epidermal-growth-factor and Carcinoma--Ductal--Breast

Several GI peptides linked to intestinal barrier function could be involved in the modification of intestinal permeability and the onset of diarrhea during adjuvant chemotherapy. The aim of the study was to evaluate the circulating levels of zonulin, glucagon-like peptide-2 (GLP-2), epidermal growth factor (EGF) and ghrelin and their relationship with intestinal permeability and chemotherapy induced diarrhea (CTD).. Sixty breast cancer patients undergoing an FEC60 regimen were enrolled, 37 patients completed the study. CTD(+) patients were discriminated by appropriate questionnaire and criteria. During chemotherapy, intestinal permeability was assessed by lactulose/mannitol urinary test on day 0 and day 14. Zonulin, GLP-2, EGF and ghrelin circulating levels were evaluated by ELISA tests at five time-points (days 0, 3, 10, 14, and 21).. During FEC60 administration, the lactulose/mannitol ratio was significantly higher on day 14 than at baseline. Zonulin levels were not affected by chemotherapy, whereas GLP-2 and EGF levels decreased significantly. GLP-2 levels on day 14 were significantly lower than those on day 0 and day 3, while EGF values were significantly lower on day 10 than at the baseline. In contrast, the total concentrations of ghrelin increased significantly at day 3 compared to days 0 and 21, respectively. Ten patients (27%) suffered from diarrhea. On day 14 of chemotherapy, a significant increase of the La/Ma ratio occurred in CTD(+) patients compared to CTD(-) patients. With regards to circulating gut peptides, the AUCg of GLP-2 and ghrelin were significantly lower and higher in CTD(+) patients than CTD(-) ones, respectively. Finally in CTD(+) patients a significant and inverse correlation between GLP-2 and La/Ma ratio was found on day 14.. Breast cancer patients undergoing FEC60 showed alterations in the intestinal permeability, which was associated with modifications in the levels of GLP-2, ghrelin and EGF. In CTD(+) patients, a different GI peptide profile and increased intestinal permeability was found in comparison to CTD(-) patients. This evidence deserves further studies for investigating the potentially different intestinal luminal and microbiota conditions.. Clinical trial NCT01382667.

Topics: Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Carcinoma, Ductal, Breast; Chemotherapy, Adjuvant; Cholera Toxin; Cyclophosphamide; Diarrhea; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Epirubicin; Female; Fluorouracil; Ghrelin; Glucagon-Like Peptide 2; Haptoglobins; Humans; Intestinal Absorption; Intestinal Mucosa; Italