation. Treatment with LY294002 abrogated these effects, suggesting the essential role of PI3-kinase. In contrast, in MCF-7 cells with wild type p53 and estrogen receptor, there was no change in Akt activation, while suppression of p53 activity by pifithrin-alpha increased phosphorylation of Akt after the treatment with copper. In MCF-7 cells, the metal treatment increased the phosphorylation of p53 at serine 15, up-regulated p21 expression, and resulted in cell-cycle arrest in G1 phase with apoptosis. These results demonstrate that copper-induced apoptosis in MCF-7 cells is p53 dependent, whereas the metal resistance in MDA-MB-231 cells may be due to activation of Akt in the absence of a functional p53.

Topics: Apoptosis; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Chromones; Copper; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Enzyme Activation; Enzyme Inhibitors; Epithelial Cells; Humans; Morpholines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proto-Oncogene Proteins; Signal Transduction; Thiazoles; Tumor Suppressor Protein p53; Zinc

Steroid hormones bind to their receptors and trans-activate target genes. Rapid non-genomic action of steroid hormones has been proposed in addition to the one at the genomic level. Estrogen has been described to activate c-Src kinase and this activation has been shown to be responsible for estrogen-dependent mitogenicity. A major substrate of c-Src kinase activity is the cytoskeletal protein p130Cas, originally identified in v-Src-transformed cells. We show that in the human breast carcinoma T47D cells, upon estrogen treatment, p130Cas rapidly and transiently associates with the estrogen receptor alpha in a multi-molecular complex containing the c-Src kinase and the p85 subunit of PI 3-kinase. Association of p130Cas with the estrogen receptor alpha occurs within 3 minutes of estrogen treatment and is dependent on c-Src kinase activation. Transient overexpression of p130Cas in T47D cells increases estrogen-dependent Src kinase and Erk1/2 MAPKs activities and accelerates their kinetics of stimulation. A similar effect was detected on estrogen-dependent cyclin D1 expression, suggesting a role for p130Cas in regulating estrogen-dependent cell cycle progression. Double-stranded small RNA interference (siRNA) by silencing endogenous p130Cas protein, was sufficient to inhibit estrogen-dependent Erk1/2 MAPKs activity and cyclin D1 induction, demonstrating the requirement of p130Cas in such events. Therefore, our data show that the adaptor protein p130Cas associates with the estrogen receptor transducing complex, regulating estrogen-dependent activation of c-Src kinase and downstream signaling pathways.

Topics: Breast Neoplasms; Carcinoma; Cell Line, Tumor; Crk-Associated Substrate Protein; Cyclin D1; Enzyme Activation; Estrogen Receptor alpha; Estrogens; Female; Humans; Kinetics; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phosphatidylinositol 3-Kinases; Proteins; Retinoblastoma Protein; Retinoblastoma-Like Protein p130; RNA, Small Interfering; Signal Transduction; src-Family Kinases

1,1-Bis(3'-indolyl)-1-(p-trifluoromethylphenyl)methane (DIM-C-pPhCF(3)) and several p-substituted phenyl analogues have been investigated as a new class of peroxisome proliferator-activated receptor gamma (PPARgamma) agonists. Structure-activity studies in PPARgamma-dependent transactivation assays in MCF-7 breast cancer cells show that 5-20 micro M concentrations of compounds containing p-trifluoromethyl, t-butyl, cyano, dimethylamino, and phenyl groups were active, whereas p-methyl, hydrogen, methoxy, hydroxyl, or halogen groups were inactive as PPARgamma agonists. Induction of PPARgamma-dependent transactivation by 15-deoxy-Delta12,14-prostaglandin J2 (PGJ2) and DIM-C-pPhCF(3) was inhibited in MCF-7 cells cotreated with the PPARgamma-specific antagonist N-(4'-aminopyridyl)-2-chloro-5-nitrobenzamide. In mammalian two-hybrid assays, DIM-C-pPhCF(3) and PGJ2 (5-20 micro M) induced interactions of PPARgamma with steroid receptor coactivator (SRC) 1, SRC2 (TIFII), and thyroid hormone receptor-associated protein 220 but not with SRC3 (AIB1). In contrast, DIM-C-pPhCF(3), but not PGJ2, induced interactions of PPARgamma with PPARgamma coactivator-1. C-substituted diindolylmethanes inhibit carcinogen-induced rat mammary tumor growth, induce differentiation in 3T3-L1 preadipocytes, inhibit MCF-7 cell growth and G(0)/G(1)-S phase progression, induce apoptosis, and down-regulate cyclin D1 protein and estrogen receptor alpha in breast cancer cells. These compounds are a novel class of synthetic PPARgamma agonists that induce responses in MCF-7 cells similar to those observed for PGJ2.

Topics: 3T3-L1 Cells; Adipocytes; Animals; Apoptosis; Breast Neoplasms; Carcinogens; Cell Cycle; Cell Differentiation; Cell Division; Cell Line, Tumor; Cell Separation; Cloning, Molecular; Cyclin D1; Dose-Response Relationship, Drug; Down-Regulation; Estrogen Receptor alpha; Female; Flow Cytometry; G1 Phase; Humans; Indoles; Ligands; Luciferases; Methane; Mice; Plasmids; Prostaglandin D2; Protein Structure, Tertiary; Rats; Rats, Sprague-Dawley; Receptors, Cytoplasmic and Nuclear; Receptors, Estrogen; Resting Phase, Cell Cycle; Structure-Activity Relationship; Transcription Factors; Transcriptional Activation; Transfection; Two-Hybrid System Techniques

Soy-based products consumed in Asian countries are minimally processed whereas in the USA many of the soy foods and soy ingredients are highly processed. Soy foods contain complex mixtures of bioactive compounds, which may interact with one another. The objective of this study was to evaluate the ability of various soy products containing genistin, the glycoside form of genistein, to affect growth of MCF-7 cells transplanted into ovariectomized athymic mice. Products investigated included soy flour, two crude extracts of soy (soy molasses and Novasoy(R)), a mixture of isoflavones and genistin in pure form. Each of the soy flour-processed products was added to the diet to provide equivalent amounts of genistein aglycone equivalents (750 p.p.m.). Tumors in the negative control animals regressed throughout the study while the tumors in the soy flour-fed animals remained basically the same size (neither grew nor regressed). In animals consuming soy molasses, Novasoy(R), mixed isoflavones or genistin alone, tumor growth was stimulated when compared with animals consuming a control diet devoid of soy. These same dietary treatments resulted in increased cellular proliferation. Changes in mRNA expression of gene targets (estrogen responsiveness, cell cycle progression, apoptosis and aromatase activity) in tumors induced by the different diets were evaluated. The relative expression of pS2, progesterone receptor and cyclin D1 was increased in animals consuming the Novasoy(R), mixed isoflavones and genistin. Bcl2 mRNA expression was low in most of the dietary treatment groups compared with positive (estradiol implant) controls. Aromatase expression was not affected in any of the treatment groups. The degree of soy flour processing affects the estrogenicity of products containing a constant amount of genistein. Collectively, these findings suggest that for postmenopausal women with estrogen-dependent breast cancer, the consumption of foods containing soy flour is more advisable than consuming isoflavones in more purified forms.

Topics: Animals; Apoptosis; Aromatase; Breast Neoplasms; Cell Cycle; Cyclin D1; Diet; Estrogens; Female; Food Handling; Genistein; Humans; Mice; Mice, Nude; Neoplasms, Hormone-Dependent; Ovariectomy; Proteins; Receptors, Progesterone; Soy Foods; Trefoil Factor-1; Tumor Cells, Cultured; Tumor Suppressor Proteins

Clinical data and samples from patients diagnosed, more than 10 years previously, with operable node-negative breast cancer (participants in the Scottish Adjuvant Tamoxifen trial), were revisited. Cases with two distinct categories of outcome were selected; more than 10 years disease-free survival ('good outcome') or distant relapse within 6 years of diagnosis ('poor outcome'). An initial set of cases was analysed for a range of putative prognostic markers and a prognostic index, distinguishing the two outcome categories, was calculated. This index was then validated by testing its predictive power on a second, independent set of cases. A combination of histological grade plus immunochemical staining for BCL-2, p27 and Cyclin D1, generated a useful prognostic index for tamoxifen-treated patients but not for those treated by surgery alone. The value of the index was confirmed in a second set of tamoxifen-treated, early stage breast cancers. Overall, it correctly predicted good and poor outcome in 79 and 74% of cases, respectively (odds ratio 11.0). Other markers assessed added little to prediction of outcome. In the case of molecular assays, sensitivity and reliability were compromised by the age of the tissue specimens and the variability of fixation protocols. In selecting patients for adjuvant systemic chemotherapy, the proposed index improves considerably on current international guidelines and matches the performance reported for 'gene-expression signature' analysis.

Topics: Biomarkers, Tumor; Breast Neoplasms; Cyclin D1; Disease-Free Survival; Female; Follow-Up Studies; Genes, bcl-2; Humans; Immunohistochemistry; Lymphatic Metastasis; Microfilament Proteins; Middle Aged; Muscle Proteins; Neoplasm Staging; Prognosis; Risk Factors

Aberrant activation of the beta-catenin signaling pathway has been implicated in several malignancies, including breast carcinoma. Recently, it was shown that p53 down-regulated beta-catenin in a complex fashion. The authors examined the expression of beta-catenin, key members of its signaling pathway, and p53 in a large cohort of breast tumors.. The authors conducted an immunohistochemical analysis of the expression of beta-catenin, upstream modulators (HER-2/neu, Met, and epidermal growth factor receptor [EGFR]), downstream target genes (cyclin D1 and matrix metalloproteinase-7 [MMP7]), and p53 on a tissue microarray of 346 lymph node-negative breast carcinomas. The results were correlated with one another and with standard clinicopathologic parameters.. beta-Catenin expression was observed in the membrane and/or cytoplasm without any significant nuclear expression. HER-2/neu and EGFR were observed on the membrane in 21% and 6% of tumors, respectively, and Met stained in a membrane/cytoplasm distribution in 28% of cases. Cyclin D1 was expressed in the nucleus and MMP7 was expressed in the cytoplasm in 26% and 75% of tumors, respectively. Nuclear expression of p53 was noted in 31% of tumors. When each marker was analyzed separately, only p53 and Met demonstrated a significant correlation with survival. However, patients who had tumors that coexpressed high levels of beta-catenin and p53 had markedly worse overall survival (P = 0.0026). In multivariate analysis, only tumor size, Met, and the coexpression of beta-catenin and p53 retained statistical significance.. The current findings support a potential synergistic effect of abnormal beta-catenin regulation and p53 status in the pathogenesis and natural history of lymph node-negative breast carcinoma. Furthermore, the results show that a combined analysis of multiple markers, notably beta-catenin and p53, may enhance the prognostic capabilities compared with individual markers.

Topics: beta Catenin; Biomarkers, Tumor; Breast Neoplasms; Cyclin D1; Cytoskeletal Proteins; ErbB Receptors; Female; Humans; Immunoenzyme Techniques; Matrix Metalloproteinase 7; Proto-Oncogene Proteins c-met; Receptor, ErbB-2; Survival Rate; Trans-Activators; Tumor Suppressor Protein p53

Recent studies have shown that p21-activated kinase 1 (Pak1) phosphorylates estrogen receptor-alpha (ER alpha) at Ser 305 and also promotes its transactivation function. Here, we sought to investigate whether substitution of serine 305 in ER with glutamic acid (ER alpha-S305E), which mimics the phosphorylation state, would influence the status of ER-target genes. To explore this possibility, we generated clones overexpressing ER alpha-S305E in ER-negative MDA-MB-231 cells and analyzed the status of ER-regulated genes using a gene array. Results indicated that the expression of ER alpha-S305E is sufficient to upregulate the expression of a few but not all ER-regulated genes, i.e., cyclin D1 and zinc finger protein 147 (estrogen-responsive finger protein), while there was no significant change in the expression of remaining genes on the array. In addition, we found an increased expression as well as nuclear accumulation of cyclin D1 protein in MDA-MB-231 cells expressing ER alpha-S305E as compared to the level of cyclin D1 in MDA-MB-231 cells expressing WT-ER alpha or pcDNA. Furthermore, ER alpha-S305E, but not mutation of ER alpha-S305 to alanine, enhanced the cyclin D1 promoter activity. These findings suggest that ER alpha activation at S305 is sufficient to upregulate the expression of cyclin D1, an ER-regulated gene that is implicated in the progression of breast cancer. Phosphorylation of ER alpha by Pak1 or its upstream regulators could upregulate the expression of a subset of ER-target genes in a ligand-independent manner and hence, might contribute toward the development of hormone independence in breast cancer cells.

Topics: Amino Acid Substitution; Breast Neoplasms; Cell Line, Tumor; Cyclin D1; Estrogen Receptor alpha; Estrogens; Female; Fluorescent Antibody Technique; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Humans; Microscopy, Confocal; Oligonucleotide Array Sequence Analysis; Promoter Regions, Genetic; Receptors, Estrogen; Recombinant Proteins; RNA; Serine; Signal Transduction; Transfection; Up-Regulation

The Ink4a/Arf locus encodes two structurally unrelated tumor suppressor proteins, p16(INK4a) and p14(ARF) (murine p19(ARF)). Invariant inactivation of either the p16(INK4a)-cyclin D/CDK-pRb pathway and/or p53-p14(ARF) pathway occurs in most human tumors. Cyclin D1 is frequently overexpressed in breast cancer cells contributing an alternate mechanism inactivating the p16(INK4a)/pRb pathway. Targeted overexpression of cyclin D1 to the mammary gland is sufficient for tumorigenesis, and cyclin D1-/- mice are resistant to Ras-induced mammary tumors. Recent studies suggest cyclin D1 and p16(INK4a) expression are reciprocal in human breast cancers. Herein, reciprocal regulation of cyclin D1 and p16(INK4a) was observed in tissues of mice mutant for the Ink4a/Arf locus. p16(INK4a) and p19(ARF) inhibited DNA synthesis in MCF7 cells. p16(INK4a) repressed cyclin D1 expression and transcription. Repression of cyclin D1 by p16(INK4a) occurred independently of the p16(INK4a)-cdk4-binding function and required a cAMP-response element/activating transcription factor-2-binding site. p19(ARF) repressed cyclin D1 through a novel distal cis-element at -1137, which bound p53 in chromatin-immunoprecipitation assays. Transcriptional repression of the cyclin D1 gene through distinct DNA sequences may contribute to the tumor suppressor function of the Ink4a/Arf locus.

Topics: Animals; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; DNA, Neoplasm; Fibroblasts; Humans; Mice; Mice, Knockout; Promoter Regions, Genetic; Repressor Proteins; Transcription, Genetic; Transfection; Tumor Suppressor Protein p14ARF

Several reports have shown that a long delay between cutting sections and immunohistochemical (IHC) staining can decrease the IHC reaction intensity. However, systematic large-scale studies to investigate to what extent this problem may influence the outcome of translational research studies are lacking. In this study, we used a tissue microarray (TMA) approach to investigate the influence of slide age on comparisons between the results of IHC analyses for estrogen receptor (ER), progesterone receptor (PR), cyclin D1, HER2 (HercepTest), and E-cadherin and clinical outcome in a series of 522 breast cancer patients. Old TMA sections stored for 6 months at 4 degrees C and freshly cut sections were analyzed under exactly identical experimental conditions. As compared to results obtained on freshly cut sections, the frequency of positivity on old sections decreased from 65 to 46% for ER (P<0.0001), from 33 to 18.5% for PR (P<0.0001), from 16.3 to 9.6% for HER2 (P=0.0047), from 45.1 to 37.7% for cyclin D1 (P=0.10), and from 58.9 to 32.9% for E-cadherin (P<0.0001). Despite the lower fraction of positive cases, most associations between IHC data and tumor phenotype that were observed in fresh section analysis were also found when old section data were analyzed. The results confirm that slide aging has a great influence on the intensity of IHC staining in individual cases, but they also suggest that many clinicopathological associations can be detected if suboptimally processed sections are used for IHC.

Topics: Biomarkers, Tumor; Breast Neoplasms; Cadherins; Cyclin D1; Humans; Immunohistochemistry; Prognosis; Quality Assurance, Health Care; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone; Sensitivity and Specificity; Specimen Handling; Survival Analysis; Time Factors

A(3) adenosine receptor (A(3)AR) activation was shown to inhibit the growth of various tumor cells via the down-regulation of nuclear factor kappaB and cyclin D1. To additionally elucidate whether A(3)AR is a specific target, a survey of its expression in tumor versus adjacent normal cells was conducted.. A(3)AR mRNA expression in various tumor tissues was tested in paraffin-embedded slides using reverse transcription-PCR analysis. A comparison with A(3)AR expression in the relevant adjacent normal tissue or regional lymph node metastasis was performed. In addition, A(3)AR protein expression was studied in fresh tumors and was correlated with that of the adjacent normal tissue.. Reverse transcription-PCR analysis of colon and breast carcinoma tissues showed higher A(3)AR expression in the tumor versus adjacent non-neoplastic tissue or normal tissue. Additional analysis revealed that the lymph node metastasis expressed even more A(3)AR mRNA than the primary tumor tissue. Protein analysis of A(3)AR expression in fresh tumors derived from colon (n = 40) or breast (n = 17) revealed that 61% and 78% had higher A(3)AR expression in the tumor versus normal adjacent tissue, respectively. The high A(3)AR expression level in the tumor tissues was associated with elevated nuclear factor kappaB and cyclin D1 levels. High A(3)AR mRNA expression was also demonstrated in other solid tumor types.. Primary and metastatic tumor tissues highly express A(3)AR indicating that high receptor expression is a characteristic of solid tumors. These findings and our previous data suggest A(3)AR as a potential target for tumor growth inhibition.

Topics: Blotting, Western; Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Line, Tumor; Colonic Neoplasms; Cyclin D1; Down-Regulation; Humans; Lung Neoplasms; Lymphatic Metastasis; Melanoma; Neoplasm Metastasis; Neoplasms; NF-kappa B; Receptor, Adenosine A3; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

E-cadherin links to the cytoskeleton via catenins and mediates cell-cell homophilic adhesion. beta-catenin not only regulates cell-cell adhesion as a protein interacting with cadherin, but also functions as an important component of the Wnt signaling pathway which has been found to be closely associated with tumor formation. This study was performed to examine the expression of E-cadherin, beta-catenin, and cyclin D1 in breast cancer in order to evaluate their possible roles in the formation and progression of breast cancer.. The alterations of E-cadherin, beta-catenin, and cyclin D1 in 60 cases of breast cancer were determined using highly sensitive SP immunohistochemical method.. Normal immunoreactivity of E-cadherin and beta-catenin were observed in 29 (48.3%) and 18 (30.0%) cases, respectively. Twenty-eight cases (46.7%) showed cyclin D1 overexpression. Thirty percent (9/29) of the cases with normal staining of E-cadherin showed overexpression of cyclin D1, while 61.3%(19/31) of the cases showed overexpression of cyclin D1 with abnormal staining of E-cadherin. Abnormal expression of E-cadherin and overexpression of cyclin D1 showed a significantly positive correlation (rs=0.303,P< 0.05). Forty-two cases showed abnormal staining of beta-catenin. Cyclin D1 overexpression was observed in 57.1% (24/42) of these cases with abnormal staining of beta-catenin, but only observed in 22.2% (4/18) of these cases with normal membranous staining of beta-catenin. There was a significantly positive correlation between the abnormal expression of beta-catenin and overexpression of cyclin D1 (rs=0.321, P< 0.05).. Down-regulation of E-cadherin and beta-catenin accumulation in the cytoplasm/nuclear may promote malignant transformation and progression by triggering cyclin D1 overexpression in breast cancer.

Topics: Adult; Aged; beta Catenin; Breast Neoplasms; Cadherins; Carcinoma, Ductal, Breast; Cyclin D1; Cytoskeletal Proteins; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Middle Aged; Trans-Activators

Phosphorylation on certain Ser/Thr-Pro motifs is a major oncogenic mechanism. The conformation and function of phosphorylated Ser/Thr-Pro motifs are further regulated by the prolyl isomerase Pin1. Pin1 is prevalently overexpressed in human cancers and implicated in oncogenesis. However, the role of Pin1 in oncogenesis in vivo is not known. We have shown that Pin1 ablation is highly effective in preventing oncogenic Neu or Ras from inducing cyclin D1 and breast cancer in mice, although it neither affects transgene expression nor mammary gland development. Moreover, we have developed an ex vivo assay to uncover that a significant fraction of primary mammary epithelial cells from Neu or Ras mice display various malignant properties long before they develop tumors in vivo. Importantly, these early transformed properties are effectively suppressed by Pin1 deletion, which can be fully rescued by overexpression of cyclin D1. Thus, Pin1 is essential for tumorigenesis and is an attractive anticancer target. Our ex vivo assay can be used to study early events of breast cancer development in genetically predisposed mice.

Topics: Animals; Breast Neoplasms; Cell Differentiation; Cell Transformation, Neoplastic; Cells, Cultured; Cyclin D1; Disease Models, Animal; Female; Gene Expression Regulation, Neoplastic; Male; Mammary Glands, Animal; Mice; Mice, Nude; Mice, Transgenic; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; Phenotype; Proto-Oncogene Proteins c-myc; ras Proteins; Receptor, ErbB-2; Sexual Abstinence

Transcriptional activation of the cyclin D1 gene (CCND1) plays a pivotal role in G(1)-phase progression, which is thereby controlled by multiple regulatory factors, including nuclear receptors (NRs). Appropriate CCND1 gene activity is essential for normal development and physiology of the mammary gland, where it is regulated by ovarian steroids through a mechanism(s) that is not fully elucidated. We report here that CCND1 promoter activation by estrogens in human breast cancer cells is mediated by recruitment of a c-Jun/c-Fos/estrogen receptor alpha complex to the tetradecanoyl phorbol acetate-responsive element of the gene, together with Oct-1 to a site immediately adjacent. This process coincides with the release from the same DNA region of a transcriptional repressor complex including Yin-Yang 1 (YY1) and histone deacetylase 1 and is sufficient to induce the assembly of the basal transcription machinery on the promoter and to lead to initial cyclin D1 accumulation in the cell. Later on in estrogen stimulation, the cyclin D1/Cdk4 holoenzyme associates with the CCND1 promoter, where E2F and pRb can also be found, contributing to the long-lasting gene enhancement required to drive G(1)-phase completion. Interestingly, progesterone triggers similar regulatory events through its own NRs, suggesting that the gene regulation cascade described here represents a crossroad for the transcriptional control of G(1)-phase progression by different classes of NRs.

Topics: Base Sequence; Breast Neoplasms; Cell Line, Tumor; Cyclin D1; Estrogen Receptor alpha; Estrogens; Female; G1 Phase; Gene Expression Regulation; Genes, Reporter; Humans; Macromolecular Substances; Models, Genetic; Progesterone; Promoter Regions, Genetic; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Receptors, Estrogen; Repressor Proteins; Response Elements; Transcription Factors; Transcription, Genetic; Transcriptional Activation

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Cell Culture Techniques; Chemokine CXCL12; Chemokines, CXC; Cyclin D1; Drug Screening Assays, Antitumor; Epithelial Cells; Female; Gene Expression Regulation, Neoplastic; Humans; Neoplasms; Oncogenes; Stromal Cells

The breast and ovarian cancer susceptibility gene BRCA1 acts as a tumor suppressor gene, its product expresses in a cell cycle-dependent manner. Inheritance of a mutant allele of BRCA1 leads to increased risk of developing breast,and ovarian cancers. Lovastatin, as a potent inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase),is the main rate-limiting enzyme of endogenous cholesterol biosynthesis pathway. This study was to evaluate the effect and mechanism of combined application of BRCA1 and lovastatin on cell cycle distribution of MCF-7 cells.. MCF-7 cells were transfected with expression vector pcDNA3-beta-HA-hsBRCA1 containing full-length BRCA1, and named MCF-7BRCA1 cells. The expression of BRCA1 gene was examined with reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis. After cultured with 8 micromol/L lovastatin, growth curve and MTT assay were used to determine cell proliferation capacity; the cell cycle distribution was measured with flow cytometry (FCM). Meanwhile,the protein expression of Cyclin D1 and retinoblastoma (Rb) were analyzed by Western blot analysis.. The pcDNA3-beta-HA-hsBRCA1 plasmids were successfully transfected into MCF-7 cells and expressed stably. The growth curve and MTT assay results showed that cell proliferation capacity decreased in the cells cultured with lovastatin for 4 days. FCM showed that S phase, and G2/M phase of BRCA1-infected cells decreased,while G0/G1 phase increased after cultured with lovastatin, the cells were blocked in G0/G1 phase at 72 h post-treatment by lovastatin. Ectopic expression of BRCA1 leads to decrease of the Rb protein and Cyclin D1 protein. Overall data showed that the effects of Lovastatin were more obviously in MCF-7BRCA1 cells than MCF-7 cells.. Overexpression of BRCA1 protein may amplify sensibility of breast cancer to lovastatin,and enhance the anti-tumor effect of lovastatin.

Topics: Antineoplastic Agents; BRCA1 Protein; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Genes, BRCA1; Genetic Vectors; Humans; Lovastatin; Retinoblastoma Protein; Transfection

Alterations in ErbB2 or fibroblast growth factor receptor-4 (FGFR-4) expression and activity occur in a significant fraction of breast cancers. Because signaling molecules and pathways cooperate to drive cancer progression, simultaneous targeting of multiple pathways is an appealing therapeutic strategy. With this in mind, we examined breast tumor cells for their sensitivity to the ErbB2 and FGFR inhibitors, PKI166 and PD173074, respectively. Simultaneous blocking of ErbB2 and FGFR-4 in MDA-MB-453 tumor cells had a stronger anti-proliferative effect than treatment with individual inhibitors. Examination of cell cycle regulators revealed a novel translation-mediated mechanism whereby ErbB2 and FGFR-4 cooperate to regulate cyclin D1 levels. Our results showed that FGFR-4 and ErbB2 via the MAPK and the phosphatidylinositol 3-kinase/protein kinase B pathways, respectively, both contribute to the maintenance of constitutive activity of the mammalian target of rapamycin translational pathway. Dual inhibition of these receptors strongly blocked S6 kinase 1 (S6K1) activity and cyclin D1 translation, as attested by a decrease in cyclin D1 mRNA association with polysomes. Ectopic expression of active protein kinase B or active S6K1 abrogated the dual inhibitor-mediated down-regulation of cyclin D1 expression, demonstrating the importance of these FGFR-4/ErbB2 signaling targets in regulating cyclin D1 translation. S6K1 has the central role in this process, since small interfering RNA-targeted S6K1 depletion led to a decrease in cellular S6K1 activity and, as a consequence, repression of cyclin D1 expression. Thus, we propose a novel mechanism for controlling cyclin D1 expression downstream of combined activity of ErbB2 and FGFR-4 that involves S6K1-mediated translation.

Topics: Breast Neoplasms; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Protein Kinases; Receptor, ErbB-2; Receptor, Fibroblast Growth Factor, Type 4; Receptors, Fibroblast Growth Factor; Ribosomal Protein S6 Kinases, 70-kDa; TOR Serine-Threonine Kinases

Expression of an estrogen receptor alpha (ER) transgene in hormone independent breast cancer and normal breast epithelial cells arrests cell cycling when estradiol is added. Although endogenously expressed ER does not typically affect estradiol-induced cell cycling of hormone dependent breast cancer cells, we observed that elevated expression of a green fluorescent protein fused to ER (GFP-ER) hindered entry of estrogen treated MCF-7 cells into S phase of the cell cycle. In analyses of key cell-cycle regulating proteins, we observed that GFP-ER expression had no affect on the protein levels of cyclin D1, cyclin E, or p27, a cyclin dependent kinase (Cdk) inhibitor. However, at 24 h, p21 (Waf1, Cip1; a Cdk2 inhibitor) protein remained elevated in the high GFP-ER expressing cells but not in non-GFP-ER expressing cells. Elevated expression of p21 inhibited Cdk2 activity, preventing cells from entering S phase. The results show that elevated levels of ER prevented the down-regulation of p21 protein expression, which is required for hormone responsive cells to enter S phase.

Topics: Breast Neoplasms; CDC2-CDC28 Kinases; Cell Cycle Proteins; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Down-Regulation; Estrogen Receptor alpha; Green Fluorescent Proteins; Humans; S Phase; Tumor Cells, Cultured; Tumor Suppressor Proteins

Success in breast cancer treatment depends greatly upon early detection, and in the employment of prognostic markers able to anticipate the evolution of the disease, allowing a more rational management of the patient. A fundamental cause of cancer is the alteration of the genetic material, which may modify the expression of proteins that play key roles in cell cycle progression. The aim of this study was to analyze the expression of cyclins D1, E, and B1 and of the CDKIs p16 and p21 in a population of uniformly treated patients with stage I or II breast tumors (n=56) compared with patients with benign breast pathology (n=23). Malignant breast tumors showed higher cyclin E and lower p21 expression than benign breast pathology (NS), determined by immunohistochemistry (IHC). In breast cancer patients, overexpression of cyclins D1 and E was associated with the presence of ER and stage respectively independently of other prognostic variables (multivariate analysis). Kaplan-Meier curves demonstrated that only overexpression of cyclin E was associated with a longer recurrence-free survival. Cox analysis showed that neither cyclins nor CDKIs were independent prognostic markers. We demonstrated that several regulators of cell cycle progression were altered in a large number of breast tumor cases, however, these abnormalities were not indicators of a worse outcome in breast cancer patients of stages I and II.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Blotting, Western; Breast Neoplasms; Carcinoma, Ductal; Carcinoma, Lobular; Cell Cycle; Cell Cycle Proteins; Cyclin B; Cyclin B1; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Female; Humans; Immunoenzyme Techniques; Middle Aged; Neoplasm Staging; Prognosis; Survival Rate; Tumor Suppressor Proteins

The development of resistance to tamoxifen, the most common antiestrogen used in the treatment of breast cancer, is a frequent and severe clinical problem. Tamoxifen-resistant tumors are still capable of responding to other hormonal therapies such as those that downregulate estrogen receptor expression. Mechanisms leading to acquisition of tamoxifen-resistant but hormone-sensitive growth are not completely understood. In tamoxifen-sensitive breast cancer cells, tamoxifen inhibits, whereas estrogen induces, expression of cyclin D1, a key cell cycle regulatory protein. Ectopic expression of cyclin D1 can lead to antiestrogen resistance. Thus, to determine whether cyclin D1 is involved in the growth of tamoxifen-resistant cells, we developed several tamoxifen-resistant variants from MCF-7 cells. These variants grow in the absence of estrogen or in the presence of tamoxifen, but their growth is inhibited by estrogen receptor downregulators. We show here that cyclin D1 expression is maintained at comparable levels in all tamoxifen-resistant variants, whereas pS2, another estrogen-regulated protein, is not. The addition of physiological levels of estrogen further stimulates cyclin D1 expression and proliferation. In contrast, treatment with estrogen receptor downregulators decreases cyclin D1 expression and proliferation. Thus, changes in cyclin D1 expression upon second-line hormonal therapy may predict hormonal sensitivity of tamoxifen-resistant tumors. These studies suggest that estrogen receptor mediates cyclin D1 expression and growth of tamoxifen-resistant tumors.

Topics: Breast Neoplasms; Cell Cycle; Cell Division; Cell Line, Tumor; Cyclin D1; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Humans; Receptors, Estrogen; Tamoxifen

Multiple different oncogenes have been described previously to be amplified in breast cancer including HER2, EGFR, MYC, CCND1, and MDM2. Gene amplification results in oncogene overexpression but may also serve as an indicator of genomic instability. As such, presence of one or several gene amplifications may have prognostic significance. To assess the prognostic importance of amplifications and coamplifications of HER2, EGFR, MYC, CCND1, and MDM2 in breast cancer, we analyzed a breast cancer tissue microarray containing samples from 2197 cancers with follow-up information. Fluorescence in situ hybridizations revealed amplifications of CCND1 in 20.1%, HER2 in 17.3%, MDM2 in 5.7%, MYC in 5.3%, and EGFR in 0.8% of the tumors. All gene amplifications were significantly associated with high grade. HER2 (P < 0.001) and MYC amplification (P < 0.001) were also linked to shortened survival. In case of HER2, this was independent of grade, pT, and pN categories. MYC amplification was almost 3 times more frequent in medullary cancer (15.9%), than in the histologic subtype with the second highest frequency (ductal; 5.6%; P = 0.0046). HER2 and MYC amplification were associated with estrogen receptor/progesterone receptor negativity (P < 0.001) whereas CCND1 amplification was linked to estrogen receptor/progesterone receptor positivity (P < 0.001). Coamplifications were more prevalent than expected based on the individual frequencies. Coamplifications of one or several other oncogenes occurred in 29.6% of CCND1, 43% of HER2, 55.7% of MDM2, 65% of MYC, and 72.8% of EGFR-amplified cancers. HER2/MYC-coamplified cancers had a worse prognosis than tumors with only one of these amplifications. Furthermore, a gradual decrease of survival was observed with increasing number of amplifications. In conclusion, these data support a major prognostic impact of genomic instability as determined by a broad gene amplification survey in breast cancer.

Topics: Breast Neoplasms; Cyclin D1; Gene Amplification; Gene Dosage; Genes, erbB-1; Genes, erbB-2; Genes, myc; Humans; In Situ Hybridization, Fluorescence; Multivariate Analysis; Neoplasm Staging; Nuclear Proteins; Prognosis; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2

Estrogen receptor alpha (ERalpha)-positive breast cancer cell lines are up to 10 times more sensitive than ERalpha-negative cell lines to the antiproliferative activity of the histone deacetylase inhibitor trichostatin A (TSA). The purpose of the study was to investigate the mechanisms underlying this differential response.. In the ERalpha-positive MCF-7 cell line, TSA repressed ERalpha and cyclin D1 transcription and induced ubiquitin dependent proteasomal degradation of cyclin D1, leading primarily to G(1)-S-phase cell cycle arrest. By contrast, cyclin D1 degradation was enhanced but its transcription unaffected by TSA in the ERalpha-negative MDA-MB-231 cell line, which arrested in G(2)-M phase. Cyclin D1 degradation involved Skp2/p45, a regulatory component of the Skp1/Cullin/F-box complex; silencing SKP2 gene expression by RNA interference stabilized cyclin D1 and abrogated the cyclin D1 down-regulation response to TSA.. Tamoxifen has been shown to inhibit ERalpha-mediated cyclin D1 transcription, and acquired resistance to tamoxifen is associated with a shift to ERalpha-independent cyclin D1 up-regulation. Taken together, our data show that TSA effectively induces cyclin D1 down-regulation through both ERalpha-dependent and ERalpha-independent mechanisms, providing an important new strategy for combating resistance to antiestrogens.

Topics: Antineoplastic Agents, Hormonal; Breast Neoplasms; Cell Cycle; Cell Proliferation; Cyclin D1; Cysteine Proteinase Inhibitors; Drug Resistance, Neoplasm; Endopeptidases; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Leupeptins; RNA Interference; S-Phase Kinase-Associated Proteins; Tamoxifen; Transcription, Genetic; Tumor Cells, Cultured; Uterine Neoplasms

The effects of 17 beta-estradiol, dihydrodydrogesterone, tamoxifen and cyclophosphamide upon parameters of cell maturation (Mucine1 expression), cell proliferation (Cyclin D1 expression) and apoptosis (loss of nuclear DNA) were studied in estrogen receptor positive (ER+) and negative (ER-) human breast cancer cells. Tamoxifen was the most potent inducer of apoptosis in ER+ and ER- breast cancer cells. 17 beta-estradiol in a concentration of 10(-6) M induced proliferation in ER+ cells after 144 h. incubation, while equimolar co-incubation with dihydrodydrogesterone prevented this effect and even induced a significant increase of cell death. It is speculated that the continuous use of combined 17 beta-estradiol plus dihydrodydrogesterone might be given as hormone replacement therapy without increased risk of breast cancer and even may reduce the relapse rate in breast cancer patients.

Topics: Antineoplastic Agents, Alkylating; Antineoplastic Agents, Hormonal; Apoptosis; Breast Neoplasms; Cell Death; Cell Division; Cell Nucleus; Cyclin D1; Cyclophosphamide; Dydrogesterone; Estradiol; Humans; Mucins; Progesterone Congeners; Receptors, Estrogen; Reverse Transcriptase Polymerase Chain Reaction; Tamoxifen; Time Factors; Tumor Cells, Cultured

Although interactions between estrogen and growth factor signaling pathways have been studied extensively, how growth factors and progesterone regulate each other is less clear. In this study, we found that IGF-I sharply lowers progesterone receptor (PR) mRNA and protein levels in breast cancer cells. Other growth factors, such as epidermal growth factor, also showed the same effect. The decrease of PR levels was associated with reduced PR activity. Unlike progestins, IGF-I does not utilize the proteasome for down-regulating PR. Instead, the IGF-I-mediated decrease in PR levels is via an inhibition of PR gene transcription. In addition, the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway was found to be specifically involved in this IGF-I effect. Our data also suggest that the IGF-I down-regulation of PR is not mediated via a reduction of estrogen receptor (ER) levels or activity. First, IGF-I induced ligand-independent ER activity while reducing ER-dependent PR levels. Second, whereas PR and cyclin D1 are both ER up-regulated, IGF-I increased cyclin D1 levels while decreasing PR levels. Third, constitutively active PI3K or Akt induced ER activity but reduced PR levels and activity. Taken together, our data indicate that IGF-I inhibits PR expression in breast cancer cells via the PI3K/Akt/mTOR pathway. Because low or absent PR in primary breast cancer is associated with poor prognosis and response to hormone therapy, our results suggest that low PR status may serve as an indicator of activated growth factor signaling in breast tumor cells, and therefore of an aggressive tumor phenotype and resistance against hormonal therapy.

Topics: Breast Neoplasms; Cyclin D1; Enzyme Inhibitors; Female; Growth Substances; Humans; Insulin-Like Growth Factor I; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Predictive Value of Tests; Protein Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Receptors, Estrogen; Receptors, Progesterone; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transcription, Genetic; Tumor Cells, Cultured

A number of studies have demonstrated that the STAT pathway is an important signaling cascade utilized by the IL-6 cytokine family to regulate a variety of cell functions. However, the downstream target genes of STAT activation that mediate the cytokine-induced cellular responses are largely uncharacterized. The aims of the current study are to determine whether the STAT signaling pathway is critically involved in the oncostatin M (OM)-induced growth inhibition and morphological changes of MCF-7 cells and to identify STAT3-target genes that are utilized by OM to regulate cell growth and morphology. We show that expression of a dominant negative (DN) mutant of STAT3 in MCF-7 cells completely eliminated the antiproliferative activity of OM, whereas expression of DN STAT1 had no effect. The growth inhibition of breast cancer cells was achieved through a concerted action of OM on cell cycle components. We have identified four cell cycle regulators including c-myc, cyclin D1, c/EBPdelta, and p53 as downstream effectors of the OM-activated STAT3 signaling cascade. The expression of these genes is differentially regulated by OM in MCF-7 cells, but is unaffected by OM in MCF-7-dnStat3 stable clones. We also demonstrate that the OM-induced morphological changes are correlated with increased cell motility in a STAT3-dependent manner. Expression analysis of extracellular matrix (ECM) proteins leads to the identification of fibronectin as a novel OM-regulated ECM component. Our studies further reveal that STAT3 plays a key role in the robust induction of fibronectin expression by OM in MCF-7 and T47D cells. These new findings provide a molecular basis for the mechanistic understanding of the effects of OM on cell growth and migration.

Topics: Breast Neoplasms; CCAAT-Enhancer-Binding Proteins; Cell Movement; Cyclin D1; DNA-Binding Proteins; Extracellular Matrix; Female; Fibronectins; Genes, cdc; Humans; Mutation; Oncostatin M; Peptides; Signal Transduction; STAT1 Transcription Factor; STAT3 Transcription Factor; Trans-Activators; Transcription Factor CHOP; Transcription Factors

Activation of estrogen receptors (ERs) by estrogens triggers both ER nuclear transcriptional activity and Src/Ras/Erks pathway-dependent mitogenic activity. The present study implicates prenylated proteins in both estrogenic actions. The farnesyltransferase and geranylgeranyltransferase I inhibitors (FTI-277 and GGTI-298, respectively) antagonize estradiol-stimulated cell cycle progression, progesterone receptor, cyclin D1, and c-Myc expression. In contrast, the inhibitors markedly stimulate transcription from two genes containing estrogen response elements, both in the absence and presence of estradiol. The pure antiestrogen ICI 182,780 inhibits by more than 85% these effects on transcription. We demonstrate that both FTI-277 and GGTI-298 increase the association of steroid receptor coactivator-1 with ER alpha and FTI-277 decreases the association of ER alpha with the histone deacetylase 1, a known transcriptional repressor. In addition, FTI-277 has no marked effect on the association of the two corepressors, nuclear receptor corepressor and silencing mediator of retinoid and thyroid receptor with ER alpha, whereas GGTI-298, similar to tamoxifen, clearly increased these associations. Together, these results demonstrate that prenylated proteins play a role in estradiol stimulation of proliferation and progesterone receptor expression. However, they antagonize the ability of ER alpha to stimulate estrogen response element-dependent transcriptional activity, acting presumably through coregulator complex formation.

Topics: Alkyl and Aryl Transferases; Benzamides; Breast Neoplasms; Cell Cycle; Cell Division; Cyclin D1; Dimethylallyltranstransferase; Enzyme Inhibitors; Estradiol; Estrogen Antagonists; Estrogen Receptor alpha; Farnesyltranstransferase; Gene Expression; Humans; Methionine; Proto-Oncogene Proteins c-myc; Receptors, Estrogen; Receptors, Progesterone; Response Elements; RNA, Messenger; Transcription, Genetic; Tumor Cells, Cultured

The amplification of cyclin D1, located on chromosome 11q13, in breast cancer patients has been found to be associated with reduced relapse-free and overall survival; however, there still exists strong controversy about these findings. In order to evaluate the prognostic value of cyclin D1 and other prognostic variables in human breast cancers, we have assessed estrogen receptor (ER) status, cyclin D1, c-erbB2 and p53 overexpression in 175 primary breast carcinomas, and investigated the relationships of prognostic variables to the patient clinical outcome and the association between cyclin D1 overexpression and other prognostic variables. There was some degree of variability in staining intensities and proportions within the same tumor. The overexpression of both cyclin D1 and ER revealed a significantly prolonged survival in univariate analysis (P = 0.020). Among the various prognostic variables, distant metastasis showed a statistically significant association with overall survival. A significant correlation was observed between cyclin D1 overexpression and small size of the primary tumor (P = 0.031), low Bloom and Richardson's histological grade (P = 0.001), and positive ER status (P = 0.000). In contrast to what was previously expected, the present study suggests that the overexpression of cyclin D1 has a tendency to have a positive clinical outcome and a potential role in identifying a subset of patients predicting a good prognosis, particularly when ER is coexpressed.

Topics: Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Cell Nucleus; Combined Modality Therapy; Cyclin D1; Disease-Free Survival; Female; Humans; Immunoenzyme Techniques; Middle Aged; Neoplasm Staging; Receptor, ErbB-2; Receptors, Estrogen; Survival Analysis; Survival Rate; Tumor Suppressor Protein p53

Estrogen receptors (ER) have been localized to the cell plasma membrane (PM), where signal transduction mediates some estradiol (E2) actions. However, the precise structural features of ER that result in membrane localization have not been determined. We obtained a partial tryptic peptide/mass spectrometry analysis of membrane mouse ERalpha protein. Based on this, we substituted alanine for the determined serine at amino acid 522 within the E domain of wild-type (wt) ERalpha. Upon transfection in CHO cells, the S522A mutant ERalpha resulted in a 62% decrease in membrane receptor number and reduced colocalization with caveolin 1 relative to those with expression of wt ERalpha. E2 was significantly less effective in stimulating multiple rapid signals from the membranes of CHO cells expressing ERalpha S522A than from those of CHO cells expressing wt ERalpha. In contrast, nuclear receptor expression and transcriptional function were very similar. The S522A mutant was also 60% less effective than wt ERalpha in binding caveolin 1, which facilitates ER transport to the PM. All functions of ERalpha mutants with other S-to-A substitutions were comparable to those of wt ER, and deletion of the A/B or C domain had little consequence for membrane localization or function. Transfection of ERalpha S522A into breast cancer cells that express native ER downregulated E2 binding at the membrane, signaling to ERK, and G1/S cell cycle events and progression. However, there was no effect on the E2 transactivation of an ERE-luciferase reporter. In summary, serine 522 is necessary for the efficient translocation and function of ERalpha at the PM. The S522A mutant also serves as a dominant-negative construct, identifying important functions of E2 that originate from activating PM ER.

Topics: Animals; Binding, Competitive; Breast Neoplasms; Caveolin 1; Caveolins; Cell Cycle; Cell Membrane; Cell Nucleus; CHO Cells; Cricetinae; Cyclin D1; Dimerization; Dose-Response Relationship, Drug; Endoplasmic Reticulum; Estrogen Receptor alpha; G1 Phase; Genes, Dominant; Kinetics; Mice; Microscopy, Fluorescence; Mitogen-Activated Protein Kinases; Mutagenesis, Site-Directed; Mutation; Myristic Acid; Palmitic Acid; Protein Binding; Protein Structure, Tertiary; Receptors, Estrogen; S Phase; Signal Transduction; Transcription, Genetic; Transcriptional Activation; Transfection; Tumor Cells, Cultured; Type C Phospholipases

Treatment of MCF-7 cells with the peroxisome proliferator-activated receptor (PPAR) gamma agonists ciglitazone or 15-deoxy-Delta 12,14-prostaglandin J2 resulted in a concentration- and time-dependent decrease of cyclin D1 and estrogen receptor (ER) alpha proteins, and this was accompanied by decreased cell proliferation and G(1)-G(0)-->S-phase progression. Down-regulation of cyclin D1 and ER alpha by PPARgamma agonists was inhibited in cells cotreated with the proteasome inhibitors MG132 and PSII, but not in cells cotreated with the protease inhibitors calpain II and calpeptin. Moreover, after treatment of MCF-7 cells with 15-deoxy-Delta 12,14-prostaglandin J2 and immunoprecipitation with cyclin D1 or ER alpha antibodies, there was enhanced formation of ubiquitinated cyclin D1 and ER alpha bands. Thus, PPARgamma-induced inhibition of breast cancer cell growth is due, in part, to proteasome-dependent degradation of cyclin D1 (and ER alpha), and this pathway may be important for other cancer cell lines.

Topics: Breast Neoplasms; Cell Division; Cyclin D1; Cysteine Endopeptidases; Down-Regulation; Estrogen Receptor alpha; G1 Phase; Humans; Multienzyme Complexes; Prostaglandin D2; Proteasome Endopeptidase Complex; Receptors, Cytoplasmic and Nuclear; Receptors, Estrogen; RNA, Messenger; S Phase; Thiazoles; Thiazolidinediones; Transcription Factors; Transcription, Genetic; Tumor Cells, Cultured; Ubiquitin

Patients diagnosed with ductal carcinoma in situ (DCIS) at a young age appear to have a different natural history and biology, including a higher local relapse rate, than patients diagnosed later in life. The current study compared various pathologic and molecular features of DCIS arising in a cohort of young women with those of DCIS arising in a cohort of older women to identify potential biologic differences between these two populations of patients.. The study population consisted of 20 patients age < 42 years and 34 patients age > 60 years who were treated at Yale University School of Medicine with breast-conserving therapy (BCT) and whose archival paraffin blocks were available and had sufficient tumor for staining. The original slides from each case were reviewed and the most representative specimen block from each case was processed for immunohistochemical staining. Pathologic characteristics evaluated for each case included histology, grade, and presence of necrosis. Paraffin-embedded sections were immunohistochemically evaluated for expression of HER-2/neu, estrogen receptor (ER), progesterone receptor (PR), bcl-2, cyclin D1, Ki-67, and p53.. Although there was no difference in pathologic features of the tumors between the two groups, HER-2/neu was found to be overexpressed in a greater percentage of the younger population (P = 0.06). There was no apparent difference in expression of the other markers. Of note, HER-2/neu expression was correlated with high nuclear grade (P = 0.004), necrosis (P = 0.06), and ER and PR negativity (P = 0.01 and P = 0.03, respectively) in the combined population.. The current study data suggested that HER-2/neu overexpression in younger patients may characterize a biologic difference in their tumor and may partially contribute to their higher risk of recurrence. Further studies are needed to assess whether this difference holds independent of grade and to evaluate the prognostic significance of HER-2/neu overexpression in DCIS.

Topics: Adult; Age Factors; Aged; Aged, 80 and over; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Cohort Studies; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Ki-67 Antigen; Middle Aged; Necrosis; Neoplasm Recurrence, Local; Proto-Oncogene Proteins c-bcl-2; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone; Risk Factors; Tumor Suppressor Protein p53

Caveolin-1 is the principal structural component of caveolae microdomains, which represent a subcompartment of the plasma membrane. Several independent lines of evidence support the notion that caveolin-1 functions as a suppressor of cell transformation. For example, the human CAV-1 gene maps to a suspected tumor suppressor locus (D7S522/7q31.1) that is frequently deleted in a number of carcinomas, including breast cancers. In addition, up to 16% of human breast cancers harbor a dominant-negative mutation, P132L, in the CAV-1 gene. Despite these genetic associations, the tumor suppressor role of caveolin-1 still remains controversial. To directly assess the in vivo transformation suppressor activity of the caveolin-1 gene, we interbred Cav-1 (-/-) null mice with tumor-prone transgenic mice (MMTV-PyMT) that normally develop multifocal dysplastic lesions throughout the entire mammary tree. Herein, we show that loss of caveolin-1 gene expression dramatically accelerates the development of these multifocal dysplastic mammary lesions. At 3 wk of age, loss of caveolin-1 resulted in an approximately twofold increase in the number of lesions (foci per gland; 3.3 +/- 1.0 vs. 7.0 +/- 1.2) and an approximately five- to sixfold increase in the total area occupied by these lesions. Similar results were obtained at 4 wk of age. However, complete loss of caveolin-1 was required to accelerate the appearance of these dysplastic mammary lesions, because Cav-1 (+/-) heterozygous mice did not show any increases in foci development. We also show that loss of caveolin-1 increases the extent and the histological grade of these mammary lesions and facilitates the development of papillary projections in the mammary ducts. Finally, we demonstrate that cyclin D1 expression levels are dramatically elevated in Cav-1 (-/-) null mammary lesions, consistent with the accelerated appearance and growth of these dysplastic foci. This is the first in vivo demonstration that caveolin-1 can function as a transformation suppressor gene.

Topics: Age Factors; Animals; Antigens, Polyomavirus Transforming; Breast Neoplasms; Caveolin 1; Caveolins; Cell Transformation, Neoplastic; Cyclin D1; DNA-Binding Proteins; Female; Fibrocystic Breast Disease; Gene Expression Regulation; Humans; Male; Mammary Glands, Animal; Mice; Mice, Transgenic; Milk Proteins; Mitogen-Activated Protein Kinases; STAT5 Transcription Factor; Trans-Activators

The use of herbal medicine is a common practice among Chinese women with breast cancer. Yunzhi (Voriolus versicolor), a substance that is regarded as a biological response modifier, is frequently used. The aim of the present study is to evaluate the anti-proliferative action of, Yunzhi, polysaccharide peptide (PSP), on breast cancer cells. Breast cancer cells (MDA-MB-231) were cultured with and without PSP for 7 days. Cell growth at 24, 72, 120 and 168 hours was measured by Cell Proliferation Reagent (WST-1). Cells treated with PSP were found to have a significant reduction in cell proliferation as compared to controls after 72 hours of incubation. This lasted for 168 hours. When the effect of PSP on apoptosis was studied by the TdT-mediated X-dUTP nick end-labeling (TUNEL) assay, we found that PSP had a significant effect upon apoptosis from 24 hours onward. Immunostaining showed that PSP increased p21 expression and decreased cyclin D1 expression. In conclusion, PSP is effective in inhibiting cell proliferation through apoptosis. The mechanism for the apoptosis may be through up-regulation of p21 and down-regulation of cyclin D1.

Topics: Apoptosis; Breast Neoplasms; Cyclin D1; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Humans; In Situ Nick-End Labeling; Proteoglycans; Time Factors; Tumor Cells, Cultured; Up-Regulation

A population-based series of 122 patients with pregnancy-associated breast carcinomas was histologically revised and the relationship between hormone receptors, c-erbB-2, BRCA1, p27, cyclin E, and cyclin D1 was studied. The 5-year overall survival was 41%; 70% had tumor size >20 mm; 72% had metastasized to regional lymph nodes; 95% were histologic grade II or III; 66% and 75% were negative for estrogen and progesterone receptor, respectively; and c-erbB-2 expression was high (44%). BRCA1 expression was reduced in 33% of the cases. The expression of p27, cyclin D1, and cyclin E was low, 11%, 9%, and 16%, respectively. Cyclin D1 was positively associated with the hormone receptors (p< or =0.01). In multivariate analysis, lymph node status, progesterone receptor, and c-erbB-2 were significant prognostic factors. In subdividing the group according to lymph node status, c-erbB-2 and progesterone receptor retained a prognostic significance in the node positive group only. In conclusion, pregnancy-associated breast carcinomas are aggressive tumors, with low expression of hormone receptors, BRCA1, p27, and cyclin E and D1, and high expression of c-erbB-2.

Topics: Adult; Biomarkers, Tumor; BRCA1 Protein; Breast Neoplasms; Carcinoma; Cyclin D1; Cyclin E; Female; Humans; Immunohistochemistry; Lymphatic Metastasis; Pregnancy; Pregnancy Complications, Neoplastic; Prognosis; Proliferating Cell Nuclear Antigen; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone

Most human tumors display inactivation of the p53 and the p16(INK4)/pRb pathway. The Ink4a/alternative reading frame (ARF) locus encodes the p16(INK4a) and p14(ARF) (murine p19(ARF)) proteins. p16(INK4a) is deleted in 40-60% of breast cancer cell lines, and p16(INK4a) inactivation by DNA methylation occurs in < or =30% of human breast cancers. In mice genetically heterozygous for p16(INK4a) or Ink4a/Arf, predisposition to specific tumor types is enhanced. Ink4a/Arf(+/-) mice have increased E micro -Myc-induced lymphomagenesis and epidermal growth factor receptor-induced gliomagenesis. ErbB2 (epidermal growth factor receptor-related protein B2) is frequently overexpressed in human breast cancer and is sufficient for mammary tumorigenesis in vivo. We determined the role of heterozygosity at the Ink4a/Arf locus in ErbB2-induced mammary tumorigenesis. Compared with mouse mammary tumor virus-ErbB2 Ink4a/Arf(+/-) mice, mouse mammary tumor virus-ErbB2 Ink4a/Arf(wt) mammary tumors showed increased p16(INK4a), reduced Ki-67 expression, and reduced cyclin D1 protein but increased mammary tumor apoptosis with no significant change in the risk of developing mammary tumors. These studies demonstrate the contribution of Ink4a/Arf heterozygosity to tumor progression is tissue specific in vivo. In view of the important role of Ink4a/Arf in response to chemotherapy, these transgenic mice may provide a useful model for testing breast tumor therapies.

Topics: Adenocarcinoma; Aneuploidy; Animals; Apoptosis; Breast Neoplasms; Cell Cycle; Cell Transformation, Neoplastic; Cell Transformation, Viral; Crosses, Genetic; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Female; Gene Expression Regulation, Neoplastic; Genes, erbB-2; Genes, p16; Genetic Predisposition to Disease; Heterozygote; Humans; Ki-67 Antigen; Mammary Neoplasms, Experimental; Mammary Tumor Virus, Mouse; Mice; Mice, Knockout; Mice, Transgenic; Organ Specificity; Transfection; Tumor Suppressor Protein p14ARF

Abnormalities in G1/S transition in cell cultures have been attributed to alterations in ErbB (erythroblastic leukaemia viral [v-erb-b] oncogene homologue, avian) signalling, cyclin D1 overexpression or disturbance of the p21(WAF1) (p21)-mediated cell cycle arrest induced by p53. To investigate the significance of these mechanisms on an early stage of human breast tumour growth, we studied the expression of EGFR (ErbB1), HER-2/neu (ErbB2), cyclin D1, p21 and p53 as well as oestrogen (ER) and progesterone receptor (PgR) in paraffin sections of 45 ductal carcinoma in situ (DCIS) by immunohistochemistry. Cell proliferation was assessed by immunohistochemical quantification of Ki-67. Five cases with cyclin D1 overexpression were analysed by FISH for CCND1 amplification. Increased proliferative activity was observed in 46% of DCIS. It was correlated with the expression of EGFR and HER-2/neu (p < 0.05), but neither with cyclin D1 and p21 overexpression nor with p53 accumulation. ErbB positive status was associated with p21 overexpression (p < 0.05). In addition we found a correlation between the overexpression of p21 and cyclin D1 restricted to ErbB-positive cases (p = 0.013). ErbB-negative tumours with increased proliferative activity were ER and cyclin D1 positive. No CCND1 amplification was detected in the analysed cases. In conclusion, our data support that EGFR and HER-2/neu play an important role in cell cycle control in DCIS. p21 appears to be a potential mediator of ErbB signalling. We propose that cyclin D1 could be indirectly induced by ErbB signalling through p21. Besides, ER-mediated upregulation of cyclin D1 seems to be a possible mechanism of maintaining cell proliferation in DCIS in case of EGFR- and HER-2/neu-negativity.

Topics: Adult; Aged; Breast; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Cell Division; Chi-Square Distribution; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; ErbB Receptors; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Ki-67 Antigen; Middle Aged; Oncogene Proteins v-erbB; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone; Tumor Suppressor Protein p53

The aim of this study was to assess the levels of cell cycle regulatory proteins p21waf1 (p21), p53, Cyclin A, Cyclin D1 and Ki-67 to see whether they correlated with recurrence-free survival (RFS). From 1982 to 1996, 50 patients aged less than 51 years underwent lumpectomy followed by radiotherapy for a pure ductal carcinoma in situ (DCIS). For each case, the following immunohistochemical stains were carried out: Ki-67, Cyclin A, Cyclin D1, p53 and p21waf1 (p21). The percentage of positive nuclei was assessed. Multiple combinations of these factors were performed; in particular, we called the sum of Ki-67 and Cyclin A a global proliferation factor (GPF). Correlations with classical clinicopathological data were assessed. After a multivariate analysis, only GPF, Van Nuys Prognostic Index (VNPI) grade and mitotic index were independent predictive factors of recurrence in the whole population. In the population with close surgical margins, when the GPF level was less than the 25th percentile or more than the 75th percentile recurrence was low. In this preliminary study, GPF seems to be of interest to help in the decision process in the post-surgical management of the patient.

Topics: Adult; Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Cycle Proteins; Cyclin A; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Disease-Free Survival; Female; Humans; Immunohistochemistry; Ki-67 Antigen; Middle Aged; Prognosis; Tumor Suppressor Protein p53

To study the expression of phosphatase and tensin homology deleted on chromosometen ten (PTEN) protein, a tumor suppressor gene in breast cancer and its correlation with p27(kip1) and cyclin D1 expression.. PTEN protein expression, p27(kip1) and cyclin D1 protein expression were detected by immunohistochemical method in paraffin sections from 61 women with primary breast cancer. PTEN protein expression was compared with clinico-pathologic parameters as related to p27(kip1) and cyclin D1.. PTEN, being shown in the cytoplasm, was negative in 6.6% (4/61), reduced in 41.0% (25/61) and positive in 52.5% (32/61) samples. PTEN expression level was correlated with axillary lymph node status, loss of estrogen receptor stain, recurrence and metastasis. On univariate analysis, the disease-free survival rate of patients with higher PTEN expression (> 50% cells stained) was better than those with lower expression (P = 0.0101). However, there was no correlation between p27(kip1), cyclin D1 expression or PTEN expression.. PTEN, its lower expression being correlated with poor outcome of breast cancer patients, plays a prominent role in breast cancer. p27(kip1) or cyclin D1 may not be the primary downstream genes of PTEN in breast cancer.

Topics: Adult; Aged; Breast Neoplasms; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Female; Humans; Immunohistochemistry; Intracellular Signaling Peptides and Proteins; Lymphatic Metastasis; Middle Aged; Prognosis; PTEN Phosphohydrolase

To study the role of IGF-I receptor signaling on cell cycle events we utilized MCF-7 breast cancer cells. IGF-I at physiological concentrations increased the level of p21CIP/WAF mRNA after 4has well as protein after 8hby 10- and 6-fold, respectively, in MCF-7 cells. This IGF-1 effect was reduced by 50% in MCF-7-derived cells (SX13), which exhibit a 50% reduction in IGF-1R expression, demonstrating that IGF-1 receptor activation was involved in this process. Preincubation with the ERK1/2 inhibitor U0126 significantly reduced the IGF-1 effect on the amount of p21CIP/WAF protein in MCF-7 cells. These results were confirmed by the expression of a dominant negative construct for MEK-1 suggesting that the increase of the abundance of p21CIP/WAF in response to IGF-1 occurs via the ERK1/2 mitogen-activated protein kinase pathway. Using an antisense strategy, we demonstrated that abolition of p21CIP/WAF expression decreased by 2-fold the IGF-1 effect on cell proliferation in MCF-7. This latter result is explained by a delay in G1 to S cell cycle progression due partly to a reduction in the activation of some components of cell cycle including the induction of cyclin D1 expression in response to IGF-1. MCF-7 cells transiently overexpressing p21 showed increased basal and IGF-I-induced thymidine incorporation. Taken together, these results define p21CIP/WAF as a positive regulator in the cell proliferation induced by IGF-1 in MCF-7 cells.

Topics: Breast Neoplasms; Cell Division; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; Female; G1 Phase; Humans; Insulin-Like Growth Factor I; Mitogen-Activated Protein Kinases; Phosphorylation; Proto-Oncogene Proteins; Retinoblastoma Protein; RNA, Antisense

Altered expression of cell-cycle regulators is prevalent in clinical breast cancer. This study was performed to analyze the impact of cyclin E expression to the outcome of breast cancer together with cyclin D1 and p27Kip1.. The correlation between cyclin D1/E and p27Kip1 expression was analyzed in tissue arrays of 175 node-negative breast cancers treated by the same chemotherapy composed of fluorouracil, cyclophosphamide, and methotrexate. Data from the immunohistochemical assays of three molecules were correlated and were analyzed with clinical outcome of the patients.. Cyclin E expression was observed in 48 (27.4%) of 175 breast carcinomas. Cyclin E expression was significantly increased in young age patients and poorly differentiate tumors. Expression of cyclin E was significantly increased in cyclin D1 expressing tumors (P = 0.034). p27Kip1 expression was preserved above the 50% level in 87 tumors (49.7%) and was inversely correlated with cyclin E expression (P = 0.042). Ki67 labeling index was significantly increased in cyclin E-expressing tumors (P = 0.033) and was inversely related with p27Kip1 expression. In multivariate survival analysis, cyclin E expression was significant for the prediction of poor survival of the patients.. Cyclin E expression was associated with poor prognosis and intimately correlated with the expression of cyclin D1 and p27Kip1. Integration of TMA technology allowed a high-throughput analysis for correlating molecular in situ findings with clinico-pathologic information.

Topics: Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cell Cycle Proteins; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase Inhibitor p27; Cyclophosphamide; Female; Fluorouracil; Humans; Lymph Nodes; Methotrexate; Oligonucleotide Array Sequence Analysis; Prognosis; Survival Analysis; Tumor Suppressor Proteins

The cyclin D1 gene is overexpressed in human breast cancers and is required for oncogene-induced tumorigenesis. Peroxisome proliferator-activated receptor gamma (PPAR gamma) is a nuclear receptor selectively activated by ligands of the thiazolidinedione class. PPAR gamma induces hepatic steatosis, and liganded PPAR gamma promotes adipocyte differentiation. Herein, cyclin D1 inhibited ligand-induced PPAR gamma function, transactivation, expression, and promoter activity. PPAR gamma transactivation induced by the ligand BRL49653 was inhibited by cyclin D1 through a pRB- and cdk-independent mechanism, requiring a region predicted to form an helix-loop-helix (HLH) structure. The cyclin D1 HLH region was also required for repression of the PPAR gamma ligand-binding domain linked to a heterologous DNA binding domain. Adipocyte differentiation by PPAR gamma-specific ligands (BRL49653, troglitazone) was enhanced in cyclin D1(-/-) fibroblasts and reversed by retroviral expression of cyclin D1. Homozygous deletion of the cyclin D1 gene, enhanced expression by PPAR gamma ligands of PPAR gamma and PPAR gamma-responsive genes, and cyclin D1(-/-) mice exhibit hepatic steatosis. Finally, reduction of cyclin D1 abundance in vivo using ponasterone-inducible cyclin D1 antisense transgenic mice, increased expression of PPAR gamma in vivo. The inhibition of PPAR gamma function by cyclin D1 is a new mechanism of signal transduction cross talk between PPAR gamma ligands and mitogenic signals that induce cyclin D1.

Topics: 3T3 Cells; Animals; Breast; Breast Neoplasms; CCAAT-Enhancer-Binding Protein-alpha; CCAAT-Enhancer-Binding Protein-beta; Cyclin D1; Ecdysterone; Epithelial Cells; Fatty Liver; Female; Gene Expression Regulation; Humans; Mice; Mice, Mutant Strains; Mice, Transgenic; Models, Molecular; Mutation; Protein Conformation; Receptors, Cytoplasmic and Nuclear; Reference Values; Repressor Proteins; Rosiglitazone; Thiazoles; Thiazolidinediones; Transcription Factors; Transcriptional Activation

Transcriptional activation of the cyclin D1 gene is a key step in cell proliferation. Accordingly, cyclin D1 overexpression is frequently an early step in neoplastic transformation, particularly in mammary epithelium. Numerous studies have linked elevated cyclin D1 promoter activity to a sustained activation of the ERK1/2 cascade. Here we show that the ERK5 cascade, a distinct mitogen-induced MAPK pathway, can also drive cyclin D1 expression. In CCL39 cells, serum induces a strong, prolonged peak of ERK1/2 and ERK5 phosphorylation, and subsequently elevates cyclin D1 mRNA and protein levels. Overexpression of constitutively active MEK5 and wt ERK5 induces a cyclin D1 reporter gene (D1 -973-luciferase) at least as well as constitutively active MEK1. Activation is blocked by kinase-dead mutants of ERK5 and ERK2, respectively. Mutation of the CRE at -50 in the cyclin D1 promoter decreases activation by the ERK5 but not the ERK1/2 cascade. Importantly, expression of kinase-dead ERK5 diminishes endogenous cyclin D1 protein induction by serum in CCL39 cells and the breast cancer cell lines MCF-7 and HS579. These data identify the cyclin D1 gene as a novel target of the ERK5 cascade, an observation with important implications in cancers involving cyclin D1 deregulation.

Topics: Breast Neoplasms; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Kinetics; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase 7; Mitogen-Activated Protein Kinases; Promoter Regions, Genetic; Transcription, Genetic; Tumor Cells, Cultured

Cyclin D1 is frequently overexpressed in human neoplasias by gene rearrangement and amplification, but no mutations in the CCND1 gene have so far been reported. However, in vitro mutagenesis of CCND1 has shown that substitutions affecting threonine 286 residue produced cyclin D1 nuclear accumulation, by interfering with protein degradation and induced neoplastic transformation in murine fibroblasts. To test whether similar genetic changes may occur in vivo, we analysed a series of 60 endometrioid endometrial carcinomas (EECs) for cyclin D1 expression and gene amplification by immunohistochemistry and FISH, respectively. Two of 17 carcinomas showing cyclin D1 expression in more than 5% of neoplastic cells, but without gene amplification, were found to harbor single-base substitutions in CCND1 that changed proline 287 into threonine and serine, respectively. Both cases expressed cyclin D1 in more than 50% of neoplastic cells. Additionally, seven tumors with cyclin D1 overexpression of an independent series of 59 EECs were also analysed, and a 12-bp in-frame deletion that eliminated amino acids 289-292 was detected in one case with cylin D1 expression in more than 50% of neoplastic cells. In contrast, no mutations of the CCND1 gene were detected in a set of breast carcinomas with cyclin D1 overexpression without gene amplification. In summary, our data indicate that mutations of CCND1, which probably render the protein insensitive to degradation, represent a previously unreported mechanism of cyclin D1 overexpression in human tumors in vivo.

Topics: Breast Neoplasms; Carcinoma; Cyclin D1; DNA Mutational Analysis; Endometrial Neoplasms; Female; Gene Amplification; Humans; Mutation; Sensitivity and Specificity

Overexpression of the HER-2/neu receptor (HER-2) is associated with a poor prognosis in patients with breast carcinoma and also in patients with head and neck squamous cell carcinoma (HNSCC). In a previous study on HNSCC cell lines, we found that epigalocathechin-3-gallate (EGCG), a major biologically active component of green tea, inhibited activation of the epidermal growth factor receptor (EGFR) and thereby inhibited EGFR-related downstream signaling pathways in HNSCC cells. In the present study, we examined the effects of EGCG on activation of the HER-2 receptor in human HNSCC and breast carcinoma cell lines that display constitutive activation of HER-2. Treatment of these cells with 10 or 30 microg of EGCG, respectively, doses that cause 50% inhibition of growth, markedly inhibited the phosphorylation of HER-2 in both cell lines. This was associated with inhibition of Stat3 activation, inhibition of c-fos and cyclin D1 promoter activity, and decreased cellular levels of the cyclin D1 and Bcl-XL proteins. Although these concentrations of EGCG are quite high, we found that concentrations of 0.1-1.0 microg/ml, which are in the range of plasma concentrations after administering a single oral dose of EGCG or a green tea extract, markedly enhanced the sensitivity of both types of cell lines to growth inhibition by Taxol, a drug frequently used in the treatment of breast carcinoma and HNSCC. These results, taken together with previous evidence that EGCG also inhibits activation of the EGFR in carcinoma cells, suggest that EGCG may be useful in treating cases of breast carcinoma and HNSCC in which activation of the EGFR and/or HER-2 plays important roles in tumor survival and growth.

Topics: Antineoplastic Agents, Phytogenic; Blotting, Western; Breast Neoplasms; Carcinoma; Catechin; Cell Division; Cell Line, Tumor; Cyclin D1; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation, Neoplastic; Genes, Reporter; Head and Neck Neoplasms; Humans; Immunoblotting; Luciferases; Paclitaxel; Phosphorylation; Prognosis; Promoter Regions, Genetic; Proto-Oncogene Proteins c-fos; Receptor, ErbB-2; Signal Transduction; Time Factors

Posttranslational modifications of prolactin (PRL), including phosphorylation, vary with physiologic state and alter biologic activity. In light of the growing evidence for a role for PRL in proliferation in mammary cancer, we examined the ability of a mimic of phosphorylated human PRL, S179D-PRL, to initiate signals to several pathways in mammary tumor cells alone and in combination with unmodified PRL. Unmodified PRL employed multiple pathways to increase cellular proliferation and cyclin D1 levels in PRL-deficient MCF-7 cells. S179D-PRL was a weak agonist compared with unmodified PRL with regard to cellular proliferation, cyclin D1 levels, and phosphorylation of signal transducer and activator of transcription 5 and ERKs. However, S179D-PRL was a potent antagonist of unmodified PRL to these endpoints. In contrast to the reduced levels of the long isoform of the PRL receptor observed in response to a 3-d incubation with unmodified PRL, S179D-PRL up-regulated expression of this isoform, 4-fold. These studies support the utility of this mutant as a PRL antagonist to proliferative signals in mammary epithelial cells, including a potential role in breast cancer therapeutics.

Topics: Animals; Breast Neoplasms; Cell Count; Cell Division; Cell Line, Tumor; CHO Cells; Cricetinae; Cyclin D1; DNA-Binding Proteins; Humans; Milk Proteins; Mitogen-Activated Protein Kinases; Molecular Mimicry; Phosphorylation; Prolactin; Receptors, Prolactin; Signal Transduction; STAT5 Transcription Factor; Trans-Activators

We have reported previously that reactivation of progesterone receptor (PR) expression in estrogen receptor (ER)- and PR-negative MDA-MB-231 breast cancer cells enabled progesterone to inhibit cell growth and invasiveness, and to induce remarkable focal adhesions. The present study addressed molecular mechanisms that mediate these anticancer effects of progesterone in the PR-transfected breast cancer cells ABC28. In response to progesterone treatment are the marked up-regulation of cyclin-dependent kinase inhibitor protein p21WAF1/CIP1 and decreased expression of cyclin A, cyclin B1, and cyclin D1 that are required for G1 progression and during cell mitosis. Progesterone also induced down-regulation of phosphorylated MAPK (p42/44 MAPK). Furthermore, this study also demonstrated that MEK inhibitor PD98059 that inhibits the phosphorylation of p42/44 MAPK also caused reduction of cyclin D1 level and inhibition of cell proliferation. These results suggest that inhibition of p42/44 MAPK pathway is part of the mechanisms mediating progesterone's growth-inhibitory effect. On the other hand, progesterone-induced focal adhesion is mediated by separate pathway. Whereas PD98059 exhibited no effects on cell adhesion, inhibitory antibody to beta1-integrin was able to reverse progesterone-induced focal adhesion and progesterone-induced increase in the phosphorylation of focal adhesion kinase. On the other hand, beta1-integrin antibody had no effect on progesterone-mediated growth inhibition and on progesterone-mediated expression of cyclins p21CIP1/WAF1 and phosphorylation of P42/P44 MAPK. In the context of complex functions of progesterone in breast cancer and reproductive organs, identification of distinct pathways offers new strategies for designing therapeutic agents to target the specific pathway so as to minimize the side effects.

Topics: Antibodies; Breast Neoplasms; Cell Division; Cell Line, Tumor; Cyclin A; Cyclin B; Cyclin B1; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Enzyme Inhibitors; Flavonoids; Focal Adhesions; Humans; Integrin beta1; MAP Kinase Kinase Kinase 1; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Phosphorylation; Progesterone; Protein Serine-Threonine Kinases; Receptors, Estrogen; Receptors, Progesterone; Signal Transduction; Transfection; Up-Regulation

Ductal carcinoma in situ (DCIS) is a preinvasive-stage breast carcinogenesis that accounts for approximately 20 approximately 25% of mammographically detected breast cancers. A significant fraction of untreated DCIS will evolve into invasive cancer. ras homologue I (ARHI) is an imprinted tumor suppressor gene that is expressed in normal breast epithelial cells but absent or down-regulated in breast cancer cells. This study investigated the relationship of ARHI expression to the progression of breast cancer.. We analyzed ARHI expression in DCIS, invasive breast carcinoma, and adjacent normal breast epithelium from 64 formalin-fixed, paraffin-embedded DCIS specimens by both immunohistochemistry and in situ hybridization. We also analyzed the correlation between ARHI expression and progression of breast cancer, as well as the correlation of ARHI expression and cyclin D1 and p21(WAF1/CIP1) expression in DCIS.. Normal breast epithelium was found in all of the specimens and invasive breast carcinoma was found in 23 specimens. ARHI mRNA and protein were detected in all of the normal breast epithelia. ARHI expression was detected mainly in cytoplasm and rarely present in the nucleus. By histochemical analysis, ARHI expression was down-regulated in 41% (26 of 64) of DCIS and 70% (16 of 23) of invasive carcinomas comparing the specimens with adjacent normal breast epithelium. When DCIS and invasive cancer were present in the same sample, ARHI was further down-regulated in 26% (6 of 23) of invasive carcinoma. In four cases [4 (17%) of 23] of invasive carcinoma, ARHI protein expression was totally lost. Consistent results were obtained with an in situ hybridization assay for ARHI at the mRNA level. Higher levels of expression of cyclin D1 and p21(WAF1/CIP1) were observed in DCIS than in the adjacent epithelia. The expression of cyclin D1 and p21(WAF1/CIP1) was inversely correlated with that of ARHI.. Our results indicate that ARHI expression is markedly down-regulated in DCIS, and a further decrease in ARHI expression is associated with progression of breast cancer.

Topics: Adult; Aged; Antibodies, Monoclonal; Blotting, Western; Breast; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Cytoplasm; Digoxigenin; Disease Progression; DNA, Complementary; Down-Regulation; Female; Genes, Tumor Suppressor; Humans; Immunohistochemistry; In Situ Hybridization; Middle Aged; Reverse Transcriptase Polymerase Chain Reaction; rho GTP-Binding Proteins; RNA; RNA, Messenger; Sensitivity and Specificity; Transfection

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a member of the nuclear hormonal receptor superfamily expressed in a large number of human cancers. Here, we demonstrate that PPARgamma is expressed and transcriptionally active in breast cancer cells independent of their p53, estrogen receptor, or human epidermal growth factor receptor 2 status. 2-Cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO), a novel synthetic triterpenoid, is a ligand for PPARgamma. We investigated the molecular mechanisms of CDDO on proliferation and apoptosis in breast cancer cells. In all breast cancer cell lines studied, CDDO transactivated PPARgamma, induced dose- and time-dependent cell growth inhibition, cell cycle arrest in G(1)-S and G(2)-M, and apoptosis. We then used differential cDNA array analysis to investigate the molecular changes induced by CDDO. After 16-h exposure of MCF-7 and MDA-MB-435 cells to CDDO, we found genes encoding the following proteins to be up-regulated in both cell lines: p21(Waf1/CIP1); GADD153; CAAT/enhancer binding protein transcription factor family members; and proteins involved in the ubiquitin-proteasome pathway. Among the down-regulated genes, we focused on the genes encoding cyclin D1, proliferating cell nuclear antigen, and the insulin receptor substrate 1. Using Western blot analysis and/or real-time PCR, we confirmed that CDDO regulated the expression of cyclin D1, p21(Waf1/CIP1), and Bcl-2. Cyclin D1 and p21(Waf1/CIP1) were additionally confirmed as important mediators of CDDO growth inhibition in genetically modified breast cancer cell lines. CDDO was able to significantly reduce the growth of MDA-MB-435 tumor cells in immunodeficient mice in vivo. The finding that CDDO can target genes critical for the regulation of cell cycle, apoptosis, and breast carcinogenesis suggests usage of CDDO as novel targeted therapy in breast cancer.

Topics: Animals; Apoptosis; Breast Neoplasms; Cell Differentiation; Cell Division; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Humans; Immunocompromised Host; Mice; Mice, Nude; Oleanolic Acid; Receptors, Cytoplasmic and Nuclear; Transcription Factors

To investigate the effects of estrogen (E2) on telomerase activity and its mechanism in human breast cancer cells, estrogen receptor positive MCF-7 cells were treated with different concentrations of E2. Telomerase activity was measured by using TRAP-ELISA method, the cell cycle phases analyzed by using flow cytometry, and the expression of Cyclin D1 detected by using immunohistochemistry method. The results showed that telomerase activity levels were increased in MCF-7 cells treated with 10(-8) mol/L E2 during the observed period (P < 0.05), and E2 increased telomerase activity levels in a dose-dependent manner(10(-10)-10(-8) mol/L); Simultaneously, the cell cycle phases of MCF-7 cells treated with 10(-8) mol/L E2 were changed significantly: G0/G1 phase decreased from 60.52% to 50.93%. S phase increased from 29.03% to 30.83%; However, the expression of Cyclin D1 was decreased. It was concluded that estrogen can upregulate telomerase activity of MCF-7 cells, and the effect can be blocked by antiestrogen tamoxifen. Its mechanism may be closely associated with modulation of cell cycle phases.

Topics: Breast Neoplasms; Cell Cycle; Cyclin D1; Estrogens; Female; Humans; Receptors, Estrogen; Telomerase; Tumor Cells, Cultured

The objective of this article was to compare five tumor markers between white women in the U.S. and native Korean women with early-onset breast carcinoma.. Sixty Korean women who were diagnosed with breast carcinoma at age 45 years or younger and 60 white women with breast carcinoma who were matched by age were selected for this study. The median age of both groups was 37 years. Paraffin embedded blocks of the primary tumor were processed for immunohistochemical staining of estrogen receptor (ER), progesterone receptor (PR), p53, cyclin D1, and HER-2/neu.. The proportion of tumors that stained positive for ER, PR, p53, and cyclin D1 in the Korean women were 47.5%, 42.4%, 28.8%, and 40.9%, respectively; in the white women, the proportions were 43.9%, 52.6%, 21.1%, and 59.1%, respectively. The differences between the white patients and the Korean patients were not statistically significant with respect to any of those variables. A significant difference was found in the expression of HER-2/neu. Specifically, positive HER-2/neu status was observed in 47.5% of Korean women, compared with overexpression in only 15.8% of white women (P < 0.001). Fluorescence in situ hybridization analysis for HER-2/neu gene amplification on all HER-2/neu positive samples that scored 2 + and 3 + demonstrated a significant difference (P = 0.007) in gene amplification between the two populations. Differences in HER-2/neu positivity were observed for the entire cohort as well as among the subsets of patients with negative and positive lymph node status. No association was found between immunoreactivity for the five markers and axillary lymph node metastasis.. The findings of high positivity of HER-2/neu expression and gene amplification in Korean women with early-onset breast carcinoma may have potential implications for local and systemic management of breast carcinoma, especially anti-HER-2/neu therapy for patients with hormone receptor negativity. Further research will be needed to identify biologic and genetic factors and their effects on the survival between different racial groups.

Topics: Adult; Asian People; Biomarkers; Breast Neoplasms; Cyclin D1; Female; Gene Amplification; Genes, erbB-2; Humans; Immunohistochemistry; In Situ Hybridization, Fluorescence; Korea; Receptors, Estrogen; Receptors, Progesterone; Tumor Suppressor Protein p53; White People

Arzoxifene (ARZ) is a selective estrogen receptor (ER) modulator with greater bioavailability than raloxifene which is being developed as treatment for breast cancer. We have used an in vivo model of hormone-sensitive breast cancer to study the growth-inhibitory and pharmacodynamic effects of ARZ in comparison with the most widely used antiestrogen, tamoxifen (TAM). We compared the abilities of ARZ and TAM to antagonize the estrogen (E2)-dependent growth of MCF-7 human breast cancer xenografts in oophorectomized athymic mice. At four different time points over 28 days, we studied their time-related pharmacodynamic effects on biomarkers of tumor growth (cell proliferation/death measured by Ki-67 and apoptosis scores), cell cycle activity (cyclin D1 and p27(kip1)), and hormone-regulated gene expression (ERalpha, progesterone receptor, and pS2). Although maximal growth inhibition was seen after E2 withdrawal, ARZ and TAM induced significant and similar inhibition of E2-stimulated tumor growth. Inhibition of growth was reflected by changes in the tumor growth index (ratio posttreatment/pretreatment Ki-67/apoptosis scores). ARZ and TAM resulted in a significant (P < 0.001) increase in ER expression and reduction in progesterone receptor expression, whereas changes in cyclin D1 score were inversely related to p27(kip1) score. A significant but delayed biological effect was observed with a 10-fold lower dose of ARZ. These results show that ARZ is an effective antagonist of E2-stimulated breast cancer growth with similar growth-inhibitory and pharmacodynamic effects to TAM in this model.

Topics: Animals; Apoptosis; Biomarkers, Tumor; Breast Neoplasms; Cell Cycle Proteins; Cell Division; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Female; Humans; Mice; Mice, Nude; Piperidines; Protein Biosynthesis; Proteins; Receptors, Estrogen; Receptors, Progesterone; Selective Estrogen Receptor Modulators; Tamoxifen; Thiophenes; Trefoil Factor-1; Tumor Suppressor Proteins; Xenograft Model Antitumor Assays

AND-34 is a murine protein that binds by a cdc25-like GDP exchange factor domain to the focal adhesion docking protein p130Cas. Overexpression of either of the human homologues of AND-34 and p130Cas, BCAR3 and BCAR1, respectively, has been reported to induce resistance to antiestrogens in breast cancer cell lines. Here we show that overexpression of AND-34 leads to activation of the Rho family GTPases Cdc42 and Rac. Consistent with these findings, BCAR3 overexpression induced alterations in F-actin distribution and augmented both autophosphorylation and kinase activity of the Cdc42/Rac-responsive serine/threonine kinase PAK1. p130Cas-associated BCAR3 protein was detected in the estrogen-independent breast cancer cell line 578-T, but not in estrogen-dependent MCF7 or ZR-75-1 cells. Stable ZR-75-1 transfectants overexpressing BCAR3, but not vector-only transfectants, grew in the presence of the pure antiestrogen ICI 182,780. Stable transfection with RacV12, a constitutively active form of Rac1, also induced antiestrogen resistance in ZR-75-1 cells. Transient transfection of BCAR3 in estrogen-dependent MCF7 cells induced activation of luciferase constructs containing the proximal 1745 or 163 bp but not 66 bp of the cyclin D1 promoter. Such cyclin D1 promoter activation was inhibited by dominant negative forms of Rac1 and PAK1. Overexpression of the PAK1 autoinhibitory domain (residues 83-149) but not an inactive PAK1 autoinhibitory domain point mutant (L107F) also blocked BCAR3-mediated cyclin D1 activation. These studies suggest that AND-34/BCAR3 induces antiestrogen resistance in breast cancer cell lines by a Rac1- and PAK1-dependent pathway.

Topics: Adaptor Proteins, Signal Transducing; Amino Acid Sequence; Animals; Breast Neoplasms; Carrier Proteins; cdc42 GTP-Binding Protein; Cell Line, Tumor; Cyclin D1; Enzyme Activation; Estradiol; Estrogen Receptor Modulators; Fulvestrant; Guanine Nucleotide Exchange Factors; Humans; Mice; Molecular Sequence Data; Neoplasms, Hormone-Dependent; NIH 3T3 Cells; p21-Activated Kinases; Promoter Regions, Genetic; Protein Biosynthesis; Protein Serine-Threonine Kinases; Proteins; Rabbits; rac1 GTP-Binding Protein; Transfection

Ductal carcinoma in situ (DCIS) of the breast constitutes about 10% of all diagnosed breast cancers and, despite surgical removal, it may recur, either as DCIS or invasive breast cancer. Nuclear grade and growth pattern according to Andersen et al as well as surgical margins are factors that have been used to predict local recurrence, but ideally a set of tumour-specific factors should be identified and used as prognostic markers. Many cell cycle regulatory gene products have been shown to be involved in the formation of tumours and are either oncogenes or suppressor genes and involved in key processes in the transformation. We therefore characterised the cell cycle regulators cyclin E, cyclin D1, p27 and p16 in a material of DCIS cases arranged in a tissue microarray. With a manual tissue arrayer, 52% of the initial 177 DCIS samples were successfully targeted allowing immunohistochemical analyses of all four proteins in 92 cases of DCIS. As also observed in invasive breast cancer, there was a trend indicating that DCIS cases with high cyclin D1 were cyclin E low and oestrogen receptor-positive, whereas cyclin E high DCIS cases were cyclin D1 low and oestrogen receptor-negative. For the 64 patients that did not receive postoperative radiotherapy, there were 16 local recurrences (eight DCIS and eight invasive breast cancer) during a mean follow-up time of 63 months. Cyclin E, p27 or p16 were not associated with local recurrence, but interestingly cyclin D1 was significantly and inversely associated with local recurrence, both using univariate and multivariate analyses. In summary, using a tissue array approach we have shown that cyclin D1, besides growth pattern, is a prognostic marker for local recurrence in DCIS.

Topics: Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Immunohistochemistry; Neoplasm Recurrence, Local; Oncogenes

To investigate the clinical significance of the protein expression of 4 cell cycle regulators in papillomatosis of the breast.. Immunohistochemistry was used to examine the protein expression of CyclinD1, p16, E2F-1 and Rb in 4 groups (196 cases) with mild papillomatosis (MP), moderate papillomatosis (MoP), serious papillomatosis (SP) and ductal carcinoma in situ (DCIS).. There were significant difference of the positive rate and the intensity of CyclinD1, p16, E2F-1 and Rb expression among the groups, respectively (chi(2) were 16.702, 20.742, 40.335, 42.317; 19.120, 29.469, 45.080, 46.920, P < 0.01. Meaning time there were significant difference in the expression of these 4 factors between MoP and SP (P < 0.05). There was significance of E2F-1 or Rb expression between SP and DCIS (P < 0.01), but there was no significance to be found in the expression of CyclinD1 or p16 between the two groups (P > 0.05). Rb was screened as the highest risk factor by Odd Risk Logistic Analysis.. CyclinD1, p16, E2F-1 and Rb played the important roles from MP into SP and then DCIS. E2F-1 and Rb can be used as the assistant indicators on the differentiation of SP and DCIS. Rb may be helpful in screening high risk cases from SP or MoP.

Topics: Breast Neoplasms; Cell Cycle; Cell Cycle Proteins; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Diagnosis, Differential; DNA-Binding Proteins; E2F Transcription Factors; E2F1 Transcription Factor; Female; Humans; Immunohistochemistry; Logistic Models; Papilloma; Retinoblastoma Protein; Transcription Factors

B-cell lymphoma gene (BCL-6) upregulation contributes to immortalization of mouse embryo fibroblast and primary B cells via upregulation of cyclin D1. As cyclin D1 overexpression is a common phenomenon in different cancers, BCL-6 protein overexpression may not be restricted to lymphomas. In this study, expression of BCL-6 was investigated by immunohistochemistry on paraffin-embedded specimens from 150 breast cancer patients and 10 specimens of normal breast tissue. The results showed BCL-6 overexpression (> or =10% of cells) in 24/150 (16%) breast cancer patients, whereas in normal breast low expression (<1%) of BCL-6 was observed. In linear regression analysis BCL-6 expression was associated with cyclin D1 (r=0.197, P=0.016). Further, in chi2 analyses, BCL-6-positivity was associated with overexpression of p53 (P=0.016), and hypoxia-inducible factor-1alpha (P<0.001). Involvement of BCL-6 in breast carcinogenesis is further underscored by comparative genomic hybridization analysis that showed gains at the BCL-6 locus (3q27) in 14/86 (16%) breast cancer tissues. The cases with amplification in BCL-6 showed an increased (25%) incidence of BCL-6 protein overexpression. Thus, this study is the first to show that BCL-6 oncogene activation plays a role in cancers other than lymphomas.

Topics: Breast Neoplasms; Carcinoma, Ductal; Carcinoma, Ductal, Breast; Cyclin D1; DNA-Binding Proteins; Female; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Immunohistochemistry; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-6; Transcription Factors

All malignant cells appear to have increased needs for glucose in vitro as well as in vivo. The enhanced glucose uptake is mediated through transporters (Gluts), whose action and expression are regulated by oncogenes and growth factors. Cyclin D1 is a nuclear protein that plays an important role in regulating the cell cycle by promoting entry of cells from the G1 to S phase. Increased expression of Glut1 glucose transporter and cyclin D1 have been reported in several neoplasms. In the present study we examined the expression of Glut1 and cyclin D1 in breast carcinomas with negative lymph nodes. We studied 78 infiltrating ductal carcinomas (25 grade 1, 36 grade 2, and 17 grade 3) with negative lymph nodes. Glut1 was expressed in 28% of grade 1, 63.8% of grade 2 and 58.7% of grade 3 carcinomas. Nuclear expression of cyclin D1 was detected in 32% of grade 1, 44.4% of grade 2, and 41.2% of grade 3 carcinomas. From our study it appears that in breast carcinomas with negative lymph nodes Glut1 expression is better correlated to the grade of the neoplasm than cyclin D1.

Topics: Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Ductal, Breast; Cyclin D1; Female; Glucose Transporter Type 1; Humans; Immunohistochemistry; Lymph Nodes; Lymphatic Metastasis; Monosaccharide Transport Proteins; Predictive Value of Tests

The biologic effect of continuing hormone replacement therapy (HRT) after a diagnosis of breast carcinoma is unclear. The goal of rhe current study was to determine the short-term effect of HRT withdrawal on invasive breast carcinoma using biologic surrogate markers of tumor response.. The study was performed between 1996 and 2000 and comprised 140 women who had been using HRT at the time of breast carcinoma diagnosis by core needle biopsy. The breast tumors were removed a median of 17 days later (range, 2-31 days). Of these women, 125 women stopped HRT at the time of core needle biopsy and 15 continued to receive HRT until surgery. In addition, 55 women with breast carcinoma from the same time period, who were not receiving HRT at diagnosis, were studied. Changes in expression of Ki-67 (a measure of epithelial cell proliferation), progesterone receptor (PR), p27KIP-1 (a cyclin-dependent kinase inhibitor), and cyclin D1 (a cell cycle-related protein) were determined by immunohistochemistry on paired sections of the core needle biopsy and surgical specimens from each patient.. In women who stopped HRT, a significant decrease in Ki-67 expression was observed between core needle biopsy and surgery in estrogen receptor (ER)-positive (n = 106; P < 0.001), but not in ER-negative tumors (n = 19; P = 0.58), with an associated reduction in PR (P < 0.001) and cyclin D1 expression (P < 0.001) and an increase in p27KIP-1 (P = 0.03). These changes in Ki-67 and PR expression occurred irrespective of c-erb-B2 status. No change was observed in any parameter in the other groups of patients.. ER-positive invasive breast carcinomas demonstrated a favorable biologic response to withdrawal of HRT. Therefore, HRT should be stopped at the time of diagnosis and was subsequently contraindicated.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Biopsy, Needle; Breast Neoplasms; Carcinoma, Ductal; Carcinoma, Lobular; Cell Cycle Proteins; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Estrogen Replacement Therapy; Female; Humans; Immunoenzyme Techniques; Ki-67 Antigen; Middle Aged; Receptors, Estrogen; Receptors, Progesterone; Risk Factors; Substance Withdrawal Syndrome; Tumor Suppressor Proteins

Paget's disease of the nipple (PDN) is characterized by Paget's cells infiltrating from an underlying breast carcinoma into the epidermis of the nipple. The tumor cells were reported to overexpress ERBB-2 protein. There is no evidence, whether the overexpression is caused by amplification of the ERBB2 gene (as is the case of invasive duct carcinomas) or by other causes. Because the Paget's cells proliferate vividly we were interested also in cyclin D1 expression and in its relation to the number of CCND1 gene copies. To address these issues, we performed a study using fluorescence in situ hybridization on interphasic nuclei (I-FISH) on formalin fixed / paraffin embedded tissue sections from twelve women with PDN. We compared immunohistochemical (IHC) expression of the ERBB-2 protein and cyclin D1 with the copy number of the gene ERBB2 and CCND1, respectively (I-FISH). In all cases of our series we found overexpression of the ERBB-2 protein (3+), and in all of them there was a strong amplification of the ERBB2 gene (more than 10 signals per a nucleus, usually about 20). Keratinocytes of the epidermis had two signals of the gene. IHC and I-FISH revealed comparable findings in successive biopsies in two patients. Cyclin D1 was positive in seven cases. We found a moderate amplification of the CCND1 gene (up to 10 signals per a nucleus) in three patients only. Any correlation between the cyclin D1 overexpression anda copy number of the CCND1 gene was observed. Combined numerical changes of chromosome 17 and of chromosome 11 were found in seven women. It is concluded that in PDN, the ERBB-2 protein overexpression is caused by amplification of the ERBB2 gene. A specific immuno-therapy with trastuzumab (Herceptin) used in patients with invasive duct carcinoma may be also effective in patients with PDN.

Topics: Adult; Aged; Aged, 80 and over; Breast Neoplasms; Cyclin D1; Female; Gene Amplification; Genes, erbB-2; Humans; Lymphatic Metastasis; Middle Aged; Neoplasm Staging; Paget's Disease, Mammary; Receptor, ErbB-2

This study was designed to comprehensively analyze the differential expression of proteins from human umbilical vein endothelial cells (HUVECs) exposed to tumor conditioned medium (TCM) and to identify the key regulator in the cell cycle progression. The HUVECs were exposed to TCM from breast carcinoma cell line MDA-MB-231, then their cell cycle distribution was measured by flow cytometer (FCM). The role of protein in cell cycle progression was detected via two-dimensional polyacrylamide gel electrophoresis (2-DE) and western blotting. Following the stimulation of TCM, HUVECs showed a more cells in the S phase than did the negative control group (ECGF-free medium with 20% FBS), but the HUVECs' level was similar to the positive control group (medium with 25 micrograms/ml ECGF and 20% FBS). Increased expression of cyclin D1/E and some changes in other related proteins occurred after incubation with TCM. From our results, we can conclude that breast carcinoma cell line MDA-MB-231 may secrete soluble pro-angiogenic factors that induce the HUVEC angiogenic switch, during which the expression of cell cycle regulator cyclin D1/E increases and related proteins play an important role in this process.

Topics: Angiogenesis Inducing Agents; Apoptosis; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cells, Cultured; Culture Media, Conditioned; Cyclin D1; Cyclin E; Electrophoresis, Gel, Two-Dimensional; Endothelial Cells; Endothelium, Vascular; Female; Flow Cytometry; Gene Expression Profiling; Humans; Hydrogen Peroxide; Mitosis; Protein Biosynthesis; Proteins; Proteomics; Subtraction Technique; Umbilical Veins

LKB1 (also called STK11) is a recently identified tumor suppressor gene in which its mutation can lead to Peutz-Jeghers syndrome, characterized by gastrointestinal polyps and cancers of different organ systems. Weak expression of this gene does occur at a certain frequency in sporadic breast cancer. This indicates that LKB1 gene may relate to the tumorigenesis of breast cancer.. To investigate the function of the LKB1 gene in sporadic breast cancer, we reintroduced LKB1 into breast cancer cell lines which lack the LKB1 gene. Also, we examined the LKB1 protein expression in human breast cancer samples.. We found that reintroducing LKB1 into breast cancer cell lines suppresses cell growth by G(1) cell cycle block. The LKB1-mediated G(1) cell cycle arrest is caused by up-regulation of the expression of p21(WAF1/CIP1) in breast cancer MDA-MB-435 cells. We also demonstrated that low LKB1 protein expression correlates with higher histological grade (P = 0.013), larger tumor size (P = 0.001), progesterone receptor status (P = 0.048), and presence of lymph node metastasis (P = 0.003). Furthermore, LKB1 low expression was associated with a higher relapse rate (P = 0.002) and a worse OS (P = 0.008).. LKB1 plays a role in tumor suppressor function in human breast cancer. LKB1 expression may be a useful prognostic marker in human breast cancer.

Topics: Adult; Aged; AMP-Activated Protein Kinase Kinases; Biomarkers, Tumor; Breast Neoplasms; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; DNA Primers; Female; Flow Cytometry; G1 Phase; Genes, Tumor Suppressor; Humans; Middle Aged; Prognosis; Protein Serine-Threonine Kinases; Receptors, Estrogen; Receptors, Progesterone; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transfection; Tumor Cells, Cultured

Previous studies have suggested that antiestrogens inhibit MCF-7 cell proliferation by alteringthe expression or activity of components of the insulin-like growth factor I (IGF-I) signaling pathway, including IGF-I receptor, insulin receptor substrate 1, and phosphatidylinositol 3-kinase. In this report, we examine the effects of the pure antiestrogen ICI 182,780 (ICI) on various targets of IGF-I signaling in MCF-7 cells. ICI treatment led to decreases in the absolute levels of cyclin D1 and cyclin A expression, retinoblastoma protein phosphorylation, and DNA synthesis in IGF-I-treated cells. However, IGF-I retained the ability to induce these events in the presence of ICI, suggesting that ICI treatment did not completely block IGF-I signaling. Consistent with this suggestion, IGF-I-induced phosphorylation of extracellular signal-regulated kinase, AKT, and insulin receptor substrate 1 was unaffected by ICI treatment. Finally, transient expression of either constitutively active phosphatidylinositol 3-kinase or AKT was unable to induce proliferation in ICI-treated MCF-7 cells. Together, these results indicate that ICI can inhibit proliferation without blocking IGF-I signaling and suggest a model in which both estrogen receptor and IGF-I signaling regulate cell cycle components and are required for MCF-7 cell proliferation.

Topics: Antineoplastic Agents, Hormonal; Breast Neoplasms; Cell Division; Cyclin D1; Drug Resistance, Neoplasm; Enzyme Activation; Estradiol; Estrogen Receptor Modulators; Fulvestrant; Humans; Insulin Receptor Substrate Proteins; Insulin-Like Growth Factor I; Mitogen-Activated Protein Kinase Kinases; Phosphatidylinositol 3-Kinases; Phosphoproteins; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Receptors, Estrogen; RNA, Messenger; Signal Transduction; Tumor Cells, Cultured

We have examined whether inhibition of phosphatidylinositol-3 kinase (PI3K) and its target, the serine/threonine kinase Akt, play a role in the antitumor effect of the HER2 antibody Herceptin. Herceptin inhibited colony formation, down-regulated cyclin D1, and increased p27 protein levels in the HER2 gene-amplified BT-474 and SKBR-3 human breast cancer cells. These effects were temporally associated with the inhibition of PI3K activity in vitro as well as Akt function as measured by steady-state levels of phospho-Ser473 Akt and kinase activity against glycogen synthase kinase (GSK)-3beta. These responses were not observed in MDA-361 and MDA-453 cells, which do not exhibit HER2 gene amplification and are relatively resistant to Herceptin. Treatment of BT-474 cells with Herceptin inhibited the constitutive tyrosine phosphorylation of HER3 and disrupted the basal association of HER3 with HER2 and of HER3 with p85alpha potentially explaining the inhibition of PI3K. Treatment with either Herceptin or the PI3K inhibitor LY294002 increased the levels of p27 in the nucleus>cytosol, thus increasing the ratio of p27:Cdk2 in the nucleus and inhibiting Cdk2 activity and cell proliferation. Antisense p27 oligonucleotides abrogated the increase in p27 induced by Herceptin and prevented the antibody-mediated reduction in S phase. Transduction of BT-474 cells with an adenovirus-encoding active (myristoylated) Akt (Myr-Akt), but not with a beta-galactosidase control adenovirus, prevented the Herceptin- or LY294002-induced down-regulation of cyclin D1 and of phosphorylated GSK-3beta and prevented the accumulation of p27 in the nucleus and cytosol. In addition, Myr-Akt prevented Herceptin-induced inhibition of the cell proliferation of BT-474 cells and Herceptin-induced apoptosis of SKBR-3 cells. These data suggest that (a) changes in cell cycle- and apoptosis-regulatory molecules after HER2 blockade with Herceptin result, at least in part, from the inhibition of Akt; and (b) disabling PI3K and Akt is required for the antitumor effect of HER2 inhibitors.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Breast Neoplasms; Cell Cycle Proteins; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Gene Amplification; Genes, erbB-2; Humans; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Receptor, ErbB-2; Signal Transduction; Trastuzumab; Tumor Cells, Cultured; Tumor Suppressor Proteins

Treatment of MCF7 human breast cancer cells with taxol induces G2/M arrest followed by mitotic death. A moderate overexpression of ectopic cyclin D1 accelerated these G2/M associated events and resulted in a reduced clonogenic survival upon taxol treatment. Taxol treatment resulted in elevated expression of p53 and of p21, which was more pronounced and persistent in cyclin D1 overexpressing cells. Overexpression of cyclin D1 altered sensitivity to taxol by modulating exit from mitosis, which is controlled by p21. These results indicate that overexpression of cyclin D1 sensitizes MCF7 cells to treatment with taxol.

Topics: Antineoplastic Agents, Phytogenic; Breast Neoplasms; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Mitosis; Paclitaxel; Tumor Cells, Cultured

Morphine is used to treat pain in several medical conditions including cancer. Here we show that morphine, in a concentration typical of that observed in patients' blood, stimulates human microvascular endothelial cell proliferation and angiogenesis in vitro and in vivo. It does so by activating mitogen-activated protein kinase/extracellular signal-regulated kinase phosphorylation via Gi/Go-coupled G protein receptors and nitric oxide in these microvascular endothelial cells. Other contributing effects of morphine include activation of the survival signal PKB/Akt, inhibition of apoptosis, and promotion of cell cycle progression by increasing cyclin D1. Consistent with these effects, morphine in clinically relevant doses promotes tumor neovascularization in a human breast tumor xenograft model in mice leading to increased tumor progression. These results indicate that clinical use of morphine could potentially be harmful in patients with angiogenesis-dependent cancers.

Topics: Analgesics, Opioid; Animals; Apoptosis; Breast Neoplasms; Cell Cycle; Cyclin D1; Endothelium, Vascular; Female; Humans; Male; MAP Kinase Signaling System; Mice; Mice, Inbred BALB C; Mice, Nude; Mitogen-Activated Protein Kinase Kinases; Morphine; Neovascularization, Pathologic; Neovascularization, Physiologic; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Stimulation, Chemical

The issue of how progesterone affects mammary gland growth is controversial, and the mechanism governing the effects of the hormone remains mostly unknown. We have previously shown that G protein-coupled receptor 30 (GPR30) is a progestin target gene whose expression correlates with progestin-induced growth inhibition in breast cancer cells. In this study, we investigate the role of GPR30 in regulating cell proliferation and mediating progestin-induced growth inhibition. When progestin failed to inhibit the growth of MCF-7 cells and instead stimulated growth, GPR30 was down-regulated. In this way, the inhibitory or stimulatory affects that progestin has on proliferation correlated with the level of expression of GPR30. Transient expression of GPR30 resulted in a marked inhibition of cell proliferation independent of estrogen treatment. GPR30 antisense was used to evaluate the role of GPR30 expression in progestin-induced growth inhibition. A diminished GPR30 mRNA expression by the antisense stimulated growth. Interestingly, GPR30 antisense abrogated the growth inhibitory effect of progestin and progesterone. Indeed, progestin induced 1) a reduction in cell proliferation, 2) G1-phase arrest, and 3) down-regulation of cyclin D1 was diminished. These data suggest that the orphan receptor, GPR30, is important for the inhibitory effect of progestin on growth.

Topics: Breast Neoplasms; Cell Cycle; Cell Division; Cyclin D1; DNA; Estradiol; Flow Cytometry; G1 Phase; Gene Expression; Humans; Medroxyprogesterone Acetate; Oligodeoxyribonucleotides, Antisense; Progestins; Receptors, Cell Surface; Receptors, Estrogen; Receptors, G-Protein-Coupled; Receptors, Progesterone; RNA, Messenger; Tetracycline; Transfection; Tumor Cells, Cultured

Because of the relatively low incidence of lobular breast carcinoma, there are very few studies on the molecular characteristics of this breast cancer. In an attempt to improve its characterization, we investigated in a large collection of invasive lobular carcinomas (ILCs) the status of markers known to be involved in the better-studied invasive ductal carcinomas (IDC). In the current study we disposed of 80 well-characterized ILC cases. Gene amplification of cyclin D1 (CCND1) and c-erbB2-encoding gene (ERBB2) and expression of their gene products were studied by differential polymerase chain reaction (PCR) and immunohistochemistry, respectively. A comprehensive point mutation study of the phosphatase and tensin homolog tumor suppressor gene (PTEN) was pursued by single strand conformation polymorphism (SSCP)/sequencing analysis. The CCND1 gene was rarely amplified in ILC in spite of showing overexpression of the protein in 41% of tumors. Hence, unlike IDC, increase in gene dosage did not account for the protein excess. PTEN mutations were detected in ILC (truncating mutations) in around 2% of the tumors. Unlike IDC, ILC did not display ERBB2 overexpression and expression of the transcription factor E2F1 correlated inversely with tumor grade. The observed discrepancy in the pattern of the human oncogenes CCND1 and ERBB2, which are involved in the process of carcinogenesis of ductal tumors, appears to suggest a different molecular basis for development and progression of ILC.

Topics: Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Lobular; Cell Cycle Proteins; Cyclin D1; DNA Mutational Analysis; DNA-Binding Proteins; E2F Transcription Factors; E2F1 Transcription Factor; Female; Gene Amplification; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Ki-67 Antigen; Neoplasm Invasiveness; Phosphoric Monoester Hydrolases; Point Mutation; Polymorphism, Single-Stranded Conformational; PTEN Phosphohydrolase; Receptor, ErbB-2; Transcription Factors; Tumor Suppressor Proteins

Most breast cancers arise from luminal epithelial cells and 25-30% of these tumours overexpress the ErbB-2 receptor. Herein, a non-transformed, immortalized cell system was used to investigate the effects of ErbB-2 overexpression in luminal epithelial cells. The phenotypic consequence of ErbB-2 overexpression is a shortening of the G1 phase of the cell cycle and early S phase entry, which leads to hyperproliferation. We show that this effect was mediated through the up-regulation of cdk6 and cyclins D1 and E, and enhanced degradation and relocalization of p27(Kip1). These changes were effected predominantly through enhanced MAPK signalling, resulting in cdk2 hyperactivation. PI3K signalling also participated in cell cycle progression, since PI3K and MAPK coordinately regulated changes in cyclin D1 and cdk6 expression. Cdk4 activity was not required for cell cycle progression in these cells, and was constitutively inhibited through its association with p16(INK4A). MAPK-dependent induction of p21(Cip1) was also necessary for G1 phase progression, although its degradation by the proteasome was required for S phase entry. These data provide new insights into the complex molecular mechanisms underlying mitogenic cell cycle control in luminal epithelial cells, the cell type relevant to primary breast cancer, and show how ErbB-2 overexpression subverts this normal control.

Topics: Breast; Breast Neoplasms; CDC2-CDC28 Kinases; Cell Cycle; Cell Division; Cell Transformation, Neoplastic; Cyclin D1; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; Epidermal Growth Factor; Epithelial Cells; Female; Humans; Microfilament Proteins; Mitogen-Activated Protein Kinases; Muscle Proteins; Phosphatidylinositol 3-Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-myc; Receptor, ErbB-2

Breast cysts are associated with an increased risk of breast cancer. Some biomarkers such as estrogen receptor alpha (ERa), progesterone receptor (PR), and cyclin D1, show similar patterns of expression in epithelial cells lining breast cysts as malignant epithelial cells in local and invasive ductal breast cancer. We have attempted to answer two questions: (1) Do epithelial cells lining breast microcysts (cysts which can only be seen with a microscope) express biomarkers in a similar pattern to breast ductal carcinoma in situ and invasive ductal carcinoma? (2) Are breast microcysts precursors of breast cancer or are they part of normal involution of the breast? Seventy two archival open breast biopsy specimens of ductal carcinoma in situ and invasive ductal carcinoma and 32 normal breast biopsies from Australian women who had breast reduction surgery were selected from hospital archives. All specimens were analysed by standard immunohistochemistry for ERa, PR, cyclin D1, bcl-2, p53 and erbB-2 expression. In the same specimens, the pattern of high biomarker expression was very similar for all the above biomarkers in epithelial cells lining microcysts and in both ductal carcinoma in situ and invasive ductal carcinoma c. ErbB-2 was not expressed in normal control specimens. ErbB-2 was expressed in the same specimens in an increasing proportion of normal breast acini, microcysts and cancer cells in 36% of specimens with breast cancer. An apparent progression was observed from normal breast acini, to proliferation of epithelial cells in microcysts, ductal carcinoma in situ and invasive ductal carcinoma in the same specimen. When these findings are considered with other reports we conclude: (1) that epithelial cells lining breast cysts highly express biomarkers in a similar pattern to ductal carcinoma in situ and invasive ductal carcinoma; (2) that some microcysts are not part of normal involution of the breast and in some women may be part of the transition from normal to cancer.

Topics: Biomarkers, Tumor; Biopsy; Breast; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Cyclin D1; Estrogen Receptor alpha; Female; Fibrocystic Breast Disease; Humans; Immunoenzyme Techniques; Middle Aged; Proto-Oncogene Proteins c-bcl-2; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone; Tumor Suppressor Protein p53

We have utilized in vitro three-dimensional epithelial cell cultures to analyze the role of apoptosis in the formation and maintenance of a hollow glandular architecture. Lumen formation is associated with the selective apoptosis of centrally located cells; this apoptosis follows apicobasal polarization and precedes proliferative suppression during acinar development. Notably, either inhibiting apoptosis (by exogenously expressing antiapoptotic Bcl family proteins) or enhancing proliferation (via Cyclin D1 or HPV E7 overexpression) does not result in luminal filling, suggesting glandular architecture is resistant to such isolated oncogenic insults. However, the lumen is filled when oncogenes that enhance proliferation are coexpressed with those that inhibit apoptosis, or when ErbB2, which induces both activities, is activated by homodimerization. Hence, apoptosis can counteract increased proliferation to maintain luminal space, suggesting that tumor cells must restrain apoptosis to populate the lumen.

Topics: Actins; Anticoagulants; Apoptosis; Biocompatible Materials; Breast; Breast Neoplasms; Cell Death; Cell Division; Collagen; Cyclin D1; Dextrans; Drug Combinations; Humans; Ki-67 Antigen; Laminin; Microscopy, Electron; Microscopy, Fluorescence; Oncogene Proteins, Viral; Papillomavirus E7 Proteins; Protein Serine-Threonine Kinases; Proteoglycans; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Receptor, ErbB-2; Retroviridae; Time Factors; Tumor Cells, Cultured

Cyclin D1, an important cell cycle regulator, is overexpressed in several human cancers including breast. Both estrogens and progestins activate the transcription of the gene; antiestrogens have been shown to reduce cyclin D1 protein levels. Cyclin D1 protein overexpression has been strongly associated with well-differentiated, estrogen receptor-positive tumors. Little is known, however, as to whether epidemiological risk factors are related to this molecularly defined subset of tumors. Using a population-based study of young women <45 years in New Jersey, we analyzed whether oral contraceptives (OCs) and other risk factors were associated with the overexpression of cyclin D1 in breast cancer tissue. We measured cyclin D1 status in paraffin-embedded, archived tissue from 78.8% of the breast cancer cases using immunohistochemistry. Cyclin D1 was overexpressed in 33.7% of the cases (123 of 365). We used unordered polytomous logistic regression to estimate the odds ratios (ORs) for two case groups--(a) breast cancer with cyclin D1 overexpression (n = 123) and (b) breast cancer without overexpression (n = 242)--compared with 462 population-based controls. The multivariate-adjusted OR for ever use of OCs was 1.6 [95% confidence interval (CI), 1.0-2.5] for cases that overexpressed cyclin D1 and 1.0 (95% CI, 0.7-1.5) for those with no overexpression. Among women who started using OCs at least 20 years before the reference date, the OR was increased 2-fold for breast cancer with cyclin D1 overexpression (OR, 2.2; 95% CI, 1.2-4.0) but not for breast cancer without cyclin D1 overexpression (OR, 1.1; 95% CI, 0.7-1.8). If replicated, these findings suggest that early OC use may be associated with the subset of mammary tumors that overexpress cyclin D1.

Topics: Adult; Age Factors; Breast Neoplasms; Case-Control Studies; Contraceptives, Oral; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Risk Factors

Glucose concentration may be an important factor in breast cancer cell proliferation, and the prevalence of breast cancer is high in diabetic patients. Leptin may also be an important factor since plasma levels of leptin correlated with TNM staging for breast cancer patients. The effects of glucose and leptin on breast cancer cell proliferation were evaluated by examining cell doubling time, DNA synthesis, levels of cell cycle related proteins, protein kinase C (PKC) isozyme expression, and peroxisome proliferator-activated receptor (PPAR) subtypes were determined following glucose exposure at normal (5.5 mM) and high (25 mM) concentrations with/without leptin in MCF-7 human breast cancer cells. In MCF-7 cells, leptin and high glucose stimulated cell proliferation as demonstrated by the increases in DNA synthesis and expression of cdk2 and cyclin D1. PKC-alpha, PPARgamma, and PPARalpha protein levels were up-regulated following leptin and high glucose treatment in drug-sensitive MCF-7 cells. However, there was no significant effect of leptin and high glucose on cell proliferation, DNA synthesis, levels of cell cycle proteins, PKC isozymes, or PPAR subtypes in multidrug-resistant human breast cancer NCI/ADR-RES cells. These results suggested that hyperglycemia and hyperleptinemia increase breast cancer cell proliferation through accelerated cell cycle progression with up-regulation of cdk2 and cyclin D1 levels. This suggests the involvement of PKC-alpha, PPARalpha, and PPARgamma.

Topics: Breast Neoplasms; CDC2-CDC28 Kinases; Cell Cycle; Cell Division; Cyclin D1; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinases; DNA; Dose-Response Relationship, Drug; Drug Resistance; Glucose; Humans; Isoenzymes; Leptin; Protein Kinase C; Protein Kinase C-alpha; Protein Serine-Threonine Kinases; Receptors, Cytoplasmic and Nuclear; Thymidine; Transcription Factors; Tumor Cells, Cultured; Up-Regulation

Intratumoral heterogeneity mirrors subclonal diversity and might affect treatment response. To investigate molecular heterogeneity of primary breast cancer specimens, we determined the amplification status of growth regulatory genes (c-erbB2, topoisomerase IIalpha, c-myc, and cyclinD1) in macroscopically and microscopically separate areas of individual tumors (n = 21). Using laser-assisted microdissection and quantitative PCR, we found marked intratumoral heterogeneity with different patterns for each gene. Molecular heterogeneity in amplification pattern could be demonstrated between both macroscopically (0.5 to several centimeters) and microscopically (10 to several hundred micrometers) distant tumor areas. C-erbB2 amplification proved to be the most stable amplification in individual tumors, with heterogeneity occurring in only 36% of amplified cases. By contrast, amplification of c-myc and cyclinD1 revealed varying patterns in the vast majority of amplified cases (100% and 83%). The constancy of c-erbB2 amplification underlines its presumed importance in breast cancer biology. We conclude that the molecular heterogeneity of breast cancer as evidenced in this study requires thorough and representative sampling of different tumor areas when the biologic significance of somatic mutations is considered. Patterns of heterogeneity can be used to trace the clonal evolution within different compartments of an individual tumor.

Topics: Breast Neoplasms; Cyclin D1; Female; Gene Amplification; Genes, myc; Humans; Polymerase Chain Reaction; Receptor, ErbB-2

Cyclin D1, the regulatory subunit for mid-G(1) cyclin-dependent kinases, controls the expression of numerous cell cycle genes. A cyclic AMP-responsive element (CRE), located upstream of the cyclin D1 mRNA start site, integrates mitogenic signals that target the CRE-binding factor CREB, which can recruit the transcriptional coactivator CREB-binding protein (CBP). We describe an alternative mechanism for CREB-driven cyclin D1 induction that involves the ubiquitous POU domain protein Oct-1. In the breast cancer cell line MCF-7, overexpression of Oct-1 or its POU domain strongly increases transcriptional activation of cyclin D1 and GAL4 reporter genes that is specifically dependent upon CREB but independent of Oct-1 DNA binding. Gel retardation and chromatin immunoprecipitation assays confirm that POU forms a complex with CREB bound to the cyclin D1 CRE. In solution, CREB interaction with POU requires the CREB Q2 domain and, notably, occurs with CREB that is not phosphorylated on Ser 133. Accordingly, Oct-1 also potently enhances transcriptional activation mediated by a Ser133Ala CREB mutant. Oct-1/CREB synergy is not diminished by the adenovirus E1A 12S protein, a repressor of CBP coactivator function. In contrast, E1A strongly represses CBP-enhanced transactivation by CREB phosphorylated on Ser 133. Our observation that Oct-1 potentiates CREB-dependent cyclin D1 transcriptional activity independently of Ser 133 phosphorylation and E1A-sensitive coactivator function offers a new paradigm for the regulation of cyclin D1 induction by proliferative signals.

Topics: Breast Neoplasms; CREB-Binding Protein; Cyclic AMP Response Element-Binding Protein; Cyclin D1; DNA-Binding Proteins; Genes, Reporter; Host Cell Factor C1; Humans; Nuclear Proteins; Octamer Transcription Factor-1; Phosphoproteins; Promoter Regions, Genetic; Protein Binding; Protein Structure, Tertiary; Recombinant Fusion Proteins; Trans-Activators; Transcription Factors; Transcription, Genetic; Tumor Cells, Cultured

Although the pharmacology and clinical application of water extracts of Ganoderma lucidum have been extensively documented, little is known regarding its alcohol extract. In the present study, the anti-tumor effect of an alcohol extract of Ganoderma lucidum was investigated using MCF-7 cells. We found that the alcohol extract of Ganoderma lucidum inhibited cell proliferation in a dose- and time-dependent manner, which might be mediated through up-regulation of p21/Waf1 and down-regulation of cyclin D1. Furthermore, this compound can directly induce apoptosis in MCF-7 cells, which might be mediated through up-regulation of a pro-apoptotic Bax protein and not by the immune system. Our findings suggest that there are multiple mechanisms underlying the anti-tumor effects of Ganoderma lucidum.

Topics: Alcohols; Apoptosis; Blotting, Western; Breast Neoplasms; Caspase 7; Caspases; Cell Cycle Proteins; Cell Division; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; DNA-Binding Proteins; Dose-Response Relationship, Drug; Down-Regulation; E2F Transcription Factors; Genes, p53; Humans; Plant Extracts; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); Reishi; Time Factors; Transcription Factors; Tumor Cells, Cultured; Up-Regulation

Cyclin E, a regulator of the cell cycle, affects the behavior of breast-cancer cells. We investigated whether levels of cyclin E in the tumor correlated with survival among patients with breast cancer.. Tumor tissue from 395 patients with breast cancer was assayed for cyclin E, cyclin D1, cyclin D3, and the HER-2/neu oncogene with the use of Western blot analysis. Full-length, low-molecular-weight, and total cyclin E were measured. Immunohistochemical assessments of cyclin E were also made of 256 tumors. We sought correlations between levels of these molecular markers and disease-specific and overall survival.. The median follow-up was 6.4 years. A high level of the low-molecular-weight isoforms of cyclin E, as detected by Western blotting, correlated strongly with disease-specific survival whether axillary lymph nodes were negative or positive for metastases (P<0.001). Among 114 patients with stage I breast cancer, none of the 102 patients with low levels of cyclin E in the tumor had died of breast cancer by five years after diagnosis, whereas all 12 patients with a high level of low-molecular-weight cyclin E had died of breast cancer within that period. In multivariate analysis, a high total cyclin E level or high levels of the low-molecular-weight forms of cyclin E were significantly correlated with poor outcome. The hazard ratio for death from breast cancer for patients with high total cyclin E levels as compared with those with low total cyclin E levels was 13.3--about eight times as high as the hazard ratios associated with other independent clinical and pathological risk factors.. Levels of total cyclin E and low-molecular-weight cyclin E in tumor tissue, as measured by Western blot assay, correlate strongly with survival in patients with breast cancer.

Topics: Adult; Aged; Aged, 80 and over; Analysis of Variance; Blotting, Western; Breast Neoplasms; Cyclin D1; Cyclin D3; Cyclin E; Cyclins; Female; Follow-Up Studies; Humans; Middle Aged; Multivariate Analysis; Prognosis; Proportional Hazards Models; Receptor, ErbB-2; Retrospective Studies; Survival Analysis

Estrogen receptor-mediated transcription is enhanced by overexpression of G1/S cyclins D1, E or A in the presence as well in the absence of estradiol. Excess of G1/S cyclins also prevents the inhibition of transactivation of estrogen receptor (ER) by the pure antiestrogen ICI 182780. Cyclin D1 mediates this transactivation independent of complex formation to its CDK4/6 partner. This raises the possibility that overexpression of G1/S cyclins renders growth of ER-positive breast cancer hormone-independent and resistant to treatment with antiestrogens. Transient transfection of ER-positive breast cancer cell lines T47D and MCF7 with G1/S cyclins could overcome the growth arrest induced by ICI 182780 treatment. The ability of various cyclin D1 mutants to overcome the ICI 182780 mediated growth arrest corresponded with their ability to stimulate cyclin A- and E2F- promoter based reporter activities in the presence of ICI 182780. Transfection of a mutant cyclin D1 (cyclin D1-KE) that was unable to bind CDK4 and was reported to transactivate ER in the presence of ICI 182780, could not stimulate proliferation in ICI 182780 treated cells. On the other hand, cyclin D1-LALA, which is unable to stimulate ERE transactivation, could overcome the ICI 182780 cell cycle arrest. Furthermore, transient transfection of T47D cells using cyclin D1 together with a catalytic inactive mutant of CDK4 (CDK4-DN) indicated that the observed effect is due to binding to CDK inhibitors. However, a moderate, sixfold overexpression of cyclin D1 in stably transfected MCF7 cells did not overcome the ICI 182780 mediated growth arrest. These results indicate that CDK-independent transactivation of the estrogen receptor by cyclin D1 is by itself, not sufficient to result in estradiol-independent growth of breast cancer cells, whereas a vast overexpression of G1/S cyclins is able to do so, most likely by capturing of CDK inhibitors.

Topics: Adenocarcinoma; Breast Neoplasms; Cell Cycle Proteins; Cyclin A; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; DNA-Binding Proteins; E2F Transcription Factors; Estradiol; Estrogen Receptor Modulators; Estrogens; Female; Fulvestrant; G1 Phase; Humans; Neoplasm Proteins; Neoplasms, Hormone-Dependent; Proto-Oncogene Proteins; Receptors, Estrogen; Recombinant Fusion Proteins; S Phase; Transcription Factors; Transcriptional Activation; Tumor Cells, Cultured

Rearrangements of chromosome 11q13 are frequently observed in human cancer. The 11q13 region harbors several chromosomal breakpoint clusters found in hematologic malignancies and exhibits frequent DNA amplification in carcinomas. DNA amplification patterns in breast tumors are consistent with the existence of at least 4 individual amplification units, suggesting the activation of more than 1 gene in this region. Two candidate oncogenes have been identified, CCND1 and EMS1/CORTACTIN, representing centrally localized amplification units. Genes involved in the proximal and distal amplicons remain to be identified. Recently we reported on a putative transforming gene, MYEOV, mapping 360 kb centromeric to CCND1. This gene was found to be rearranged and activated concomitantly with CCND1 in a subset of t(11;14)(q13;q32)-positive multiple myeloma (MM) cell lines. To evaluate the role of the MYEOV gene in the proximal amplification core, we tested 946 breast tumors for copy number increase of MYEOV relative to neighboring genes or markers. RNA expression levels were studied in a subset of 72 tumors for which both RNA and DNA were available. Data presented here show that the MYEOV gene is amplified in 9.5% (90/946) and abnormally expressed in 16.6% (12/72) of breast tumors. Amplification patterns showed that MYEOV was most frequently coamplified with CCND1 (74/90), although independent amplification of MYEOV could also be detected (16/90). Abnormal expression levels correlated only partially with DNA amplification. MYEOV DNA amplification correlated with estrogen and progesterone receptor-positive cancer, invasive lobular carcinoma type and axillary nodal involvement. In contrast to CCND1 amplification, no association with disease outcome could be found. Our data suggest that MYEOV is a candidate oncogene activated in the amplification core located proximal to CCND1.

Topics: Breast Neoplasms; Centromere; Chromosomes, Human, Pair 11; Cyclin D1; Female; Gene Amplification; Gene Expression Regulation, Neoplastic; Humans; Oncogene Proteins; Oncogenes; Prognosis; Proto-Oncogene Proteins; RNA, Neoplasm; Tumor Cells, Cultured

Inflammatory breast cancer (IBC) is a rare but particularly aggressive form of primary breast cancer. In contrast to noninflammatory breast cancer (non IBC), the molecular alterations underlying IBC are poorly known. We postulated that the kind and frequency of these alterations might differ between IBC and non IBC and account for its particular aggressiveness. We investigated allelic losses associated with primary breast cancer (on chromosome arms 1p, 3p, 6p, 6q, 7q, 8p, 9p, 11p, 11q, 16q, 17p and 17q) by analyzing 71 microsatellite markers in 66 cases of IBC. Loss of heterozygosity (LOH) was frequent, with a mean fractional allelic loss (FAL) index of 52%. Relative to published data on non IBC, allelic loss was particularly frequent at 3p21-p14, 6p, 8p22, 11q, 13q14 and 17q21, suggesting the presence of genes that are markedly altered in IBC. In contrast, the DNA amplification levels of ERBB2, MYC and CCND1, as measured by real-time quantitative PCR, did not differ between IBC and non IBC.

Topics: Breast Neoplasms; Cyclin D1; Female; Genes, erbB-2; Genes, myc; Humans; Inflammation; Loss of Heterozygosity

Cyclin D1 and cyclin E are overexpressed in approximately 45% and 30% of breast cancers, respectively, and adverse associations with patient outcome have been reported. The potential roles of cyclin D1 and cyclin E expression as markers of therapeutic responsiveness to the pure steroidal antiestrogen ICI 182780 were investigated using T-47D breast cancer cell lines constitutively overexpressing cyclin D1 or cyclin E. Measurement of S phase fraction, phosphorylation states of the retinoblastoma protein, and cyclin E-cyclin-dependent kinase (Cdk) 2 activity demonstrated that overexpression of cyclin D1 decreased sensitivity to antiestrogen inhibition at 24 and 48 h. Overexpression of cyclin E produced a less pronounced early cell cycle effect indicating only partial resistance to antiestrogen inhibition in the short-term. In ICI 182780-treated cyclin D1-overexpressing cells, sufficient Cdk activity was retained to allow retinoblastoma protein phosphorylation and cell proliferation, despite an increase in the association of p21 and p27 with cyclin D1-Cdk4/6 and cyclin E-Cdk2 complexes. After longer-term (>7 days) treatment, antiestrogens inhibited colony growth in cyclin D1- or cyclin E-overexpressing breast cancer cells, but with an approximately 2-2.5-fold decrease in dose sensitivity. This was associated with a fall in cyclin D1 levels, a reduction in the half-life of cyclin D1 protein and a decline in cyclin E-Cdk2 activity in cyclin D1-overexpressing cells, and the maintenance of cyclin E-p27 association in the cyclin E-overexpressing cells. These data confirm that cyclin D1 expression and cyclin E-p27 association play important roles in antiestrogen action, and suggest that cyclin D1 or cyclin E overexpression has subtle effects on antiestrogen sensitivity. Additional studies to elucidate the contribution of alterations in cyclin D1 stability to antiestrogen action and to assess the relationship between antiestrogen sensitivity and expression of cyclin D1, cyclin E, or p27 in a clinical setting are required.

Topics: Breast Neoplasms; CDC2-CDC28 Kinases; Cell Cycle Proteins; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Cyclins; Down-Regulation; Drug Resistance, Neoplasm; Estradiol; Estrogen Receptor Modulators; Fulvestrant; Humans; Phosphorylation; Protein Serine-Threonine Kinases; Retinoblastoma Protein; S Phase; Transfection; Tumor Cells, Cultured; Tumor Suppressor Proteins

(-)-Epigallocatechin (EGC), one of green tea polyphenols, has been shown to inhibit growth of cancer cells. However its mechanism of action is poorly known. We show here that EGC strongly inhibited the growth of breast cancer cell lines (MCF-7 and MDA-MB-231) but not that of normal breast epithelial cells. The inhibition of breast cancer cell growth was due to an induction of apoptosis, without any change in cell cycle progression. MCF-7 cells are known to express a wild-type p53 whereas MDA-MB-231 cells express a mutated p53. The fact that EGC induced apoptosis in both these cell lines suggests that the EGC-triggered apoptosis is independent of p53 status. Moreover, neutralizing antibodies against the death receptor Fas and inhibitors of caspases, such as caspase-8 and -10, efficiently inhibited the EGC-triggered apoptosis. In addition, immunoblotting revealed that EGC treatment was correlated with a decrease in Bcl-2 and an increase in Bax level. These results suggest that EGC-triggered apoptosis in breast cancer cells requires Fas signaling.

Topics: Apoptosis; bcl-2-Associated X Protein; Breast; Breast Neoplasms; Caspases; Catechin; Cell Cycle; Cyclin D1; Dose-Response Relationship, Drug; Fatty Acid Synthases; Female; Humans; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured

Cyclin D1 is essential for Neu-induced cell growth and is induced by growth factors through Ras-dependent and Ras-independent signaling pathways (1). Because flavopiridol, a novel flavanoid cyclin-cyclin-dependent kinase inhibitor, may function through Ras-dependent and/or -independent pathways, we hypothesized that treatment of breast cancer cells with inhibitors of Neu signaling and flavopiridol might synergize to inhibit proliferation. Human breast cancer cell lines, which express high levels of endogenous Neu receptor, were treated with the anti-Neu antibody, trastuzumab, together with flavopiridol and subject to MTT assay. Cell lines were assayed for alterations in cell cycle by fluorescence-activated cell sorter and signaling proteins by Western blot. Flavopiridol and trastuzumab synergistically inhibited DNA synthesis, cellular proliferation, and contact-dependent growth. Cytotoxic synergy was observed independent of the sequence of addition of the two drugs to cultured cells. In SKBR3 cells, a combination of trastuzumab and flavopiridol inhibited the Ras-MAPK-Akt pathway, decreased cyclin D1 abundance, and kinase activity to a greater extent than either drug alone. Compared with single-agent treatment, combination treatment selectively inhibited Akt and pRB phosphorylation. Cytotoxic synergy was observed with flavopiridol plus LY294002 (selective phosphatidylinositol 3-kinase inhibitor) but not with PD98059 (selective mitogen-activated protein kinase kinase 1 inhibitor) suggesting that Akt inhibition may be important in synergy. Zinc-induced overexpression of cyclin D1 in T-47D deltaMTcycD1 cells were more resistant to drug-induced cell death when compared with vector-transfected T-47D deltaMT cells. Cyclin D1 overexpression reverses drug treatment induced cell cycle arrest in SKBR3 cells. Flavopiridol and trastuzumab yield cytotoxic synergy in human breast cancer cells overexpressing Neu. Cyclin D1 overexpression results in combination drug resistance. In addition, inhibition of Akt may prove to be a useful therapeutic strategy in combination with flavopiridol.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Blotting, Western; Breast Neoplasms; Cell Cycle; Cell Division; Cell Separation; Chromones; Cyclin D1; Dose-Response Relationship, Drug; Enzyme Inhibitors; Flavonoids; Flow Cytometry; Humans; Mitogen-Activated Protein Kinases; Morpholines; Piperidines; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; S Phase; Signal Transduction; Time Factors; Transfection; Trastuzumab; Tumor Cells, Cultured

The mechanisms by which prolactin controls proliferation of mammary epithelial cells (MECs) and morphogenesis of the breast epithelium are poorly understood. We show that cyclin D1(-/-) MECs fail to proliferate in response to prolactin and identify IGF-2 as a downstream target of prolactin signaling that lies upstream of cyclin D1 transcription. Ectopic IGF-2 expression restores alveologenesis in prolactin receptor(-/-) epithelium. Alveologenesis is retarded in IGF-2-deficient MECs. IGF-2 and prolactin receptor mRNAs colocalize in the mammary epithelium. Prolactin induces IGF-2 mRNA and IGF-2 induces cyclin D1 protein in primary MECs. Thus, IGF-2 is a mediator of prolactin-induced alveologenesis; prolactin, IGF-2, and cyclin D1, all of which are overexpressed in breast cancers, are components of a developmental pathway in the mammary gland.

Topics: Animals; Breast Neoplasms; Carcinoma; Carrier Proteins; Cell Division; Cells, Cultured; Cyclin D1; Epithelial Cells; Female; Gene Expression Regulation, Developmental; Genes; Genetic Testing; Insulin-Like Growth Factor II; Mammary Glands, Animal; Membrane Glycoproteins; Mice; Mice, Knockout; Oligonucleotide Array Sequence Analysis; Progesterone; Prolactin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Progesterone; RNA, Messenger; Signal Transduction

Overexpression of oncogenic proteins may be caused by gene amplifications. Cyclin D1 participates in regulation of the cell cycle. Relations between cyclin D1 expression and amplification of CCND1 gene encoding this protein in invasive duct breast carcinomas (IDC) are not fully elucidated. An increased interest is also focused on relations to the estrogen receptor (ER alpha).. We investigated copy numbers of the CCND1 gene, expression of cyclin D1 and expression of ER alpha in a group of 60 females and 1 male with IDC. The age range varied from 33 to 89 years (median 57 years). The number of CCND1 gene copies and the number of chromosome 11 was evaluated using FISH, the expression of cyclin D1 and ER alpha was investigated by IHC. We detected a strong amplification of CCND1 gene (> 10 copies per tumor cell nuclei) in 9 patients, weak amplification (< or = copies) in 16 patients. Amplification of the CCND1 gene correlated well with the overexpression of cyclin D1. We observed the overexpression of cyclin D1 also in 13 of 36 patients without the gene amplification; therefore, the mechanism of the protein overexpression is different than that caused by the gene amplification in a proportion of patients. Amplification of the CCND1 gene was associated with a high histologic grade of IDC, whereas cases with cyclin D1 overexpression only were not. 24 of 31 patients with overexpression of cyclin D1 coexpressed ER alpha. We did not find correlation between expression/amplification of cyclin D1/CCND1 gene and the size of carcinomas and with metastases to the axillary lymph nodes.. Amplification of the CCND1 gene is associated with overexpression of cyclin D1 in a majority of IDC. Overexpression of cyclin D1 is related to an increased expression of ER alpha. Interaction between cyclin D1 and ER alpha may explain low response to anti-estrogen therapy of some patients.

Topics: Adult; Aged; Aged, 80 and over; Breast Neoplasms; Breast Neoplasms, Male; Carcinoma, Ductal, Breast; Chromosomes, Human, Pair 11; Cyclin D1; Estrogen Receptor alpha; Female; Gene Amplification; Humans; In Situ Hybridization, Fluorescence; Male; Middle Aged; Receptors, Estrogen

A maxizyme is dimmer of minimized ribozymes (minizymes) and can specifically cleave two target sites. The maxizyme also can allosterically cleave the target RNA only when it recognizes two target sites. In this study, for a cancer gene therapy, we focused two distinct oncogenes, cyclinD1 and hst-1, which are overexpressed in breast cancer cells. If we use conventional ribozymes for suppression of expression of those genes, these ribozymes affect not only these mRNAs in cancer cells but also those in normal cells because those genes are necessary for a growth factor-dependent signal transduction and a cell cycle in normal cells. To overcome this problem, we tried to design the trans-maxizyme that can cleave these mRNAs only in the breast cancer cells.

Topics: Allosteric Regulation; Base Sequence; Breast Neoplasms; Cyclin D1; Fibroblast Growth Factor 4; Fibroblast Growth Factors; Humans; Nucleic Acid Conformation; Oncogenes; Proto-Oncogene Proteins; RNA, Catalytic

The MCF-7 cell line is a model of estrogen-dependent, antiestrogen-sensitive human breast cancer. Antiestrogen treatment of MCF-7 cells causes dramatic decreases in both Cdk4 and Cdk2 activities, which leads to a G(1) phase cell cycle arrest. In this report, we investigate the mechanism(s) by which Cdk4 activity is regulated in MCF-7 cells. Through time course analysis, we demonstrate that changes in Cdk4 activity in response to estrogen or antiestrogen treatment do not correlate directly with cyclin D1 protein levels or association. In contrast, Cdk4 activity does correlate with changes in the level of the Cdk inhibitor p21(WAF1/Cip1). Furthermore, we show that extracts of antiestrogen-treated cells contain a factor capable of inhibiting the Cdk4 activity present in extracts of estrogen-treated cells, and immunodepletion experiments identify this factor as p21(WAF1/Cip1). These results identify p21(WAF1/Cip1) as an important physiological regulator of Cdk4 complexes in human breast cancer cells.

Topics: Antineoplastic Agents; Blotting, Western; Breast Neoplasms; Cell Cycle Proteins; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Cyclins; Dose-Response Relationship, Drug; Estradiol; Fulvestrant; Humans; Precipitin Tests; Protein Binding; Proto-Oncogene Proteins; Time Factors; Tumor Cells, Cultured; Tumor Suppressor Proteins

PRL is essential for normal lobulo-alveolar growth of the mammary gland and may contribute to mammary cancer development or progression. However, analysis of the mechanism of action of PRL in these processes is complicated by the production of PRL within mammary epithelia. To examine PRL actions in a mammary cell-specific context, we selected MCF-7 cells that lacked endogenous PRL synthesis, using PRL stimulation of interferon-gamma-activated sequence-related PRL response elements. Derived clones exhibited a greater proliferative response to PRL than control cells. To understand the mechanism, we examined, by Western analysis, levels of proteins essential for cell cycle progression as well as phosphorylation of retinoblastoma protein. The expression of cyclin D1, a critical regulator of the G1/S transition, was significantly increased by PRL and was associated with hyperphosphorylation of retinoblastoma protein at Ser(780). Cyclin B1 was also increased by PRL. In contrast, PRL decreased the Cip/Kip family inhibitor, p21, but not p16 or p27. These studies demonstrate that PRL can stimulate the cell cycle in mammary epithelia and identify specific targets in this process. This model system will enable further molecular dissection of the pathways involved in PRL-induced proliferation, increasing our understanding of this hormone and its interactions with other factors in normal and pathogenic processes.

Topics: Adenocarcinoma; Base Sequence; Breast Neoplasms; Cell Cycle Proteins; Cyclin B; Cyclin B1; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Enzyme Inhibitors; Epidermal Growth Factor; Epithelial Cells; Female; Humans; Interferon-gamma; MAP Kinase Signaling System; Molecular Sequence Data; Oligonucleotides, Antisense; Phosphorylation; Prolactin; Retinoblastoma Protein; Signal Transduction

Frizzled-related protein (Frp) is a new family of secreted proteins that contain a region homologous to the extracellular cysteine-rich domain (CRD) of the frizzled family proteins. The role of Frp protein is far from clear. To explore the role of Frp and its relationship to the Wnt-signalling pathway in breast cancer, in situ hybridization and immunohistochemical analyses of Frp, Wnt-1, APC, beta-catenin, and its target genes c-myc and cyclin D1 were conducted in 70 specimens of invasive ductal carcinomas of the human breast. Frp mRNA was down-regulated in 62 and elevated in eight tumour specimens, compared with adjacent normal tissues. In the course of tumour progression, however, Frp mRNA steadily increased in both tumour and the adjacent tissues. Interestingly, the number of cases with axillary lymph node metastasis was significantly lower in the group with elevated Frp than in the group with decreased Frp, suggesting that Frp may contribute as a prognostic factor in invasive breast cancer. Wnt-1, a gene implicated in human breast cancer, was markedly elevated in grade 1 tumours, but declined as tumour grade declined. The level of Wnt-1 was linearly correlated with its downstream target beta-catenin (p<0.05), but was inversely correlated with Frp (p<0.05), suggesting a possible negative regulatory role of Frp with regard to Wnt-1. APC was inversely correlated with beta-catenin (p<0.05). Beta-catenin, a key transcriptional activator responsible for the activation of both c-myc and cyclin D1 in colorectal tumours, was detected at high levels in the plasma membranes of cells in normal tissue. In tumour masses, however, beta-catenin lost its tight association with the membrane and diffused into the cytoplasm. Surprisingly, it clearly did not penetrate the nuclei, despite the fact that both c-myc and cyclin D1 were markedly elevated in all tumour tissues. As revealed in this study, Wnt-1/beta-catenin plays very different roles in the oncogenesis of breast and colon cancers. This first systemic analysis of the Frp and the Wnt-signalling pathway in human breast cancer provides a springboard for further work on the role of Frp in the development of breast cancer.

Topics: Adenomatous Polyposis Coli Protein; beta Catenin; Breast; Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Membrane; Cyclin D1; Cytoskeletal Proteins; Female; Follistatin-Related Proteins; Glycoproteins; Humans; Immunohistochemistry; In Situ Hybridization; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-myc; Signal Transduction; Trans-Activators; Wnt Proteins; Wnt1 Protein; Zebrafish Proteins

We have analyzed the mechanism by which the combination of insulin-like growth factor I (IGF-I) and 17 beta-estradiol (E2) induces cell cycle progression in MCF-7S cells. This cell line differs from many other breast cancer-derived cell lines in that E2 (1 nM) does not induce cell cycle progression, whereas the combination of submitogenic concentrations of IGF-I (2 ng/ml) and E2 does. We find that addition of IGF-I to MCF-7S cells leads to a dose-dependent activation of the IGF type I receptor and of the MAP kinase and PI3-kinase signaling pathways. No synergy of IGF-I and E2 was detected in the activation of these signaling cascades. In terms of cell cycle-related molecules, we find that IGF-I dose-dependently raises cyclin D1 levels in serum-starved cells. Subsequent activation of cyclin E/CDK2, hyperphosphorylation of pRb, and DNA synthesis are only induced by mitogenic concentrations of IGF-I (> or =20 ng/ml). Treatment of the cells with E2 also results in the induction of cyclin D1, but in the absence of IGF-I the cells remain arrested in G1 phase. We conclude that in MCF-7S cells, the synergistic action of E2 and IGF-I derives from the ability of both hormones to induce cyclin D1 expression. The action of IGF-I is required in these cells to induce activity of the cyclin D1/CDK4 complex, which triggers progression through the cell cycle.

Topics: Breast Neoplasms; CDC2-CDC28 Kinases; Cell Cycle; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinases; Drug Synergism; Estradiol; Female; Humans; Insulin; Insulin-Like Growth Factor I; Luciferases; MAP Kinase Signaling System; Phosphatidylinositol 3-Kinases; Precipitin Tests; Protein Serine-Threonine Kinases; Receptors, Estrogen; Signal Transduction; Thymidine; Transfection; Tumor Cells, Cultured

Topics: Breast; Breast Neoplasms; Cell Transformation, Neoplastic; Cyclin D1; Epithelial Cells; Female; Humans; Models, Biological; NF-kappa B; Pregnancy

Ansamycin antibiotics, such as 17-allylaminogeldanamycin (17-AAG), bind to Hsp90 and regulate its function, resulting in the proteasomal degradation of a subset of signaling proteins that require Hsp90 for conformational maturation. HER2 is a very sensitive target of these drugs. Ansamycins cause RB-dependent G1 arrest that is associated with loss of D-cyclins via a PI3 kinase, Akt dependent pathway. Downregulation of D-cyclin was due, in part, to loss of Akt expression in response to drug. Moreover, in HER2 overexpressing breast cancer cells, 17-AAG caused rapid inhibition of Akt activity prior to any change in Akt protein. Ansamycins caused rapid degradation of HER2 and a concomitant loss in HER3 associated PI3 kinase activity. This led to a loss of Akt activity, dephosphorylation of Akt substrates, and loss of D-cyclin expression. Introduction into cells of a constitutively membrane bound form of PI3 kinase prevented the effects of the drug on Akt activity and D-cyclins. Thus, in breast cancer cells with high HER2, Akt activation by HER2/HER3 heterodimers is required for D-cyclin expression. In murine xenograft models, non-toxic doses of 17-AAG markedly reduced the expression of HER2 and phosphorylation of Akt and inhibited tumor growth. Thus, pharmacological inhibition of Akt activation is achievable with ansamycins and may be useful for the treatment of HER2 driven tumors.

Topics: Animals; Antibiotics, Antineoplastic; Benzoquinones; Blotting, Western; Breast Neoplasms; Cell Division; Cyclin D; Cyclin D1; Cyclin D3; Cyclins; Dimerization; Dose-Response Relationship, Drug; Enzyme Activation; Female; G1 Phase; Gene Expression Regulation, Neoplastic; Humans; Lactams, Macrocyclic; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Receptor, ErbB-2; Rifabutin; Transplantation, Heterologous; Tumor Cells, Cultured

We have studied the effects of purvalanol A on the cell cycle progression, proliferation and viability. In synchronized cells, purvalanol A induced a reversible arrest the progression in G1 and G2 phase of the cell cycle, but did not prevent the completion of DNA synthesis in S-phase cells. The specificity of action of the drug was supported by the selective inhibition of the phosphorylation of cyclin-dependent kinase (cdk) substrates such as Rb and cyclin E. The cell contents of cyclins D1 and E were lower in cells incubated with purvalanol A compared to controls, but the level of the cdk inhibitory protein p21(WAF1/CIP1) was increased, indicating that the drug did not cause a general inhibition of gene expression. Purvalanol A did not inhibit transcription under cell-free conditions. This compound, however, caused an inhibition of the estradiol-induced expression of an integrated luciferase gene, suggesting that cdk or related enzymes may participate in the regulation of the activity of certain promoters. When exponentially growing cells, both mouse fibroblasts and human cancer cell lines, were incubated with purvalanol A for prolonged periods of time (24 hr), a lasting inhibition of cell proliferation as well as cell death were observed. In contrast, a 24 hr incubation of quiescent (non-transformed) cells with purvalanol A did not prevent their resumption of cell cycle after removal of the drug.

Topics: Breast Neoplasms; Cell Cycle; Cell Division; Cell Survival; Colonic Neoplasms; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; Enzyme Inhibitors; Female; HeLa Cells; HT29 Cells; Humans; Methionine; Microfilament Proteins; Muscle Proteins; Phosphorylation; Retinoblastoma Protein; Transcription, Genetic; Tumor Cells, Cultured; Uterine Cervical Neoplasms

The CCND1 gene, a key cell-cycle regulator, is often altered in breast cancer, but the mechanisms underlying CCND1 dysregulation and the clinical significance of CCND1 status are unclear. We used real-time quantitative PCR and RT-PCR assays based on fluorescent TaqMan methodology to quantify CCND1 gene amplification and expression in a large series of breast tumours. CCND1 overexpression was observed in 44 (32.8%) of 134 breast tumour RNAs, ranging from 3.3 to 43.7 times the level in normal breast tissues, and correlated significantly with positive oestrogen receptor status (P=0.0003). CCND1 overexpression requires oestrogen receptor integrity and is exacerbated by amplification at 11q13 (the site of the CCND1 gene), owing to an additional gene dosage effect. Our results challenge CCND1 gene as the main 11q13 amplicon selector. The relapse-free survival time of patients with CCND1-amplified tumours was shorter than that of patients without CCND1 alterations, while that of patients with CCND1-unamplified-overexpressed tumours was longer (P=0.011). Only the good prognostic significance of CCND1-unamplified-overexpression status persisted in Cox multivariate regression analysis. This study confirms that CCND1 is an ER-responsive or ER-coactivator gene in breast cancer, and points to the CCND1 gene as a putative molecular marker predictive of hormone responsiveness in breast cancer. Moreover, CCND1 amplification status dichotomizes the CCND1-overexpressing tumors into two groups with opposite outcomes.

Topics: Adult; Aged; Aged, 80 and over; Breast Neoplasms; Cyclin D1; DNA Primers; DNA, Neoplasm; Female; Genes, erbB-2; Genes, myc; Humans; Middle Aged; Prognosis; Receptors, Estrogen; Reference Values; Retinoblastoma Protein; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Survival Rate

CyclinD1 plays a critical role in regulating cell cycle progression. CyclinD1 mRNA and protein are overexpressed in approximately 50% of primary breast cancer cases. However, its clinical significance as a predictive factor remains unclear. One hundred and seventy-three female patients diagnosed with invasive ductal carcinoma who had undergone a mastectomy (161 patients) or breast-conserving surgery (12 patients) were followed up for 6-119 months (median 86 months) postoperatively. Immunoreactivity for monoclonal anti-cyclinD1 antibody (clone DCS-6) with paraffin-embedded carcinoma tissues was investigated using a labeled streptavidin-biotin method. Overexpression of cyclinD1 was found in 42% (73 of 173), and strongly correlated with estrogen receptor (ER) expression (p < 0.000001). Univariate analysis revealed no association between overexpression of cyclinD1 and overall survival or relapse-free survival in all patient groups. However, in the ER-negative subgroup (n = 75), overexpression of cyclinD1 was significantly correlated with shorter overall survival (p = 0.018) and relapse-free survival (p = 0.014) as well as the lymph node status and tumor size. In contrast, there were no significant associations between overexpression of cyclinD1 and clinical outcome in the ER-positive subgroup. According to Cox's multivariate analysis in the ER-negative subgroup, overexpression of cyclinD1 had the most significant effect on overall survival (p = 0.02) and relapse-free survival (p = 0.0058), followed by nodal status and histologic grade. These findings suggest that overexpression of cyclinD1 is an independent prognostic indicator in ER-negative breast cancer patients.

Topics: Adult; Aged; Aged, 80 and over; Breast Neoplasms; Carcinoma, Ductal, Breast; Cyclin D1; Female; Humans; Immunoenzyme Techniques; Lymph Nodes; Mastectomy; Middle Aged; Neoplasm Staging; Prognosis; Receptors, Estrogen; Receptors, Progesterone; Survival Rate; Treatment Outcome

In a previous study of phyllodes tumours, it has been shown that both the stroma and the epithelium can exhibit distinct molecular changes, suggesting that both are part of the neoplastic process. In view of this finding, it was decided to study stromal-epithelial interactions in these tumours by examining the Wnt-APC-beta-catenin pathway. Beta-catenin and cyclin D1 immunohistochemistry was performed on 119 phyllodes tumours. Eighty-six (72%) showed stromal nuclear beta-catenin localization and in 57% the staining was moderate or strong; however, of the eight malignant tumours in the series, seven showed absent or weak nuclear staining (p<0.025). In no tumour was nuclear beta-catenin staining seen in the epithelial component. Moderate or strong stromal cyclin D1 staining correlated with nuclear stromal beta-catenin staining (p<0.05). Forty-five of the tumours, including two malignant lesions, were screened for beta-catenin exon 3 mutations using SSCP and sequencing, but none was found. Loss of heterozygosity (LOH) of the marker D5S346 was used to infer APC mutation, but only one (benign) tumour showed LOH. Wnt2 and Wnt5a mRNA was localized by in situ hybridization in 13 cases (three malignant) chosen to reflect the different beta-catenin staining patterns. There was an association between strong nuclear beta-catenin staining of stromal cells and epithelial Wnt5a expression (p<0.0015). These data suggest that stromal proliferation in benign phyllodes tumours relies on abnormalities in the Wnt pathway which result not from mutation, but from Wnt5a expression in the epithelium. In the progression to malignancy, the stromal proliferation appears to become independent of the Wnt pathway and, presumably, of the epithelial component of these tumours.

Topics: beta Catenin; Breast Neoplasms; Cyclin D1; Cytoskeletal Proteins; Disease Progression; Epithelium; Female; Humans; Immunoenzyme Techniques; Loss of Heterozygosity; Neoplasm Proteins; Phyllodes Tumor; Proto-Oncogene Proteins; RNA, Messenger; RNA, Neoplasm; Stromal Cells; Trans-Activators; Wnt Proteins; Wnt2 Protein; Zebrafish Proteins

PRL promotes cell growth and differentiation in the mammary gland, which has implications for breast cancer as well as normal development. Our data demonstrate that PRL significantly increases proliferation of mammary carcinoma cells. PRL also increases cyclin D1 levels 2-fold, which can be inhibited by actinomycin D, suggesting that transcriptional increases in cyclin D1 are important. Using a defined Chinese hamster ovary cell model system, we demonstrate that the activity of a cyclin D1 promoter-luciferase construct increases after PRL treatment. Furthermore, this increase in promoter activity is predominantly mediated by the Jak2/Stat5 signaling pathway. The cyclin D1 promoter contains two consensus sequences for PRL-induced Stat binding (GAS sites). Disruption of Stat binding to the distal GAS site destroys PRL-induced promoter activity, whereas disruption of the proximal site has no effect. We have shown by EMSA that PRL induces Stat5a and 5b to bind to the distal GAS site, and immunoprecipitation and subsequent Western analysis of nuclear extracts from PRL-treated cells indicate that Stat5a and 5b can interact as a heterodimer in this system. These data suggest that cyclin D1 may be a target gene for PRL in normal lobuloalveolar development, as well as in the development and/or progression of mammary cancer.

Topics: Amino Acid Sequence; Animals; Breast Neoplasms; Cell Division; CHO Cells; Consensus Sequence; Cricetinae; Cyclin D1; DNA Mutational Analysis; DNA-Binding Proteins; Epithelial Cells; Female; Humans; Janus Kinase 2; Milk Proteins; Molecular Sequence Data; Prolactin; Promoter Regions, Genetic; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Signal Transduction; STAT5 Transcription Factor; Trans-Activators; Tumor Cells, Cultured; Tumor Suppressor Proteins

Evidence indicates that overexpression of cyclin D1 is an important event in malignant transformation of breast cancer cells. Therefore, cyclin D1 is a potential target for mechanistically-based chemoprevention/treatment of breast cancer. Treatment of serum-stimulated quiescent MCF-7 breast cancer cells with cyclopentenone (2-cyclopenten-1-one) blocked progression through G1 and into S phase. Growth arrest of the cyclopentenone-treated cells in G1 was associated with changes in the levels of several proteins that control the cell cycle, including a dramatic decrease in cyclin D1 protein expression. Cyclopentenone also decreased the abundance of cyclin D1 mRNA and nuclear transcripts, indicating that it regulated cyclin D1 expression at the transcriptional level. Cyclopentenone selectively inhibited the activity of the cyclin D1 and cyclin A promoters but not the activity of several other control promoters. Deletion analysis indicated that the cyclopentenone response element was located in the cyclin D1 core promoter. Additional functional studies showed that a sequence within the core promoter (CycY, located downstream from the initiator element) played an important role in activation of the cyclin D1 promoter in MCF-7 cells. Electrophoretic mobility shift assays demonstrated specific binding of the transcription factor BTEB to the CycY site. The cyclopentenone response element did not correspond to the CycY site but rather mapped to the initiator element itself. The overall results suggest that cyclopentenone interferes with the transcription initiation complex that assembles over the cyclin D1 initiator element, leading to selective inhibition of cyclin D1 gene transcription.

Topics: Animals; Base Sequence; Binding Sites; Blotting, Western; Breast Neoplasms; Cell Cycle; Cyclin D1; Cyclopentanes; Electrophoretic Mobility Shift Assay; Flow Cytometry; G1 Phase; Gene Expression Regulation, Neoplastic; Genes, bcl-1; Humans; Molecular Sequence Data; Promoter Regions, Genetic; Protein Binding; Response Elements; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; S Phase; Sequence Deletion; Sequence Homology, Nucleic Acid; Transcription Initiation Site; Tumor Cells, Cultured

Cyclin D1 is downstream of erbB2 and is required for erbB2 transformation. Here we report thatcyclin D1 functions are essential, rate limiting for erbB2 transformation, and reciprocally increase erbB2 levels. This interaction depends on three cyclin D1 activities: cyclin-dependent kinase 4-dependent kinase activity, titration of p27, and an intrinsic transcriptional activity of cyclin D1. Drugs active against erbB2 and cyclin D1 (Herceptin and flavopiridol) were synergistically cytotoxic against erbB2-positive breast cancer cell lines. Addition of flavopiridol to Herceptin synergistically lowered erbB2 levels in these cells. Our data suggest the potential use of combinations of cyclin-dependent kinase inhibitors and Herceptin in breast cancer.

Topics: 3T3 Cells; Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cell Survival; Cell Transformation, Neoplastic; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; Drug Synergism; Flavonoids; Humans; Mice; Piperidines; Proto-Oncogene Proteins; Receptor, ErbB-2; Trastuzumab

The use of dietary isoflavone supplements by postmenopausal women with breast cancer is increasing. We investigated interactions between the soy isoflavone, genistein, and an antiestrogen, tamoxifen (TAM), on the growth of estrogen (E)-dependent breast cancer (MCF-7) cells implanted in ovariectomized athymic mice. We hypothesized that weakly estrogenic genistein negate/overwhelm the inhibitory effect of TAM on the growth of E-dependent breast tumors. Six treatment groups were used: control (C); 0.25 mg estradiol (E2) implant (E); E2 implant + 2.5 mg TAM implant (2.5 TE); E2 implant + 2.5 mg TAM implant + 1000 ppm genistein (2.5 TEG); E2 implant + 5 mg TAM implant (5 TE), and E2 implant +5 mg TAM implant +1000 ppm genistein (5 TEG). Treatment with TAM (2.5 TE and 5 TE) suppressed E2-stimulated MCF-7 tumor growth in ovariectomized athymic mice. Dietary genistein negated/overwhelmed the inhibitory effect of TAM on MCF-7 tumor growth, lowered E2 level in plasma, and increased expression of E-responsive genes (e.g., pS2, PR, and cyclin D1). Therefore, caution is warranted for postmenopausal women consuming dietary genistein while on TAM therapy for E-responsive breast cancer.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cell Division; Cyclin D1; Diet; Drug Interactions; Estradiol; Female; Genistein; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasms, Hormone-Dependent; Receptors, Progesterone; Tamoxifen; Xenograft Model Antitumor Assays

Multiple cellular effects of human growth hormone (hGH) are mediated by an indirect mechanism requiring transcriptional activation of genes encoding protein effector molecules such as insulin-like growth factor-1. Such protein effector molecules then act directly to mediate the cellular functions of hGH. We report here that autocrine hGH production by mammary carcinoma cells specifically results in the transcriptional repression of the p53-regulated placental transforming growth factor-beta (PTGF-beta) gene. Transcriptional repression of the PTGF-beta gene does not require the p53-binding sites in the PTGF-beta promoter, and autocrine hGH also desensitized the response of the PTGF-beta promoter to p53 overexpression. Transcriptional repression of the PTGF-beta gene is accompanied by consequent decreases in its protein product, Smad-mediated transcription, and its cellular effects that include cell cycle arrest and apoptosis. PTGF-beta specifically inhibited the autocrine hGH-stimulated expression of cyclin D1 required for autocrine hGH-stimulated mammary carcinoma cell cycle progression. Thus, one mechanism by which autocrine hGH promotes an increase in mammary carcinoma cell number is by transcriptional repression of protein effector molecules that promote cell cycle arrest and apoptosis. Such transcriptional repression of negative regulatory factors, such as PTGF-beta, may also be requisite for direct stimulation of mammary carcinoma cell mitogenesis by hGH.

Topics: Apoptosis; Binding Sites; Blotting, Northern; Breast Neoplasms; Cell Cycle; Cyclin D1; Female; Genes, Reporter; Growth Substances; Human Growth Hormone; Humans; Insulin-Like Growth Factor I; Luciferases; Oligonucleotides, Antisense; Plasmids; Pregnancy Proteins; Reverse Transcriptase Polymerase Chain Reaction; Transcription, Genetic; Tumor Cells, Cultured; Tumor Suppressor Protein p53

To account for the accumulation of genomic alterations required for tumor progression, it has been suggested that the genomes of cancer cells are unstable and that this instability results from defective mutators (the "mutator phenotype" theory). To examine the hypothesis that abnormal cell-cycle regulators act as the mutators contributing to genomic instability, the present study, based on primary tumor tissues from 71 patients with breast cancer, was performed to determine whether there was an association between aberrant expression of cell-cycle regulators (cyclin A, cyclin D1, cyclin E, RB1, p21, and p27) and chromosomal instability. Comparative genomic hybridization was used to measure chromosomal changes, reflecting genomic instability in individual tumors, whereas immunohistochemistry was used to detect aberrant expression of cell-cycle regulators. Overexpression of cyclin D1 was found to be significantly correlated with increased chromosomal instability (defined as harboring more than 7 chromosomal changes), with 63% of tumors overexpressing and 27% of tumors not overexpressing, with cyclin D1 showing chromosomal instability (P < 0.05). Interestingly, this relationship was independent of cell outgrowth (as detected by the proliferation marker Ki-67) and was particularly significant in tumors not expressing p27 or in tumors with detectable RB1. These results suggest that cyclin D1 plays an alternative role in the regulation of genomic stability.

Topics: Adult; Aged; Aged, 80 and over; Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Cycle Proteins; Chromosome Aberrations; Chromosome Deletion; Cyclin D1; Female; Gene Amplification; Genome, Human; Humans; Middle Aged; Nucleic Acid Hybridization

The proto-oncogene c-myc is up-regulated by estrogen stimulation of hormone-dependent breast cancer cells and is frequently overexpressed in breast and other cancers. Therapeutic interventions that inhibit c-Myc expression have been extensively investigated, including antisense oligonucleotides that have high specificity and potential clinical application. This investigation compared antiestrogen-mediated growth arrest with the molecular events after repression of c-Myc expression in MCF-7 breast cancer cells using an antisense oligonucleotide. We show that the decreased cellular proliferation of MCF-7 cells after direct inhibition of c-Myc is a consequence of inhibition of cyclin D1 expression, subsequent redistribution of p21(WAF1/CIP1) from cyclin D1-Cdk4 to cyclin E-Cdk2 complexes, and a decline in cyclin E-Cdk2 enzymatic activity. Simultaneous repression of p21(WAF1/CIP1) can attenuate the growth-inhibitory effects of reduced c-Myc expression emphasizing the importance of this cyclin-dependent kinase (CDK) inhibitor in growth arrest. These molecular events are similar to the initial changes in cyclin gene expression, CDK complex formation and CDK activity seen after antiestrogen (ICI 182780)-mediated growth inhibition of MCF-7 cells, which suggests that the down-regulation of c-Myc by ICI 182780 is a primary event that culminates in cell cycle arrest.

Topics: Breast Neoplasms; CDC2-CDC28 Kinases; Cell Cycle Proteins; Cell Division; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Cyclins; Estradiol; Estrogen Receptor Modulators; Fulvestrant; Gene Expression Regulation, Neoplastic; Genes, myc; Humans; Oligonucleotides, Antisense; Protein Serine-Threonine Kinases; Proto-Oncogene Mas; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-myc; RNA, Messenger; Tumor Suppressor Proteins; Up-Regulation

In an attempt to identify subtypes of breast cancer and pinpoint patterns of cell cycle regulatory defects associated with clinical behaviour, proliferation and other transformation associated events, a multitude of cell cycle regulatory proteins were analysed in a material of 113 primary breast cancers. Increased proliferation was observed in two different scenarios; (1) with high cyclin D1 and elevated retinoblastoma protein (pRb) phosphorylation, (cyclin D1(high) tumours) or (2) with high cyclin E protein but low cyclin D1 and lack of corresponding pRb phosphorylation (cyclin E(high) tumours) indicative of an interrupted pRb pathway. Characteristic for cyclin E(high) tumours were further defects in p53, p27 and bcl-2, while c-erbB2 overexpression and c-myc amplification was found in both cyclin D1(high) and E(high) tumours. Using transfected cell lines overexpressing cyclin E, cyclin E(high) and D1(high) tumours were mimicked and the cyclin D1(high) cell line normalized the cyclin E kinase activity by an induction and redirection of p21 and p27 to the cyclin E complex whereas cyclin E(high) cell lines obtained increased kinase activity without redirection of inhibitors. Based on differences in genetic aberrations as well as function of the pRb node we therefore propose a model in which cyclin D1(high) and cyclin E(high) tumours represent two alternative mechanisms to inactivate the pRb pathway and thereby achieve unrestrained growth in the tumorogenesis of breast cancer.

Topics: Blotting, Western; Breast Neoplasms; Cell Division; Cyclin D1; Cyclin E; Cyclin-Dependent Kinases; Follow-Up Studies; Humans; In Situ Hybridization, Fluorescence; Models, Biological; Receptors, Estrogen; Retinoblastoma Protein; Signal Transduction; Survival Rate; Tumor Cells, Cultured

To understand with greater clearness the effect of overexpression of cyclins gene and the potential implications of it for tumorgenesis in breast.. We assayed the expression of cyclins D1, E and A in different types of human breast diseases by immunohistochemical staining.. Significant difference was seen among the malignant tumor, benign tumor and dysplasis(P < 0.001). The expressive intensity of above three kind of cyclins in malignant tumor was the highest, that in benign tumor was higher, and that in dysplasis was low.. These data suggest that the expressive intensity of cyclins gene may serve as an indicator for malignant intensity of tumors. The expressive ratio difference of cyclins E and A between infiltrating ductal carcinoma and infiltrating lobular carcinoma may imply that there are different mechanisms involving the occurrence of different histological types of breast tumor.

Topics: Breast Neoplasms; Carcinoma, Ductal, Breast; Cyclin A; Cyclin D1; Cyclin E; Female; Humans

The role of CyclinD1 and estrogen receptor (ER) in the process of proliferation and metastasis of breast neoplasm and their relationship were studied. The expression levels of CyclinD1 and ER in the tissue samples were detected by using flow cytometry and L SAB immunohistochemistry staining, respectively. The results showed that CyclinD1 and ER expression levels in breast cancer were significantly higher than in benign breast neoplasm (P < 0.05). The CyclinD1 expression levels in stage I was much lower than in stages II, III, IV (P < 0.05). The positive rate of ER was not related with tumor size, lymph node metastasis and TNM stage (P > 0.05), but the CyclinD1 expression level in ER (+) group was significantly higher than in ER (-) group (P < 0.05). It was concluded that CyclinD1 expression level might be obviously related with the proliferation and metastasis of breast neoplasm and ER.

Topics: Adenocarcinoma; Adolescent; Adult; Aged; Breast Neoplasms; Carcinoma, Lobular; Cyclin D1; Female; Fibroadenoma; Humans; Middle Aged; Neoplasm Metastasis; Neoplasm Staging; Receptors, Estrogen

PC-cell-derived growth factor (PCDGF) is an 88-kDa glycoprotein corresponding to the granulin precursor. We have reported that PCDGF was expressed in human breast cancer cells. In estrogen-receptor positive cells, 17-beta-estradiol (E(2)) transcriptionally stimulated PCDGF expression in a dose- and time-dependent fashion. We demonstrate here that PCDGF mediates the mitogenic effect of E(2) in MCF-7 cells. PCDGF substituted for E(2) to stimulate DNA synthesis. The E(2) mitogenic effect was inhibited in a dose-dependent fashion by anti-PCDGF neutralizing antibody. Inhibition of PCDGF expression by antisense transfection also inhibited the E(2) mitogenic effect. In contrast, overexpression of PCDGF in MCF-7 cells resulted in cells that were able to proliferate in the absence of estrogen and were tamoxifen resistant. The PCDGF signaling pathway was examined. Like E(2), PCDGF stimulated mitogen-activated protein kinase activity. PCDGF could substitute for E(2) in stimulating cyclin D1 expression. The cyclin D1 stimulation by E(2) was 50% inhibited by anti-PCDGF antibody. In contrast, PCDGF did not stimulate c-myc expression, another molecular target of E(2). We conclude that autocrine PCDGF mediates the E(2) mitogenic effect via stimulation of cyclin D1. These studies provide information on estrogen action and identify an autocrine molecular target in human breast cancer cells.

Topics: Antibodies; Breast Neoplasms; Cell Division; Cyclin D1; DNA; Drug Resistance, Neoplasm; Enzyme Activation; Estradiol; Estrogen Receptor Modulators; Gene Expression Regulation; Glycoproteins; Growth Substances; Humans; Intercellular Signaling Peptides and Proteins; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Mitogens; Oligonucleotides, Antisense; Progranulins; Proto-Oncogene Proteins c-myc; Tamoxifen; Transfection; Tumor Cells, Cultured

Cell cycle inhibitor p27 is variously expressed in breast carcinoma. A possible association between the expression of p27 and the prognosis of breast carcinoma remains to be elucidated. We investigated the expression of p27 and cyclin D1 in a retrospective series of 216 breast carcinomas immunohistochemically. Expression of p27 (p27 LI) ranged from 0 to 93.6% (median 62.4%). There was a positive association between p27 LI and cyclin D1 (p < 0.01) and between p27 LI and ER (p < 0.0001). In the combination study of p27 LI and cyclin D1 expression, the patients classified as low p27 LI/cyclin D1 negative had a poorer prognosis than those in other categories. p27 was identified as an independent prognostic factor by the multivariate Cox proportional hazard model with a relative risk of death of disease of 4.1 (p < 0.05; vs. high p27 LI compared to the median). Assessment of p27 expression and examination of both p27 LI and cyclin D1 expression may identify breast carcinoma patients who would benefit from adjuvant therapy.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Breast Neoplasms; Cell Cycle; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Genes, pX; Humans; Middle Aged; Predictive Value of Tests; Prognosis; Proportional Hazards Models; Receptors, Estrogen

Estrogens induce proliferation of estrogen receptor (ER)-positive MCF-7 breast cancer cells by stimulating G(1)/S transition associated with increased cyclin D1 expression, activation of cyclin-dependent kinases (Cdks), and phosphorylation of the retinoblastoma protein (pRb). We have utilized blockade of cyclin D1-Cdk4 complex formation through adenovirus-mediated expression of p16(INK4a) to demonstrate that estrogen regulates Cdk inhibitor expression and expression of the Cdk-activating phosphatase Cdc25A independent of cyclin D1-Cdk4 function and cell cycle progression. Expression of p16(INK4a) inhibited G(1)/S transition induced in MCF-7 cells by 17-beta-estradiol (E(2)) with associated inhibition of both Cdk4- and Cdk2-associated kinase activities. Inhibition of Cdk2 activity was associated with delayed removal of Cdk-inhibitory activity in early G(1) and decreased cyclin A expression. Cdk-inhibitory activity and expression of both p21(Cip1) and p27(Kip1) was decreased, however, in both control and p16(INK4a)-expressing cells 20 h after estrogen treatment. Expression of Cdc25A mRNA and protein was induced by E(2) in control and p16(INK4a)-expressing MCF-7 cells; however, functional activity of Cdc25A was inhibited in cells expressing p16(INK4a). Inhibition of Cdc25A activity in p16(INK4a)-expressing cells was associated with depressed Cdk2 activity and was reversed in vivo and in vitro by active Cdk2. Transfection of MCF-7 cells with a dominant-negative Cdk2 construct inhibited the E(2)-dependent activation of ectopic Cdc25A. Supporting a role for Cdc25A in estrogen action, antisense CDC25A oligonucleotides inhibited estrogen-induced Cdk2 activation and DNA synthesis. In addition, inactive cyclin E-Cdk2 complexes from p16(INK4a)-expressing, estrogen-treated cells were activated in vitro by treatment with recombinant Cdc25A and in vivo in cells overexpressing Cdc25A. The results demonstrate that functional association of cyclin D1-Cdk4 complexes is required for Cdk2 activation in MCF-7 cells and that Cdk2 activity is, in turn, required for the in vivo activation of Cdc25A. These studies establish Cdc25A as a growth-promoting target of estrogen action and further indicate that estrogens independently regulate multiple components of the cell cycle machinery, including expression of p21(Cip1) and p27(Kip1).

Topics: Adenoviridae; Base Sequence; Breast Neoplasms; Carrier Proteins; CDC2-CDC28 Kinases; cdc25 Phosphatases; Cell Cycle; Cell Cycle Proteins; Cyclin D1; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Cyclins; Estrogens; Female; Humans; Microtubule-Associated Proteins; Models, Biological; Neoplasms, Hormone-Dependent; Oligodeoxyribonucleotides, Antisense; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Receptors, Estrogen; Retinoblastoma Protein; Transduction, Genetic; Tumor Cells, Cultured; Tumor Suppressor Proteins

We have compared the anti-proliferative effects of ursodeoxycholic acid (UDCA), chenodeoxycholic acid (CDCA) and their derivatives, HS-1183, HS-1199 and HS-1200, on MCF-7 (wild-type p53) and MDA-MB-231 (mutant p53) cells. While UDCA and CDCA exhibited no significant effect, their novel derivatives inhibited the proliferation of both cell lines in a concentration-dependent manner, concomitant with apoptotic nuclear changes and the increase of a sub-G1 population and DNA fragmentation. Furthermore, we also observed an increase in the ratio of pro-apoptotic protein Bax to anti-apoptotic protein Bcl-2 and cleavages of lamin B and poly(ADP-ribose) polymerase (PARP) in MCF-7 and MDA-MB-231 cells. Cell cycle related proteins, cyclin D1 and D3, as well as retinoblastoma protein (pRb) were down-regulated, while the level of cyclin-dependent kinase inhibitor p21(WAF1/CIP1) was increased in both cancer cells after treatment with novel bile acids. These findings suggest that these cytotoxic effects of novel bile acid derivatives on human breast carcinoma cells were mediated via apoptosis through a p53-independent pathway.

Topics: Apoptosis; Blotting, Western; Breast Neoplasms; Cell Survival; Chenodeoxycholic Acid; Cyclin D1; Cyclin D3; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; DNA Fragmentation; DNA, Neoplasm; Dose-Response Relationship, Drug; Humans; Phosphorylation; Retinoblastoma Protein; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Ursodeoxycholic Acid

p21Waf1 (p21), p27Kip1 (p27) and cyclin D1 have recently been reported as useful prognostic markers for patients with breast carcinoma. However, studies on these cell cycle regulators in ductal carcinoma in situ (DCIS) have been extremely limited. Therefore, we studied the immunohistochemical expression of p21, p27 and cyclin D1 proteins in 49 DCIS cases and compared the findings with the clinicopathologic parameters (age, tumor size, gross type, histologic type, histologic grade, necrosis and mitotic index), p53 and estrogen receptor (ER) status. A significant correlation was found between positive p21 immunoreactivity (67.3% of the cases) and well-differentiated histologic grade, non-comedo type, ER-positive and p53-negative (p53-) status. DCIS with p21+/p53- is likely to be the non-comedo type. The overexpression of cyclin D1 (59.2% of the cases) correlated positively with the ER expression (P = 0.001). The p27 protein expression (46.9% of the cases) correlated with the cyclin D1 immunopositivity (P = 0.0003) and ER expression (P = 0.005). No significant associations were seen in the p27 or cyclin D1 expression and other clinicopathologic parameters. Our results suggest that p21 might be more related to the useful biologic markers in DCIS than p27 or cyclin D1. The significant positive association between p21, p27 or cyclin D1 and ER status, and close association of p27 and cyclin D1 expression might be implicated in the tumor biology of DCIS.

Topics: Adult; Aged; Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Female; Humans; Immunoenzyme Techniques; Microfilament Proteins; Middle Aged; Mitotic Index; Muscle Proteins; Neoplasm Recurrence, Local; Prognosis; Receptors, Estrogen; Tumor Suppressor Protein p53

Breast carcinoma is thought to arise because of multiple successive changes in the genome of the normal epithelial cells. However, little is known of the order of appearance of different types of genetic aberrations We studied the ERBB2 (Her-2/neu) and CCND1 (cyclin D1) oncogene amplification in flow cytometrically sorted diploid and nondiploid tumor cell populations by fluorescence in situ hybridization (FISH). The purity of the cell sorting was confirmed by static DNA image cytometry. Spectral karyotyping was used to define differences in a genome-wide manner between two distinctly different aneuploid cell clones found in each of two breast cancer cell lines. FISH indicated the presence of gene amplification both in diploid and nondiploid cell clones in 17 of the 21 amplification-containing tumors analyzed. The oncogene copy numbers remained unchanged throughout aneuploidization in 11 of 17 tumors. The remaining six tumors showed an increase in oncogene copy number as well as the number of chromosome 11 or 17 centromeres (the original location of CCNDI and ERBB2, respectively). Breast carcinoma cell lines MDA-157 and MDA-436 showed a significant number of chromosomal rearrangements in the near-diploid clones, which were present in duplicate in the corresponding aneuploid (polyploid) clones. These results indicate that ploidy shift, ie., aneuploidization, in breast cancer is a late genetic event which is preceded by both oncogene amplifications as well as many chromosomal rearrangements.

Topics: Aneuploidy; Breast Neoplasms; Chromosome Aberrations; Cyclin D1; Diploidy; Flow Cytometry; Gene Amplification; Gene Dosage; Gene Rearrangement; Genes, erbB-2; Humans; In Situ Hybridization, Fluorescence; Karyotyping

A number of epidemiological and clinical studies have revealed that excess body weight increases the risk of postmenopausal breast cancer and also adversely affects subsequent malignant progression. To elucidate the molecular mechanisms underlying these observations, we examined mRNA expression of various genes in normal (non-cancerous) mammary gland and cancer tissue of Japanese patients with primary breast cancer, in association with their body mass index (BMI). On the basis of analysis of 106 breast cancer patients, we found that mRNA expression of insulin-like growth factor I receptor (IGF-IR) and insulin-like growth factor II (IGF-II) in the normal mammary gland showed a significant and positive association with increased BMI among postmenopausal patients. Furthermore, the positive association of increased BMI with IGF-IR mRNA expression was also found in postmenopausal breast cancer tissue, while this association was not observed among premenopausal patients. In addition, increased mRNA expression of cyclin D1 and bcl-2 was observed in association with increased mRNA levels of IGF-IR among the patients regardless of menopausal status. These findings suggest that the molecular consequence of the increased BMI is the increased expression of IGF-II and IGF-IR, resulting in development of postmenopausal breast cancer and its progression mediated through modulation of the cell cycle and apoptosis.

Topics: Adult; Aged; Body Mass Index; Breast; Breast Neoplasms; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Middle Aged; Obesity; Postmenopause; Proto-Oncogene Proteins c-bcl-2; Receptor, IGF Type 1; Risk Factors; RNA, Messenger

The tumour suppressor gene PTEN/MMAC1/TEP1 encodes a dual-specificity phosphatase that recognizes phosphatidylinositol-3,4,5-triphosphate and protein substrates. We have shown previously that over-expression of PTEN in a tetracycline-controlled inducible system blocks cell cycle progression and induces apoptosis in MCF-7 breast cancer cells. Here, we demonstrate that over-expression of wild-type PTEN leads to the suppression of cell growth through the blockade of cell cycle progression, an increase in the abundance of p27, a decrease in the protein levels of cyclin D1 and the inhibition of Akt phosphorylation. In contrast, expression of the phosphatase-dead mutant, C124S, promotes cell growth and has the opposite effect on the abundance of p27, cyclin D1 levels and the phosphorylation of Akt. The G129E mutant, which does not have lipid phosphatase activity but retains protein phosphatase activity, behaves like C124S except that the former causes decreases in cyclin D1 levels similar to wild-type PTEN. Therefore, PTEN exerts its growth suppression through lipid phosphatase-dependent and independent activities and most likely, via the coordinate effect of both protein phosphatase and lipid phosphatase activities. Addition of either estrogen or insulin abrogates PTEN-mediated up-regulation of p27 and partially blocks PTEN-mediated growth suppression, whereas the combination of estrogen and insulin eliminates the alterations of p27 and cyclin D1 and completely blocks PTEN-mediated growth suppression. Our findings demonstrate that PTEN blocks cell cycle progression differentially through down-regulating the positive cell cycle regulator, cyclin D1, by its protein phosphatase activity, and up-regulating the negative cell cycle regulator, p27, by its lipid phosphatase activity.

Topics: Amino Acid Substitution; Breast Neoplasms; Cyclin D1; Down-Regulation; Drug Interactions; Estrogens; G1 Phase; Gene Expression Regulation; Humans; Insulin; Microfilament Proteins; Muscle Proteins; Phosphatidate Phosphatase; Phosphoprotein Phosphatases; Phosphoric Monoester Hydrolases; PTEN Phosphohydrolase; Tumor Cells, Cultured; Tumor Suppressor Proteins; Up-Regulation

Zearalenone is a naturally occurring estrogenic contaminant of moldy feeds and is present in high concentrations in dairy products and cereals. Zearalenone was postulated to contribute to the overall estrogen load of women, but the mechanisms of its action are not known. We demonstrated that zearalenone could stimulate the growth of estrogen receptor-positive human breast carcinoma cell line MCF-7. In addition, zearalenone functioned as an antiapoptotic agent by increasing the survival of MCF-7 cell cultures undergoing apoptosis caused by serum withdrawal. Treatment of these cells with 100 nM zearalenone induced cell-cycle transit after increases in the expression of c-myc mRNA and cyclins D1, A, and B1 and downregulation of p27(Kip-1). G(1)/G(2)-phase kinase activity and phosphorylation of the retinoblastoma gene product was also evident. Flow cytometric analysis demonstrated entry of cells into the S and G(2)/M phases of the cell cycle, and phosphorylation of histone H3 occurred 36 h after zearalenone treatment. Ectopic expression of a dominant-negative p21(ras) completely abolished the zearalenone-induced DNA synthesis in these cells, and the specific inhibitor PD98059 for mitogen/extracellular-regulated protein kinase kinase arrested S-phase entry induced by zearalenone. These data suggest that the mitogen-activated protein kinase signaling cascade is required for zearalenone's effects on cell-cycle progression in MCF-7 cells. Given the presence of this mycotoxin in cereals, milk, and meat, the possibility that zearalenone is a potential promoter of breast cancer tumorigenesis should be investigated further. Mol. Carcinog. 30:88-98, 2001.

Topics: Adenoviridae; Annexin A5; Blotting, Northern; Blotting, Western; Breast Neoplasms; Bromodeoxyuridine; Cell Cycle; Cell Cycle Proteins; Cell Survival; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Down-Regulation; Estrogens, Non-Steroidal; Female; Flow Cytometry; Formazans; Genes, myc; Humans; Luciferases; Microtubule-Associated Proteins; Mitogen-Activated Protein Kinases; Mitosis; Phosphorylation; Proto-Oncogene Proteins p21(ras); Signal Transduction; Tetrazolium Salts; Tumor Cells, Cultured; Tumor Suppressor Proteins; Zearalenone

We have previously demonstrated that the activation of p53 signaling may contribute to tumor growth inhibition by the CRE-decoy oligonucleotide containing CRE sequence (5'-TGACGTCA-3') (Lee et al., Biochemistry 39, 4863-4868, 2000). However, growth inhibition by CRE-decoy treatment was also observed in tumor cells containing a mutant p53 (Park et al., J. Biol. Chem. 274, 1573-1580, 1999). To understand additional mechanisms of the decoy oligonucleotide, we investigated the effect on cyclin D1 expression and a cyclin D1/Cdk4/retinoblastoma protein (pRB) signaling pathway. Here we show that in MCF7 breast cancer cells the CRE-decoy competed with cyclin D1-CRE (5'-TAACGTCA-3') for binding transcription factors and reduced cyclin D1 gene expression (in reporter gene assay, Northern blotting and Western blotting) to modulate cyclin D1/Cdk4/pRB signaling and G1-S progression in a steady state and/or under estrogen stimulation. Decrease of cyclin D1 protein level by CRE-decoy treatment was also observed in p53-mutated cancer cells. Cyclin D1 expression was also diminished in MCF7 cells stably expressing dominant negative mutant CREB indicating that the nonspecific effect of oligonucleotide or its degradation products could be excluded. These data suggest that inhibition of cyclin D1 expression contributes to the growth inhibition induced by the decoy oligonucleotide in MCF7 cells through a cyclin D1/Cdk4/pRB signaling pathway. Downregulation of cyclin D1 expression also provides a mechanism of CRE-decoy-induced growth inhibition in tumor cells having p53 mutation.

Topics: Breast Neoplasms; Cell Division; Cyclic AMP Response Element-Binding Protein; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; Estrogens; Female; Humans; Oligonucleotides; Promoter Regions, Genetic; Proto-Oncogene Proteins; Response Elements; Retinoblastoma Protein; RNA, Messenger; Signal Transduction; Transcriptional Activation; Transfection; Tumor Cells, Cultured

The estrogen receptor (ER) status of breast tumors is used to identify patients who may respond to endocrine agents such as tamoxifen. However, ER status alone is not perfectly predictive, and there is a pressing need for more reliable markers of endocrine responsiveness. Here, we identified the well-known CGA gene (coding for the alpha subunit of glycoprotein hormones) as a new ERalpha-responsive gene in human breast cancer cells. We used a real-time quantitative reverse transcription-PCR assay to quantify CGA mRNA copy numbers in a large series of breast tumors. CGA overexpression (> 10 SD above the mean for normal breast tissues) was observed in 44 of 131 (33.6%) breast tumor RNAs, ranging from 20 to 16,500 times the level in normal breast tissues; the highest levels of CGA gene expression were close to those observed in placenta. Significant links were observed between CGA gene overexpression and Scarff-Bloom-Richardson histopathological grade I+II (P = 0.015), and progesterone (P = 0.0009) and estrogen (P < 10(-7)) receptor positivity, which suggested that CGA is a marker of low tumor aggressiveness. We observed CGA mRNA overexpression in 44 of 90 (48.9%) ERalpha-positive tumors and in none of the 41 ERalpha-negative tumors. Immunohistochemical studies demonstrated that human chorionic gonadotropin alpha protein was strictly limited to ERalpha-positive tumor cells. Overexpression of the CGA gene was not accompanied by overexpression of the CGB gene. Our results also suggest that CGA could be a more reliable marker than PS2 and PR for ERalpha functionality and, thus, for endocrine responsiveness. Moreover, the CGA marker has the added value of dichotomizing ERalpha-positive patients into two subgroups of similar size. Specific antibodies directed to secreted human chorionic gonadotropin alpha protein are commercially available, thus facilitating the future application of this marker to the clinical management of breast cancer.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents, Hormonal; Biomarkers, Tumor; Breast Neoplasms; Chorionic Gonadotropin, beta Subunit, Human; Cyclin D1; Cytoplasm; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Genes, erbB-2; Glycoprotein Hormones, alpha Subunit; Humans; Middle Aged; Prognosis; Proteins; Proto-Oncogene Proteins c-myc; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone; RNA, Messenger; Trefoil Factor-1; Tumor Suppressor Proteins

DETA-NONOate, a nitric oxide (NO) donor, induced cytostasis in the human breast cancer cells MDA-MB-231, and the cells were arrested in the G(1) phase of the cell cycle. This cytostatic effect of the NO donor was associated with the down-regulation of cyclin D1 and hypophosphorylation of the retinoblastoma protein. No changes in the levels of cyclin E or the catalytic partners of these cyclins, CDK2, CDK4, or CDK6, were observed. This NO-induced cytostasis and decrease in cyclin D1 was reversible for up to 48 h of DETA-NONOate (1 mM) treatment. DETA-NONOate (1 mM) produced a steady-state concentration of 0.5 microM of NO over a 24-h period. Synchronized population of the cells exposed to DETA-NONOate remained arrested at the G(1) phase of the cell cycle whereas untreated control cells progressed through the cell cycle after serum stimulation. The cells arrested at the G(1) phase after exposure to the NO donor had low cyclin D1 levels compared with the control cells. The levels of cyclin E and CDK4, however, were similar to the control cells. The decline in cyclin D1 protein preceded the decrease of its mRNA. This decline of cyclin D1 was due to a decrease in its synthesis induced by the NO donor and not due to an increase in its degradation. We conclude that down-regulation of cyclin D1 protein by DETA-NONOate played an important role in the cytostasis and arrest of these tumor cells in the G(1) phase of the cell cycle.

Topics: Breast Neoplasms; Cell Cycle; Cell Division; Cyclin D1; Down-Regulation; G1 Phase; Growth Inhibitors; Humans; Nitric Oxide; Nitric Oxide Donors; Nitroso Compounds; Tumor Cells, Cultured

Cyclin D1 amplification and/or protein overexpression have been observed not only in breast cancer but also in the putative early stages of breast neoplasia. In a case-control study nested within a cohort of 4888 women, we investigated whether the occurrence of cyclin D1 gene amplification and/or protein overexpression in benign breast tissue might identify women at increased risk of subsequent breast cancer development. Cases were 92 women with a histological diagnosis of benign breast disease who subsequently developed breast cancer. Five controls (women with benign breast disease who had not developed breast cancer by the date of diagnosis of the corresponding case) were selected randomly for each case from those non-cases available within strata defined by screening centre, National Breast Screening Study (NBSS) study arm, year of birth and age at diagnosis of benign breast disease. Paraffin blocks of benign tissue were suitable for immunostaining for 71 cases and 293 controls. Sufficient DNA for analysis was obtained from a total of 356 subjects (69 cases, 287 controls). The benign breast tissues and breast cancers were immunostained for cyclin D1 and also analysed for the presence of cyclin D1 gene amplification by differential polymerase chain reaction (PCR). Fifteen cases and 60 controls showed evidence of cyclin D1 immunostaining, and 12 cases and 29 controls showed cyclin DL gene amplification. There was essentially no association between cyclin D1 protein overexpression in benign breast tissue and risk of subsequent breast cancer (adjusted odds ratio (OR) 1.06; 95% confidence interval (CI) 0.56-2.02). After adjustment for potential confounding, there was a statistically non-significant 40% increase in risk of breast cancer in association with cyclin D1 gene amplification (adjusted OR 1.41; 95% CI 0.62-3.22). As multiple genetic changes are required to develop breast cancer, it may not be until the cascade of molecular alterations leading to breast cancer development is understood that identification of biomarkers of breast cancer risk will be possible.

Topics: Adult; Breast; Breast Neoplasms; Case-Control Studies; Cyclin D1; Female; Gene Amplification; Gene Expression; Genes, bcl-1; Humans; Immunohistochemistry; Middle Aged; Polymerase Chain Reaction; Risk Factors

Inhibition of cyclin dependent kinases (Cdks) is of pivotal importance in tumor cell biology as these kinases are the drivers of cell proliferation. This inhibition can be achieved either by naturally occurring biological proteins or by small molecule compounds. They cause cell cycle arrest and/or apoptosis depending upon the specificity and efficacy of the inhibitor in question. We have reported earlier that specific pyridopyrimidines (novel Cdk inhibitors) cause cell cycle arrest in mink lung epithelial cells and the arrest is abrogated by over-expression of Cdk4. In contrast, we show here that one of these inhibitors effectively maintains cell cycle arrest in a leukemic or a breast cancer cell line even after the respective cells over-express an oncogene, either Bcl-2 or cyclin D1. However, in the leukemic cells, Bcl-2 over-expression suppresses apoptosis induced by the pyridopyrimidine. Thus, novel Cdk inhibitors can prove to be useful chemical genetics tools for understanding the underlying mechanisms of growth arrest and/or apoptosis in normal versus tumor cells. This could also lead to the development of improved inhibitors of cell proliferation.

Topics: Apoptosis; Blotting, Western; Breast Neoplasms; Cell Cycle; Cell Division; Cyclin D1; Cyclin-Dependent Kinases; Enzyme Inhibitors; Female; Flow Cytometry; Humans; Neoplasm Proteins; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma; Proto-Oncogene Proteins c-bcl-2; Pyrimidines; Tumor Cells, Cultured

Cyclin D1 is associated with cell cycle regulation and has more recently been shown to stimulate the transcriptional functions of the oestrogen receptor (ER). Furthermore, in normal breast there is a negative association between expression of ER and the proliferation marker Ki67 indicating that either ER positive cells are non-dividing or that the receptor is down-regulated as cells enter cycle. This important relationship breaks down in many ER-positive cancers and precancerous breast lesions where the receptor is often detected on proliferating cells. The aims of the present study were to determine the interplay between ER, Ki67 and cyclin D(1)in individual cells within the spectrum of human breast lesions ranging from normal to invasive carcinoma by using dual staining immunofluorescence. We found that in normal breast there was a strong positive association between ER and cyclin D(1)expression. In contrast there was a strong negative association between cyclin D(1)and Ki67 expression. Similar findings were seen for the other precancerous and cancerous breast lesions. Thus immunodetectable cyclin D(1)within individual cells does not appear to be associated with cell cycle progression in the benign or malignant breast but instead may have important interactions with ER.

Topics: Antibodies, Monoclonal; Antibody Specificity; Breast; Breast Neoplasms; Cyclin D1; Humans; Hyperplasia; Immunohistochemistry; Ki-67 Antigen; Mitosis; Neoplasm Invasiveness; Precancerous Conditions; Receptors, Estrogen

In order to understand the intricate relationship of cell proliferation and apoptosis in tumour development, proliferation markers (Ki-67 and c-myc), apoptosis, cell-cycle inducers cyclin D1 and D3, and cell-cycle inhibitors p16(INK4), p21(CIP1), and p27(KIP1) were evaluated in ductal breast carcinoma. The heterogeneous nature of breast tumours provides a system by which the changes in cell-cycle genes can be explored under a wide range of proliferation and apoptotic indices. To address the above issues, immunohistochemical studies were conducted in 40 pairs of tumours and adjacent normal ductal tissues. The TUNEL method was used to identify apoptotic cells. Except for p27/KIP1, the proliferation (Ki-67, c-myc) and the apoptotic indexes together with levels of p16/INK4a, p21/CIP1, cyclin D1, and cyclin D3, were clearly elevated among tumour tissues, while absent in the adjacent normal tissues. Spearman correlation analysis indicated strong associations among apoptotic index, Ki-67, c-myc, and tumour grade. In addition, p21/CIP1 and cyclin D3 were positively correlated, while p16/INK4a, p27/KIP1, and cyclin D1 were negatively correlated with tumour grade. There was clear decoupling between p21 and p27, as well as decoupling between cyclin D1 and cyclin D3, in terms of their relationship to cell proliferation and apoptosis, indicating differential roles in tumour progression.

Topics: Apoptosis; Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Cycle Proteins; Cell Division; Cyclin D1; Cyclin D3; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclins; Disease Progression; Female; Humans; In Situ Nick-End Labeling; Microtubule-Associated Proteins; Neoplasm Proteins; Tumor Suppressor Proteins

The proliferation of many estrogen receptor (ER)-positive breast cancer cells depends on estradiol, and tumors arising from these cells are often responsive initially to treatment with selective ER modulators, which produce an antiestrogen effect. However, tumors that are refractory to the antiestrogenic effects of selective ER modulators often reemerge, and the prognosis for these patients is poor because of the lack of additional effective therapy. Accordingly, deciphering the cellular events associated with estrogen-dependent growth and the subsequent outgrowth of tumors with an estrogen-independent phenotype is of considerable interest. Here we show that the expression of PP5, an evolutionarily conserved Ser/Thr phosphatase that functions as an inhibitor of glucocorticoid- and p53-induced signaling cascades leading to growth suppression, is responsive to 17beta-estradiol (E(2)) in ER-positive human breast carcinoma cells (MCF-7). Northern analysis revealed that E(2)-induced PP5 expression is blocked by treatment with tamoxifen, and a consensus ER recognition element was identified in the PP5 promoter. The PP5-ER recognition element associates with human ERs and confers E(2)-induced transcriptional activation to reporter plasmids. The specific inhibition of PP5 expression ablates E(2)-mediated proliferation in MCF-7 cells without having an apparent effect on E(2)-induced expression of c-myc or cyclin D1. Thus, although critical for cell growth, PP5 likely acts either downstream or independently of c-Myc and Cyclin D1. To further characterize the role of PP5 in E(2)-regulated growth control, we constructed stable MCF-7 cell lines in which the expression of PP5 was placed under the control of tetracycline-regulated transactivator and operator plasmids. Studies with these cells revealed that the constitutive overexpression of PP5 affords E(2)-dependent MCF-7 cells with the ability to proliferate in E(2)-depleted media. Together, these studies indicate that E(2)-induced PP5 expression functions to enhance E(2)-initiated signaling cascades leading to cell division and that aberrant PP5 expression may contribute to the development of MCF-7 cells with an estrogen-independent phenotype.

Topics: Base Sequence; Breast Neoplasms; Cell Division; Cyclin D1; DNA; Estrogens; Genes, myc; Humans; Molecular Sequence Data; Nuclear Proteins; Phenotype; Phosphoprotein Phosphatases; Promoter Regions, Genetic; Receptors, Glucocorticoid; RNA, Messenger; Signal Transduction; Tumor Cells, Cultured

AIB1 was isolated as a gene amplified in breast cancer and encodes a protein that acts as a steroid receptor coactivator. The role of steroid receptor coactivators such as AIB1 in breast cancer development is not clear. It is possible that AIB1 cooperates with estrogen receptor alpha in regulating estrogen-dependent cell proliferation. Ectopic expression of the estrogen receptor alpha in different cell lines does not confer estrogen-induced proliferation. This inability of the estrogen receptor to drive proliferation has been recently correlated with a lack of estrogen-dependent cyclin D1 expression in cells engineered to express the estrogen receptor. In this study, we evaluated whether high levels of AIB1 enable the estrogen receptor to direct the transcription of cyclin D1. We show here that AIB1 and other steroid receptor coactivators can enhance the functional interaction of the estrogen receptor with the cyclin D1 promoter. Increases of AIB1 levels in breast cancer cells by amplification and/or overexpression may represent one way to confer estrogen-dependent mitogenic stimulation to breast cancer cells.

Topics: Breast Neoplasms; Carrier Proteins; Cell Line; Cyclin D1; Estrogens; Gene Expression Regulation; Genes, bcl-1; Histone Acetyltransferases; Humans; Keratinocytes; Nerve Tissue Proteins; Nuclear Receptor Coactivator 1; Nuclear Receptor Coactivator 3; Promoter Regions, Genetic; Receptors, Estrogen; Receptors, Steroid; Transcription Factors; Transcription, Genetic; Transfection; Tumor Cells, Cultured

Local recurrence (LR) after breast-conserving therapy (BCT) is associated with an increased risk for the development of distant metastasis. We studied risk factors for distant metastasis risk (DMR) and poor prognosis within a group of patients with LR as first event.. From a cohort of 1481 breast carcinomas treated with BCT in the period 1980-1994, a total of 68 pT1-3 N0-1 patients developed LR as first event. We have studied risk factors for the development of distant metastasis within this group of patients with LR. In addition to clinical factors (age at BCT and LR, mode of detection, location of LR, and treatment of LR), the histology slides of the primary and the recurrent tumor were reviewed. Immunohistochemical staining was performed for the following proteins: bcl-2, cyclin D1, E-cadherin, EGF receptor, ER, PR, Ki-67, c-erbB-2/neu, and p53. Statistical analyses were performed using conditional logistic regression.. At a median follow-up after LR of 5.6 years, the 5-year DMR was 53%. In univariate analysis, none of the factors of the primary tumor was found to be associated with DMR after LR. Of the recurrent tumor the following factors were found to be risk factors for high DMR after LR: interval between treatment of the primary tumor and LR at 2 years or less (relative risk, 2.38; 95% confidence interval, 1.22-4.76; p = 0.008) and high mitotic count (relative risk, 2.51; 95% confidence interval, 1.03-6.15; p = 0.04). All patients with noninvasive recurrent tumor were alive at the time of analysis. Patients with an interval of greater than 2 years and a recurrent tumor with high mitotic count were found to have an equally poor prognosis compared to patients with LRs detected after a short interval.. LR after BCT is associated with higher DMR and poor prognosis. Patients with LR within 2 years after BCT are especially at high risk. Late recurrences with high mitotic count have the same poor prognosis as early recurrences. For these patients, systemic treatment at time of the detection of LR should be considered.

Topics: Analysis of Variance; Breast Neoplasms; Cadherins; Cyclin D1; ErbB Receptors; Female; Humans; Immunohistochemistry; Ki-67 Antigen; Mastectomy, Segmental; Middle Aged; Mitosis; Neoplasm Metastasis; Neoplasm Recurrence, Local; Neoplasm Staging; Proto-Oncogene Proteins c-bcl-2; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone; Risk Factors; Tumor Suppressor Protein p53

Preoperative assessment of the biologic characteristics of primary breast carcinoma is important because neoadjuvant medical therapy is being used increasingly. In the current study, the authors attempted to evaluate the validity of cyclin D1 assay in fine-needle aspiration (FNA) samples from patients with primary breast carcinoma.. FNA samples were obtained prior to therapy and multiple slides were stored at -80 degrees C for subsequent immunocytochemical analysis (ICA). ICA for cyclin D1 protein was performed on FNA samples from 51 breast carcinoma patients and 20 samples from patients with benign breast disease. In 45 breast carcinoma patients who had undergone surgery, sections were taken from paraffin blocks and stained by ICA for cyclin D1 validation. Possible correlations between cyclin D1 expression in the FNA samples and the biologic data of the patients also were analyzed.. Cyclin D1 expression was detected in 37 FNA samples from 51 breast carcinomas (72.5%) whereas expression of cyclin D1 was detected in 8 FNA samples from 20 patients with benign breast disease (40%). In histologic sections after surgery, 26 cases of breast carcinoma (65%) showed a positive reaction to cyclin D1. Concordance for the presence of cyclin D1 between FNA samples and histologic samples was 75%. Cyclin D1 expression was high in patients with the tumors that expressed estrogen receptor (ER) (30 of 34 vs. 5 of 11; P = 0.028) and progesterone receptor (PR) (33 of 38 vs. 2 of 7; P = 0.007). There was no significant relation found between cyclin D1 expression and tumor size or lymph node metastasis. Cyclin D1 expression within invasive ductal carcinoma was observed in > 80% of low or intermediate nuclear grade tumors but its expression decreased to 61.5% (8 of 13 cases) in tumors with high nuclear grade (P = 0.023). All 14 breast carcinomas in which the S-phase fraction was 15% showed cyclin D1 expression. Cyclin D1 expression was found to be correlated inversely with proliferative activity in breast carcinoma (P = 0.023).. The results of the current study show that cyclin D1 expression can be measured by ICA in FNA samples with reasonable concordance with the results of histologic section. Cyclin D1 expression was found to be associated with ER/PR status and cell differentiation. The results of the current study indicate that the measurement of novel molecular markers could be performed adequately in FNA samples as well as in histologic sections.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Biopsy, Needle; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Cyclin D1; Female; Humans; Immunoenzyme Techniques; Middle Aged; Neoplasm Invasiveness

Cyclin D1 gene expression is induced by 17beta-estradiol (E2) in human breast cancer cells and is important for progression of cells through the G(1) phase of the cell cycle. The mechanism of activation of cyclin D1 is mitogen- and cell context-dependent, and this study describes the role of multiple promoter elements required for induction of cyclin D1 by E2 in estrogen receptor (ER)-positive ZR-75 breast cancer cells. Transcriptional activation of cyclin D1 by E2 was dependent, in part, on a proximal cAMP-response element at -66, and this was linked to induction of protein kinase A-dependent pathways. These results contrasted to a recent report showing that induction of cyclin D1 by E2 in ER-positive MCF-7 and HeLa cells was due to up-regulation of c-jun and subsequent interaction of c-Jun-ATF-2 with the CRE. Moreover, further examination of the proximal region of the cyclin D1 promoter showed that three GC-rich Sp1-binding sites at -143 to -110 were also E2-responsive, and interaction of ERalpha and Sp1 proteins at these sites was confirmed by electromobility shift and chromatin immunoprecipitation assays. Thus, induction of cyclin D1 by E2 in ZR-75 cells is regulated through nuclear ERalpha/Sp1 and epigenetic protein kinase A activation pathways, and our results suggest that this mechanism may be cell context-dependent even among ER-positive breast cancer cell lines.

Topics: Breast Neoplasms; Cyclin D1; Enhancer Elements, Genetic; Estrogens; Female; Gene Expression Regulation; Humans; Promoter Regions, Genetic; Response Elements; Tumor Cells, Cultured

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Genes, erbB-2; Genes, ras; Genetic Predisposition to Disease; Humans; Mammary Neoplasms, Experimental; Mice; Promoter Regions, Genetic

Breast cancer is the most common malignancy among women. Most of these cancers overexpress cyclin D1, a component of the core cell-cycle machinery. We previously generated mice lacking cyclin D1 using gene targeting. Here we report that these cyclin D1-deficient mice are resistant to breast cancers induced by the neu and ras oncogenes. However, animals lacking cyclin D1 remain fully sensitive to other oncogenic pathways of the mammary epithelium, such as those driven by c-myc or Wnt-1. Our analyses revealed that, in mammary epithelial cells, the Neu-Ras pathway is connected to the cell-cycle machinery by cyclin D1, explaining the absolute dependency on cyclin D1 for malignant transformation in this tissue. Our results suggest that an anti-cyclin D1 therapy might be highly specific in treating human breast cancers with activated Neu-Ras pathways.

Topics: Animals; Animals, Genetically Modified; Antineoplastic Agents; Breast; Breast Neoplasms; Cell Transformation, Neoplastic; Crosses, Genetic; Cyclin D1; Female; Genes, bcl-1; Genes, erbB-2; Genes, myc; Genes, ras; Genetic Predisposition to Disease; Humans; Male; Mammary Neoplasms, Experimental; Mammary Tumor Virus, Mouse; Mice; Proto-Oncogene Proteins; Tumor Cells, Cultured; Wnt Proteins; Wnt1 Protein; Zebrafish Proteins

The complex insulin-like growth factor network of ligands, receptors and binding proteins has been shown to be disturbed in breast cancer. In addition to defects in proteins controlling cell cycle checkpoints, this type of aberrations could affect tumor growth and survival thereby influencing both tumor aggressiveness and potential response to treatments. We have previously identified the T1A12/mac25 protein, which is identical to the IGFBP-rP1, as a differentially expressed gene product in breast cancer cells compared with normal cells. Here we compare the expression of IGFBP-rP1 in 106 tumor samples with known status of cell cycle aberrations and other clinicopathological data. This was done using a tumor tissue section array system that allows for simultaneous immunohistochemical staining of all samples in parallel. Cytoplasmic staining of variable intensity was observed in most tumors, 15% lacked IGFBP-rP1 staining completely, 20% had weak staining, 32% intermediate and 33% showed strong staining. Low IGFBP-rP1 was associated with high cyclin E protein content, retinoblastoma protein (pRb) inactivation, low bcl-2 protein, poorly differentiated tumors and higher stage. There was a significantly impaired prognosis for patients with low IGFBP-rP1 protein tumors. Interestingly, IGFBP-rP1 showed an inverse association with proliferation (Ki-67%) in estrogen receptor negative tumors as well as in cyclin E high tumors suggesting a separate cell cycle regulatory function for IGFBP-rP1 independent of interaction with the estrogen receptor or the pRb pathway.

Topics: Aneuploidy; Biomarkers, Tumor; Breast Neoplasms; Carrier Proteins; Cell Cycle; Cyclin D1; Cyclin E; Diploidy; Female; Gene Expression Regulation, Neoplastic; Genes, bcl-2; Genes, erbB-2; Genes, p53; Genes, Tumor Suppressor; Humans; Immunohistochemistry; Insulin-Like Growth Factor Binding Proteins; Lymphatic Metastasis; Menopause; Neoplasm Invasiveness; Neoplasm Staging; Polymorphism, Single-Stranded Conformational; Prognosis; Receptors, Estrogen; Receptors, Progesterone; Retinoblastoma Protein; Telomerase; Tumor Suppressor Protein p53

Phosphorylation on serines or threonines preceding proline (Ser/Thr-Pro) is a major signaling mechanism. The conformation of a subset of phosphorylated Ser/Thr-Pro motifs is regulated by the prolyl isomerase Pin1. Inhibition of Pin1 induces apoptosis and may also contribute to neuronal death in Alzheimer's disease. However, little is known about the role of Pin1 in cancer or in modulating transcription factor activity. Here we report that Pin1 is strikingly overexpressed in human breast cancers, and that its levels correlate with cyclin D1 levels in tumors. Overexpression of Pin1 increases cellular cyclin D1 protein and activates its promoter. Furthermore, Pin1 binds c-Jun that is phosphorylated on Ser63/73-Pro motifs by activated JNK or oncogenic Ras. Moreover, Pin1 cooperates with either activated Ras or JNK to increase transcriptional activity of c-Jun towards the cyclin D1 promoter. Thus, Pin1 is up-regulated in human tumors and cooperates with Ras signaling in increasing c-Jun transcriptional activity towards cyclin D1. Given the crucial roles of Ras signaling and cyclin D1 overexpression in oncogenesis, our results suggest that overexpression of Pin1 may promote tumor growth.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Breast; Breast Neoplasms; Carcinoma in Situ; Cells, Cultured; Cyclin D1; Female; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Humans; JNK Mitogen-Activated Protein Kinases; Middle Aged; Mitogen-Activated Protein Kinases; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; Phosphorylation; Promoter Regions, Genetic; Proto-Oncogene Proteins c-jun; ras Proteins; Receptor, ErbB-2; Receptors, Estrogen; Signal Transduction; Transcription, Genetic; Tumor Cells, Cultured

The biochemical mechanisms underlying the inhibitory effects of lycopene, the main tomato carotenoid, on the growth of cancer cells are largely unknown. It has been hypothesized that lycopene derivatives may act as ligands for a nuclear receptor in analogy to retinoic acid, the hormone derived from beta-carotene. The inhibition of human mammary cancer (MCF-7) cell growth and the transactivation of the retinoic acid receptor (RAR) reporter gene by synthetic acyclo-retinoic acid, the open chain analog of retinoic acid, was compared to the effects of lycopene and retinoic acid in the same systems. Acyclo-retinoic acid activated the DR-5 retinoic acid response element with a approximately 100-fold lower potency than retinoic acid. This effect was independent of cotransfection with the RARalpha receptor. Lycopene exhibited only very modest activity in this system. In contrast to the results from the transactivation studies, acyclo-retinoic acid, retinoic acid, and lycopene inhibited cell growth with a similar potency. Preincubation with each of the three compounds slowed down cell cycle progression from G1 to S phase. In summary, acyclo-retinoic acid inhibited cancer cell growth and interacted with RAR. However, it exhibited low affinity for RAR and a correspondingly low efficacy in activating this receptor, indicating that RAR does not mediate the growth inhibitory effect of the compound. In addition, the concentrations of acyclo-retinoic acid and of lycopene required for inducing inhibition of cell growth were similar, suggesting that acyclo-retinoic acid is unlikely to be the active metabolite of lycopene.

Topics: Antineoplastic Agents; Breast Neoplasms; Carotenoids; Cell Cycle; Cell Cycle Proteins; Cell Division; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclins; Humans; Lycopene; Receptors, Retinoic Acid; Retinoids; Tumor Cells, Cultured; Tumor Suppressor Proteins

There is increasing evidence that there are different progression routes leading to invasive breast cancer, depending on histology and differentiation grade. The aim of this study was to determine alterations in the expression of proteins involved in proliferation and apoptosis in non-invasive and invasive ductal breast lesions. Immunohistochemistry was performed on 106 usual ductal hyperplasias (UDH), 61 DCIS lesions and 53 invasive ductal breast carcinomas. Increased proliferation (Ki67), overexpression of cyclin D1, HER-2/neu, p21 and p53, and decreased expression of bcl-2 and p27 could already be found in UDH. Significant differences between UDH and DCIS lesions were found for only one protein when UDH was compared with well-differentiated DCIS (p27), for three proteins when compared with intermediately differentiated DCIS (p21, cyclin D1, Ki-67), and for all proteins when compared with poorly-differentiated DCIS. Comparing DCIS with invasive lesions of same differentiation grade, proliferation was elevated in the invasive lesions. Altered expression of the other proteins was in general only slightly increased in the invasive lesion compared with DCIS. The number of proteins with altered expression per lesion was highest in poorly-differentiated lesions and was comparable between DCIS and invasive cancer of the same differentiation grade. In conclusion, the biggest changes in expression of these proliferation and apoptosis related proteins appear to occur during the transition from hyperplasia to DCIS; they probably play a minor role in the transition from DCIS to invasive breast lesion of same differentiation grade. Well-differentiated in situ and invasive breast lesions share many of the aberrations in expression of these proteins, as do poorly-differentiated in situ and invasive lesions. However, there are many differences between the well and poorly-differentiated lesions. This further supports the existence of different progression routes leading to breast cancer.

Topics: Apoptosis; Breast Neoplasms; Carcinoma in Situ; Carcinoma, Ductal, Breast; Cell Cycle Proteins; Cyclin D1; Female; Humans; Immunohistochemistry; Ki-67 Antigen; Proto-Oncogene Proteins c-bcl-2; Receptor, ErbB-2; Tumor Suppressor Protein p53

Topics: Animals; Breast Neoplasms; Cyclin D1; Humans; Mice; Mice, Knockout

Breast tumors of BRCA1 mutation carriers and those of early onset breast cancer cases share similar histological features, being generally high-grade, highly proliferative, aneuploid tumors that are predominantly estrogen- and progesterone-receptor negative. Because histological features of tumors of premenopausal women differ from those of tumors of older women, we sought to determine whether the immunophenotype of breast tumors of BRCA1 mutation carriers was influenced by age at diagnosis.. We examined 31 breast tumors from BRCA1 mutation carriers and compared them with 81 tumors of age-matched (plus or minus 5 years) breast cancer patients unselected for family history. Tumors were further matched for histology, grade, and size. Paraffin-embedded tumor tissues were examined for protein expression of estrogen receptor (ER), PR, Ki-67, cyclin D1, TP53, HER2, beta-catenin, and cyclin E using immunohistochemical approaches.. ER (P = 0.01), PR (P = 0.06), and cyclin D1 (P = 0.002) were less frequently expressed and Ki-67 (P = 0.01) and beta-catenin (P = 0.04) were more frequently expressed in tumors of BRCA1 mutation carriers than controls. After age stratification, we found a significant difference in the frequency of tumors of BRCA1 mutation carriers diagnosed before 50 years of age compared with age-matched controls that stained positive for ER (P = 0.01), PR (P = 0.03), Ki-67 (P = 0.008), cyclin D1 (P < 0.001), HER2 (P = 0.04), and beta-catenin (P = 0.05). However, no significant differences were observed in tumors of BRCA1 mutation carriers diagnosed at age 50 or older compared with age-matched controls.. These data suggest that age at diagnosis, possibly related to menopausal status, may be an important factor in the expression of specific proteins in breast tumors of BRCA1 mutation carriers.

Topics: Adult; Age of Onset; Aged; Aged, 80 and over; beta Catenin; BRCA1 Protein; Breast Neoplasms; Cyclin D1; Cyclin E; Cytoskeletal Proteins; DNA Mutational Analysis; DNA, Neoplasm; Family Health; Female; Heterozygote; Humans; Immunohistochemistry; Ki-67 Antigen; Middle Aged; Mutation; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone; Registries; Trans-Activators; Tumor Suppressor Protein p53

Human solid tumors undergo clonal evolution as they progress, but evidence for specific sequences of genetic changes that occur in individual tumors and are recapitulated in other tumors is difficult to obtain.. Patterns of amplification of Her-2/neu, c-myc, and cyclin D1 were determined by fluorescence in situ hybridization (FISH) in relation to the presence of p53 dysfunction and ploidy in 60 primary human breast cancers.. We show that there are clusters of genophenotypic abnormalities that distinguish lobular breast cancers from nonlobular tumors; that cyclin D1 amplification occurs prior to the divergence of lobular breast cancers from nonlobular cancers; that p53 dysfunction, Her-2/neu amplification, and c-myc amplification are characteristic features of nonlobular breast cancers, but not of lobular breast cancers; and that the frequencies of amplification of all three oncogenes examined increase progressively with increasing aneuploidy, but that each gene exhibits a different profile of increasing amplification in relation to tumor progression. Early amplification of c-myc appears to be an especially prominent feature of hypertetraploid/hypertetrasomic tumors.. The data suggest that in tumors containing multiple abnormalities, these abnormalities often accumulate in the same cells within each tumor. Furthermore, the same patterns of accumulation of multiple genophenotypic abnormalities are recapitulated in different tumors.

Topics: Alleles; Aneuploidy; Breast Neoplasms; Carcinoma, Ductal, Breast; Chromosomes, Human, Pair 17; Cyclin D1; Disease Progression; Genes, p53; Genotype; Humans; In Situ Hybridization, Fluorescence; Loss of Heterozygosity; Phenotype; Ploidies; Proto-Oncogene Proteins c-myc; Receptor, ErbB-2

Molecular markers are increasingly being analyzed in tumor specimens because of their relevance to both prognosis and choice of therapy. Paget disease of the breast is an uncommon form of breast cancer, in which molecular markers have not been well characterized. The objective of this study was to investigate the expression of c-erbB-2, p53, Ki-67, Cyclin D1, Bcl-2, estrogen receptors (ER), and progesterone receptors (PR) in mammary Paget disease.. Archival tumor tissues from 14 patients diagnosed between 1990 and 1999 with Paget disease of the breast were analyzed for these molecular markers by using an automated immunohistochemical assay. Both the intraepidermal Paget cells and the underlying carcinoma were assessed for these markers.. The majority of Paget cells were positive for c-erbB-2 (92.9%), Cyclin D1 (100%), and Ki-67 (85.7%), but very few were positive for Bcl-2 (14.3%). p53 was overexpressed in 42.9% of the cases, and only 28.6% were positive for ER and PR. The rate of expression of these biologic markers was similar in both the Paget cells and the underlying intraductal and/or ductal carcinoma cells.. Tumors from patients with Paget disease of the breast were positive for c-erbB-2, Cyclin D1, and Ki-67, molecular markers commonly associated with more aggressive tumor behavior and poorer survival in breast cancer patients. Few of these tumors expressed Bcl-2 or ER and PR, which are generally associated with a better prognosis. Similar expression of these markers in both Paget cells and the underlying carcinoma supports the theory that these cells are the result of an intraepidermal spread of ductal carcinoma.

Topics: Biomarkers, Tumor; Breast Neoplasms; Cyclin D1; Female; Humans; Immunohistochemistry; Ki-67 Antigen; Paget's Disease, Mammary; Prognosis; Proto-Oncogene Proteins c-bcl-2; Receptor, ErbB-2; Receptors, Estrogen; Tumor Suppressor Protein p53

It was recently reported that significantly fewer breast tumours of BRCA1 mutation carriers overexpressed cyclin D1 and HER2 protein than tumours of age matched breast cancer cases unselected for family history. This study aimed to examine the genetic basis of this reduction by determining the frequency of tumours within this cohort showing DNA amplification of these genes.. Paraffin wax embedded sections of breast tumours from BRCA1 mutation carriers and age, grade, histological type, and tumour size matched non-familial controls that had previously been stained for cyclin D1 and HER2 protein overexpression were analysed for CCND1 and HER2 gene amplification using fluorescence in situ hybridisation.. CCND1 amplification was detected in none of the 30 tumours of the BRCA1 mutation carriers and in 19 of 74 tumours of the matched controls. Of those samples previously determined to overexpress the HER2 protein, HER2 amplification was detected in one of three tumours from BRCA1 mutation carriers and in 13 of 17 tumours of the age matched non-familial cases.. None of the tumours of BRCA1 mutation carriers showed CCND1 amplification and only one tumour showed HER2 amplification. In contrast, a large proportion of cyclin D1 and HER2 overexpression in tumours of non-familial breast cancer cases could be accounted for by amplification of these genes. These data suggest that breast tumorigenesis in BRCA1 mutation carriers occurs by a molecular mechanism distinct from that of age matched non-familial cases.

Topics: Breast Neoplasms; Case-Control Studies; Cyclin D1; Female; Gene Amplification; Genes, BRCA1; Genes, erbB-2; Heterozygote; Humans; In Situ Hybridization, Fluorescence; Mutation

The aromatic fatty acid phenylacetate (PA) and its analogs have come under intense investigation due to their ability to cause the growth arrest of a variety of neoplasia, including human breast cancer. We have determined that PA and its halide derivative 4-chlorophenylacetate (4-CPA) showed marked antiproliferative activity on 3 of 6 human breast cancer cell lines tested. Interestingly, the 3 cell lines that were growth inhibited by PA and 4-CPA were estrogen receptor (ER) positive (T47-D, MCF-7 and ZR-75-1) whereas those that were little affected by these compounds were ER-negative (MDA-MB-157, MDA-MB-231 and SK-Br-3). Dose response studies indicated that 4-CPA inhibited the growth of the sensitive (ER+) cell lines with a potency 3-4 times that of PA. These findings suggest that there is "cross-talk" between the PA and estrogen signaling pathways such that PA can directly inhibit estrogen-dependent events. This hypothesis was directly tested in vitro using ER+ MCF-7 cells that were stably transfected with a luciferase reporter construct driven by the full length (1745 bp) cyclin D1 promoter (MCF-7-D1). Our experiments with MCF-7-D1 cells indicated that PA and 4-CPA inhibited basal and estrogen-induced reporter gene activity by up to 90%, resulting in almost complete elimination of estrogen-dependent cyclin D1 gene activation. Using a reporter gene construct (ERE(V)-tk-Luc) containing a canonical estrogen response element that was transiently transfected into MCF-7 and MDA-MB-231 cells, we have also demonstrated inhibition of promoter activity by PA and 4-CPA that was directly mediated by blockage of activity through the ERE. Taken together, these findings indicate that PA analogs possess potent antiestrogen properties that may, at least partly, account for their antiproliferative effects on ER+ breast cancer cells. The data suggests a novel mechanism of action that might bypass some of the limitations of conventional antiestrogen therapy.

Topics: Antimetabolites, Antineoplastic; Breast Neoplasms; Cell Division; Cyclin D1; Dose-Response Relationship, Drug; Drug Interactions; Drug Screening Assays, Antitumor; Estrogens; Gene Expression; Humans; Phenylacetates; Promoter Regions, Genetic; Tumor Cells, Cultured

Estrogen can increase insulin-like growth factor-I receptor (IGF-IR) and insulin receptor substrate-1 (IRS-1) expression, two key components of IGF-I-mediated signaling. The result is sensitization of breast cancer cells to IGF-I and synergistic growth in the presence of estrogen and IGF-I. We hypothesized that loss of estrogen receptor alpha (ERalpha) would result in reduced IGF-mediated signaling and growth. To test this hypothesis, we examined IGF-I effects in MCF-7 breast cancer cell sublines that have been selected for loss of ERalpha (C4 and C4-12 cells are ERalpha-negative) by long-term estrogen withdrawal. C4 and C4-12 cells had reduced IGF-IR and IRS-1 mRNA and protein expression (compared with MCF-7 cells) that was not inducible by estrogen. Furthermore, C4 and C4-12 cells showed reduced IGF-I signaling and failed to show any growth response to either estrogen or IGF-I. To prove that loss of IGF and estrogen-mediated signaling and growth was a consequence of loss of ERalpha, we re-expressed ERalpha in C4-12 cells by stable transfection with HA-tagged ERalpha. Three independent C4-12 ERalpha-HA clones expressed a functional ERalpha that (a) was down-regulated by estrogen, (b) conferred estrogen-induction of cyclin D1 expression, and (c) caused estrogen-mediated increase in the number of cells in S phase. All of the effects were completely blocked by antiestrogens. Interestingly, ERalpha-HA expression in C4-12 cells did not restore estrogen induction of progesterone receptor expression. However, ERalpha-positive C4-12 cells now exhibited estrogen-induction of IGF-IR and IRS-1 levels and responded mitogenically to both estrogen and IGF-I. These data show that ERalpha is a critical requirement for IGF signaling, and to our knowledge this is the first report of functional ERalpha expression that confers estrogen-mediated growth of an ER-negative breast cancer cell line.

Topics: Breast Neoplasms; Cell Division; Cyclin D1; Estradiol; Estrogen Receptor alpha; Hemagglutinins; Humans; Insulin Receptor Substrate Proteins; Insulin-Like Growth Factor I; Phosphoproteins; Phosphorylation; Receptor, IGF Type 1; Receptors, Estrogen; Signal Transduction; Transfection; Tumor Cells, Cultured

To assess the relation between growth factors, growth-factor receptors, p53, bcl-2 and bax expression, and the rate of apoptosis in invasive breast cancer patients.. Tumors from 45 patients were assessed by immunohistochemistry for expression patterns of five growth factors and their receptors; platelet-derived growth factor A chain (PDGF-AA) and PDGF-receptor alpha (PDGFalphaR), PDGF-BB and PDGFbetaR, transforming growth-factor alpha (TGFalpha) and its receptor-epidermal growth factor receptor (EGFR) and vascular-endothelial growth factor (VEGF) and its receptors vascular-endothelial growth factor receptor I (FLT-1) and vascular-endothelial growth factor receptor II (FLK-1/KDR), two growth-inhibiting factors; transforming-growth factor beta I (TGFbeta1) and TGFbeta2 and their receptor couple TGFbeta receptor I (TGFbetaR-I) and TGFbetaR-II, and basic-fibroblast growth factor (bFGF). Besides, the expression patterns of the bcl-2, bax and p53 gene products were investigated. Expression patterns were correlated to the number of apoptotic cells assessed by light microscopy.. PDGF-BB and bFGF showed a positive correlation with the AI (p = 0.006 and p = 0.030, respectively). EGFR expression was associated with a high number of apoptotic cells but did not reach significance (p = 0.10). None of the other individual growth factors, growth-inhibiting factors or receptors showed a significant relation with the AI. The presence of a possible auto- or paracrine loop of the TGFalpha/EGFR combination was associated with a high number of apoptotic cells but did not reach significance (p = 0.20). PDGF-AA, bFGF and EGFR expression showed a significant relation to p53 overexpression. TGFbeta2 expression showed an inverse correlation with p53 overexpression.. We found an association between several growth factors and growth-factor receptors with number of apoptotic cells. This underlines the importance of growth factors and their receptors not just in proliferation, but also, directly and/or indirectly, in regulating the rate of apoptosis in invasive breast cancer. Growth factors and their receptors may therefore be useful as targets of anti-cancer therapy by inducing apoptosis or increasing the sensitivity of cells for chemo- or hormonal therapy induced apoptosis.

Topics: Apoptosis; Breast Neoplasms; Cell Division; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Growth Substances; Humans; Immunohistochemistry; Neoplasm Invasiveness; Receptors, Growth Factor; Tumor Suppressor Protein p53

The effect of a kinase inhibitor Go6796 on growth of epidermal growth factor (EGF)-stimulated estrogen receptor negative (ER-) breast cancer cells in vivo and role of nuclear factor kappa B (NF-kappaB) on tumorogenesis have been investigated. This was studied in an animal model by implanting ER- mouse mammary epithelial tumor cells (CSMLO) in syngeneic A-J mice. (i) Local administration of Go6976 an inhibitor of protein kinases C alpha and beta inhibited growth of tumors and caused extensive necrotic degeneration and regression of the tumors without causing any microscopically detectable damage to the vital organs liver and lung. (ii) Stable expression of dominant-negative mutants of the beta subunit (dnIkkbeta) of the inhibitory kappa B (IkappaB) kinase (dnIkk) that selectively blocked activation of NF-kappaB caused loss of tumorigenic potential of CSMLO cells. Stable expression of dnIkkbeta also blocked phorbol 12-myristate 13-acetate (PMA)-induced activation of NF-kappaB and overexpression of cyclin D1, concomitantly with the loss or reduced tumorigenic potential of these cells. Thus, results from in vivo and in vitro experiments strongly suggest the involvement of NF-kappaB in ER- mammary epithelial cell-mediated tumorigenesis. We propose that blocking NF-kappaB activation not only inhibits cell proliferation, but also antagonizes the antiapoptotic role of this transcription factor in ER- breast cancer cells. Thus, NF-kappaB is a potential target for therapy of EGFR family receptor-overexpressing ER- breast cancers.

Topics: Animals; Base Sequence; Breast Neoplasms; Carbazoles; Cell Division; Cyclin D1; DNA Primers; Enzyme Inhibitors; Female; Humans; I-kappa B Kinase; Indoles; Mammary Neoplasms, Experimental; Mice; Mice, Inbred A; Mutation; NF-kappa B; Protein Kinase C; Protein Serine-Threonine Kinases; Receptors, Estrogen; Tetradecanoylphorbol Acetate; Transfection; Tumor Cells, Cultured

Overexpression of the cyclin D1 (CCND1) gene, encoding a downstream effector of mitogenic signals that plays a central role in G1 phase progression, is often found in cancerous cells. In sporadic breast cancer (BC), this is one of the most frequent and early genetic lesions identified so far, found in more than 50% of the tumors. Inhibitors of the mevalonate/protein prenylation pathway belong to a new family of cancer therapeutic agents that act by blocking intracellular mitogenic signal transduction pathways, thereby preventing expansion of pre-cancerous foci and inhibiting growth of transformed cells. It is not known at present whether constitutively high intracellular levels of cyclin D1 might interfere with the cytostatic actions of mevalonate/protein prenylation inhibitors. This possibility was investigated here by assessing the cell cycle effects of Simvastatin, a non-toxic upstream inhibitor of the mevalonate pathway, on human BC MCF-7 cells expressing either normal or enhanced levels of cyclin D1 from of a stably transfected, tet-inducible expression vector. Results show that constitutive overexpression of this protein, such as that found in sporadic BCs, does not influence the growth inhibitory effects of Simvastatin in vitro. In addition, D1-overexpressing embryo fibroblasts were also found to be responsive to the cell cycle effects of mevalonate/protein prenylation pathway blockade, further suggesting that high intracellular levels of cyclin D1 do not prevent the cytostatic actions of compounds targeting this metabolic pathway.

Topics: Animals; Breast Neoplasms; Cell Cycle; Cell Division; Cyclin D1; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Mevalonic Acid; Neoplasm Proteins; Protein Prenylation; Rats; Simvastatin

Estrogens are direct mitogens for hormone-responsive human breast cancercells, where they promote cell cycle progression and induce transcriptional activation of "immediate early" and cyclin genes. Nongenomic signaling by estrogens, including rapid changes of mitogen-activated protein(MAP) kinase and other signal-transduction-cascades activity, has been proposed to be essential for the mitogenic actions of these hormones and their nuclear receptors. Because regulation of gene transcription is considered a key step in cell cycle control by mitogenic protein kinase cascades, here we investigated the possibility that estrogen might induce the activation of extracellular signal-regulated kinase (Erk) 1/2-, c-Jun NH(2)-terminal kinase-, p38- or protein kinase A-responsive transcription factors in the cell nucleus during stimulation of early G(1) progression, a timing coincident with the maximum effects of these hormones on such enzyme activity. No significant changes in protein kinase-mediated transcription factor activity could be detected here after estrogen stimulation of either MCF-7 or ZR-75.1 cells. Furthermore, these steroids were able to induce activation of the human CCND1 gene promoter, accumulation of cyclin D1 and pRb phosphorylation, all key events in cell cycle stimulation by mitogens, even in the presence of Erk1/2 activation blockade by a MAP kinase-activating kinase (Mek)1/2 inhibitor. Thus, estrogens do not appear to convey significant protein kinase-dependent signaling to the cell nucleus during the early phases of human breast cancer cell stimulation. Furthermore, hormonal regulation of G(1) gene transcription can occur even without additional activation of the Mek-Erk1/2 pathway by estrogen receptors.

Topics: Breast Neoplasms; Cell Nucleus; Cyclic AMP-Dependent Protein Kinases; Cyclin D1; Estradiol; G1 Phase; Gene Expression Regulation, Neoplastic; Humans; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase 4; MAP Kinase Kinase Kinase 1; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Serine-Threonine Kinases; Retinoblastoma Protein; Tumor Cells, Cultured

It is well established that ErbB1 and ErbB2 can cooperate in mammary epithelial cell transformation. Therefore, to understand how ErbB1/ErbB2 signaling contributes to this process, we used the ErbB kinase inhibitor AG1478in ErbB2-dependent BT-474 and SKBR-3 human breast cancer cells. These cells overexpress ErbB2 and also display moderate levels of ErbB1. Treatment with AG1478 resulted in rapid ErbB2 dephosphorylation, reversible G(1) arrest, and interruption of constitutive mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/Akt signaling. Consequently, both MAPK-dependent transcription of cyclin D1 and phosphorylation of the cyclin-dependent kinase (Cdk) inhibitor p27 were inhibited. The inhibition of PI3K/Akt resulted in increased activity of glycogen synthase kinase-3beta, which phosphorylated cyclin D1, potentially reducing its steady-state levels. The loss of cyclin D1 reduced the amount of cyclin D1/Cdk4 complexes that can sequester p27 in the cytosol. This plus the reduced phosphorylation of p27 by MAPK enhanced the stability of p27 that associated with nuclear Cdk2 at high stoichiometry and inhibited its kinase activity. Antisense p27 oligonucleotides decreased p27 levels and abrogated the G(1) arrest induced by AG1478. Similarly, infection with an adenovirus encoding inducible cyclin D1 also counteracted the antiproliferative effect of AG1478. These data imply that: (a) modulation of both p27 and cyclin D1 are required for the growth arrest that results from blockade of the ErbB2 kinase; and (b) ErbB2 overexpressing cells use both MAPK and PI3K/Akt to modulate p27 and cyclin D1 and, hence, subvert the G(1)-to-S transition.

Topics: Breast Neoplasms; CDC2-CDC28 Kinases; Cell Cycle; Cell Cycle Proteins; Cell Division; Cyclin D1; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Enzyme Activation; Enzyme Inhibitors; G1 Phase; Humans; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Quinazolines; Receptor, ErbB-2; Signal Transduction; Transcription, Genetic; Transfection; Tumor Cells, Cultured; Tumor Suppressor Proteins; Tyrphostins; Up-Regulation

Peroxisome proliferator activated receptor gamma (PPARgamma) is a member of the nuclear receptor superfamily. Ligand activation of PPARgamma has been shown to cause growth arrest in several human tumor cell types, but the underlying molecular mechanism has not been elucidated. We report here that the PPARgamma ligand troglitazone (TRO) inhibited MCF-7 cell proliferation by blocking events critical for G1 --> S progression. Flow cytometry demonstrated that TRO at 20 microM increased the percentage of cells in G1 from 51 to 69% after 24 h. Accumulation of cells in G1 was accompanied by an attenuation of Rb protein phosphorylation associated with decreased CDK4 and CDK2 activities. Inhibition of CDK activity by TRO correlates with decreased protein levels for several G1 regulators of Rb phosphorylation (cyclin D1, and CDKs 2, 4, and 6). Overexpression of cyclin D1 partially rescued MCF-7 cells from TRO-mediated G1 arrest. Targeting of G1 regulatory proteins, particularly cyclin D1, and the resulting induction of G1 arrest by TRO may provide a novel antiproliferative therapy for human breast cancer.

Topics: Annexin A5; Antineoplastic Agents; Apoptosis; Blotting, Western; Breast Neoplasms; CDC2-CDC28 Kinases; Cell Cycle; Cell Division; Cell Separation; Cell Survival; Chromans; Cyclin D1; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; Dose-Response Relationship, Drug; Ethanol; Flow Cytometry; G1 Phase; Humans; Ligands; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-jun; Receptors, Cytoplasmic and Nuclear; Retinoblastoma Protein; Thiazoles; Thiazolidinediones; Time Factors; Transcription Factors; Transfection; Troglitazone; Tumor Cells, Cultured

Little is known of the function and clinical significance of the androgen receptor (AR) in human breast cancer. Paradoxically, synthetic progestins, such as medroxyprogesterone acetate, are used for second line hormone therapy of breast cancer following tamoxifen failure. A sensitive and accurate assay for AR expression in breast tumors is thus required. Here we have developed and validated a real-time RT-PCR assay to quantify AR gene expression at the mRNA level in a series of 131 patients with unilateral invasive primary breast tumors. AR expression varied widely in tumor tissues (by at least 3 orders of magnitude), being underexpressed in 24/131 (18.3%) and overexpressed in 45/131 (34.4%) relative to normal breast tissues. We observed links (or trends) between AR status and age, menopausal status, Scarff-Bloom-Richardson histopathological grade, lymph node status and estrogen receptor alpha and progesterone receptor status. High AR mRNA levels were negatively linked to MYC gene overexpression (P = 8 x 10(-6)), confirming previous in vitro studies. Our results also suggest a role of the ARA70 gene (which encodes a major AR co-activator) in the AR pathway dysregulation observed in breast cancer. This simple, rapid and semi-automated method will be useful for screening cancer patients for altered AR expression and for predicting the response to androgen therapy in AR-related cancer patients.

Topics: Acid Phosphatase; Adult; Aged; Aged, 80 and over; Breast Neoplasms; Cyclin D1; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; Estrogen Receptor alpha; Estrogen Receptor beta; Female; Gene Expression Regulation, Neoplastic; Genes, erbB-2; Genes, myc; Genes, Retinoblastoma; Humans; Middle Aged; Nuclear Receptor Coactivators; Oncogene Proteins; Prostate-Specific Antigen; Protein Tyrosine Phosphatases; Receptors, Androgen; Receptors, Estrogen; Receptors, Progesterone; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Trans-Activators; Transcription Factors

RRR-alpha-Tocopheryl succinate (vitamin E succinate, VES) is a potent antitumor agent, inducing DNA synthesis arrest, differentiation, and apoptosis. Because little is known about VES-induced differentiation, studies reported here characterize VES effects on the differentiation status of human breast cancer cell lines and investigate possible molecular mechanisms involved. VES-induced differentiation of human MCF-7 and MDA-MB-435 breast cancer cells was characterized by morphological changes, induction of lipid droplets, induction of beta-casein mRNA expression, and down-regulation of Her2/neu protein. In contrast, VES treatment of normal human mammary epithelial cells, MCF-10A cells, and T-47D cells did not induce differentiation. Studies addressing mechanisms showed that neither antibody neutralization of the transforming growth factor-beta signaling pathway nor expression of a dominant-negative mutant of c-Jun N-terminal kinase blocked the ability of VES to induce differentiation; however, treatment of cells with PD 98059, a chemical inhibitor of mitogen-activated protein kinase kinase (MEK1/2), blocked the ability of VES to induce differentiation.

Topics: Antibodies; beta Catenin; Biomarkers; Breast Neoplasms; Caseins; Cell Differentiation; Cyclin D1; Cytoskeletal Proteins; Humans; Intercellular Adhesion Molecule-1; JNK Mitogen-Activated Protein Kinases; Keratins; Lipid Metabolism; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Neutralization Tests; Receptor, ErbB-2; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; Signal Transduction; Tocopherols; Trans-Activators; Transcription Factors; Transforming Growth Factor beta; Tumor Cells, Cultured; Vitamin E

A member of the CCAAT Enhancer Binding Proteins (C/EBPs) family of transcription factors, C/EBPbeta, has recently proven to be an important player in both growth and differentiation of the epithelial cells in the mammary gland. When the gene for C/EBPbeta is disrupted in mice, these mice fail to either develop normal mammary ducts during puberty or pregnancy, or to lactate upon parturition. C/EBPbeta can be present in cells in three isoforms: C/EBPbeta-1, -2, and -3. These isoforms have the same carboxy terminus but different N-termini due to alternative translational initiation at three different initiator codons within the C/EBPbeta mRNA. Using a commercially available antibody specific to the C-terminus of C/EBPbeta and a novel antibody specific to the N-terminus of C/EBPbeta-1, we have uncovered a striking difference in the forms of C/EBPbeta present in normal mammary epithelial cells versus breast cancer cell lines. C/EBPbeta- 1 is found exclusively in normal mammary epithelial cells, whereas C/EBPbeta- 2 is found only in dividing cells, both normal and neoplastic. Our preliminary data suggest that the prevalent form of C/EBPbeta in cancer cells, C/EBPbeta- 2, can activate genes which push the cell to divide, such as cyclin D1.

Topics: Antibodies; Breast; Breast Neoplasms; Carcinoma; CCAAT-Enhancer-Binding Protein-beta; Cell Nucleus; Cells, Cultured; Cyclin D1; Epithelial Cells; Female; Humans; Promoter Regions, Genetic; Protein Isoforms; Tumor Cells, Cultured

Topics: Animals; Breast Neoplasms; Cyclin D1; Female; Gene Expression; Humans; Mammary Neoplasms, Animal; Mice; Neoplasm Proteins

Long-term growth inhibition, arrest in G(1) phase and reduced activity of both cyclin D1-Cdk4 and cyclin E-Cdk2 are elicited by progestin treatment of breast cancer cells in culture. Decreased cyclin expression, induction of p18(INK4c) and increased association of the CDK inhibitors p21(WAF1/Cip1) and p27(Kip1) with cyclin E-Cdk2 have been implicated in these responses. To determine the role of decreased cyclin expression, T-47D human breast cancer cells constitutively expressing cyclin D1 or cyclin E were treated with the progestin ORG 2058. Overexpression of cyclin E had only a modest effect on growth inhibition. Although cyclin E expression was maintained during progestin treatment, cyclin E-Cdk2 activity decreased by approximately 60%. This was accompanied by p27(Kip1) association with cyclin E-Cdk2, indicating that both cyclin E down-regulation and p27(Kip1) recruitment contribute to the decrease in activity. In contrast, overexpression of cyclin D1 induced progestin resistance and cell proliferation continued despite decreased cyclin E-Cdk2 activity. Progestin treatment of cyclin D1-overexpressing cells was associated with increased p27(Kip1) association with cyclin E-Cdk2. Thus the ability of cyclin D1 to confer progestin resistance does not depend on sequestration of p27(Kip1) away from cyclin E-Cdk2, providing evidence for a critical function of cyclin D1 other than as a high-capacity "sink" for p27(Kip1). These data indicate that regulation of cyclin D1 is a critical element of progestin inhibition in breast cancer cells and suggest that breast cancers overexpressing cyclin D1 may respond poorly to progestin therapy.

Topics: Animals; Blotting, Western; Breast Neoplasms; CDC2-CDC28 Kinases; Cell Cycle Proteins; Cell Division; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Dose-Response Relationship, Drug; Down-Regulation; Drug Resistance; Drug Resistance, Neoplasm; Flow Cytometry; G1 Phase; Phosphorylation; Precipitin Tests; Pregnenediones; Progesterone Congeners; Progestins; Prognosis; Protein Binding; Protein Serine-Threonine Kinases; Retroviridae; S Phase; Time Factors; Tumor Suppressor Proteins

PTP-S4/TC48 protein tyrosine phosphatase is localized in the nuclear and cytoplasmic membranes. To investigate the role of PTP-S4 in cell growth, adhesion, and transformation, normal and a catalytically inactive mutant form of this phosphatase were expressed in polyoma virus-transformed F111 fibroblast cell line, PyF. Expression of mutant PTP-S4 in PyF cells resulted in strong inhibition of anchorage-independent growth in soft agar but had no significant effect on growth in liquid culture. Tumor formation in nude mice was also reduced by mutant PTP-S4. Expression of normal PTP-S4 in PyF cells significantly increased anchorage-independent cell growth and tumor formation in nude mice. Overexpression of catalytically inactive mutant of PTP-S2/TC45 (a splice variant of PTP-S4 that is nuclear) did not inhibit anchorage-independent growth of PyF cells. Mutant PTP-S4-expressing cells were inhibited in adhesion and spreading on tissue culture plates compared to control cells. Expression of mutant PTP-S4 in PyF cells reduced the levels of cyclin D1 and cyclin A mRNA, whereas cyclin D2 mRNA level was not affected significantly. Expression of antisense cyclin D1 strongly inhibited anchorage-independent growth. Inhibition of anchorage-independent growth by mutant PTP-S4 was overcome to a large extent by coexpression of cyclin D1. These results suggest that mutant PTP-S4 inhibits anchorage-independent growth and adhesion of polyoma virus-transformed cells by interfering with the normal function of PTP-S4 upstream of cyclin D1 gene expression.

Topics: Animals; Breast Neoplasms; Cell Adhesion; Cell Culture Techniques; Cell Division; Chlorocebus aethiops; COS Cells; Culture Media; Cyclin A; Cyclin D1; Gene Expression; Humans; Mice; Mice, Nude; Mutagenesis; Neoplasms, Experimental; Protein Tyrosine Phosphatases; Tumor Cells, Cultured

The p85-associated phosphatidylinositol (PI) 3-kinase/Akt pathway mediates the oestradiol-induced S-phase entry and cyclin D1 promoter activity in MCF-7 cells. Experiments with Src, p85alpha and Akt dominant-negative forms indicate that in oestradiol-treated cells these signalling effectors target the cyclin D1 promoter. Oestradiol acutely increases PI3-kinase and Akt activities in MCF-7 cells. In NIH 3T3 cells expressing ERalpha, a dominant-negative p85 suppresses hormone stimulation of Akt. The Src inhibitor, PP1, prevents hormone stimulation of Akt and PI3-kinase activities in MCF-7 cells. In turn, stimulation of Src activity is abolished in ERalpha-expressing NIH 3T3 fibroblasts by co-transfection of the dominant-negative p85alpha and in MCF-7 cells by the PI3-kinase inhibitor, LY294002. These findings indicate a novel reciprocal cross-talk between PI3-kinase and Src. Hormone stimulation of MCF-7 cells rapidly triggers association of ERalpha with Src and p85. In vitro these proteins are assembled in a ternary complex with a stronger association than that of the binary complexes composed by the same partners. The ternary complex probably favours hormone activation of Src- and PI3-kinase-dependent pathways, which converge on cell cycle progression.

Topics: Breast Neoplasms; Cell Division; Cyclin D1; Enzyme Activation; Estradiol; Estrogen Receptor alpha; Female; Humans; Phosphatidylinositol 3-Kinases; Protein Serine-Threonine Kinases; Protein Subunits; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Receptors, Estrogen; S Phase; Signal Transduction; src-Family Kinases; Tumor Cells, Cultured

The recent cloning of a second estrogen receptor (ER), designated ERbeta, has prompted a reevaluation of the role of ERs in breast cancer. We have developed and validated a real-time RT-PCR assay to quantify ERalpha and ERbeta gene expression at the mRNA level in a series of 131 patients with unilateral invasive primary breast cancer. Although ERbeta expression showed wide variations in tumor tissues, its range (nearly three orders of magnitude) was smaller than that of ERalpha (nearly four orders of magnitude), suggesting that ERbeta is more tightly controlled than ERalpha. We observed a negative correlation between ERalpha and ERbeta expression. 'ERalpha-negative' tumors (containing very low ERalpha mRNA levels) were associated with SBR histopathological grade III, RB1 underexpression and ERBB2 overexpression, confirming that ERalpha negativity delineates poorly differentiated tumors. The amount of ERalpha mRNA (but not that of ERbeta mRNA) increased with age and was consequently higher in postmenopausal patients' tumors. Expression of ERalpha (but not that of ERbeta) also correlated strongly with progesterone receptor (PR) and PS2 expression, suggesting that ERalpha has stronger transcriptional activity than ERbeta towards genes containing an ERE (estrogen response element) in their promoters. Interestingly, we found a negative correlation between the expression of ERbeta (but not ERalpha) and CCND1, which contains an AP1 element but not an ERE in its promoter. Taken together, these data confirm that ERalpha and ERbeta play different roles in breast cancer, partly by mediating the transcription of various genes via different types of DNA enhancer. PR and PS2 seem to be mainly ERalpha-responsive genes, whereas CCND1 may be mainly ERbeta-responsive. Our findings also underline the need for a reliable method, providing full range of quantitative values, to determine ERalpha and ERbeta status in the clinical setting.

Topics: Breast Neoplasms; Cyclin D1; Estrogen Receptor alpha; Estrogen Receptor beta; Female; Humans; Middle Aged; Protein Biosynthesis; Proteins; Proto-Oncogene Proteins c-myc; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone; Retinoblastoma Protein; Reverse Transcriptase Polymerase Chain Reaction; RNA, Neoplasm; Trefoil Factor-1; Tumor Suppressor Proteins

Inheritance of a mutant allele of the breast cancer susceptibility gene BRCA1 confers increased risk of developing breast and ovarian cancers. Likewise, inheritance of a mutant allele of the retinoblastoma susceptibility gene (RB1) results in the development of retinoblastoma and/or osteosarcoma, and both alleles are often mutated or inactivated in sporadic forms of these and other cancers. We now demonstrate that the product of the RB1 gene, Rb, regulates the expression of the murine Brca1 and human BRCA1 genes through its ability to modulate E2F transcriptional activity. The Brca1 gene is identified as an in vivo target of E2F1 in a transgenic mouse model. The Brca1 promoter contains E2F DNA-binding sites that mediate transcriptional activation by E2F1 and repression by Rb. Moreover, ectopic expression of cyclin D1 and Cdk4 can stimulate the Brca1 promoter in an E2F-dependent manner, and this is inhibited by coexpression of the p16(INK4a) cyclin-dependent kinase inhibitor. The human BRCA1 promoter also contains a conserved E2F site and is similarly regulated by E2F1 and Rb. This functional link between the BRCA1 and Rb tumor suppressors may provide insight into the mechanism by which BRCA1 inactivation contributes to cancer development.

Topics: Animals; BRCA1 Protein; Breast Neoplasms; Carrier Proteins; Cell Cycle Proteins; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinases; DNA-Binding Proteins; E2F Transcription Factors; E2F1 Transcription Factor; Female; Gene Expression Regulation, Neoplastic; Keratinocytes; Mice; Mice, Transgenic; Ovarian Neoplasms; Promoter Regions, Genetic; Proto-Oncogene Proteins; Repressor Proteins; Retinoblastoma; Retinoblastoma Protein; Retinoblastoma-Binding Protein 1; RNA, Messenger; Skin; Transcription Factor DP1; Transcription Factors; Transcriptional Activation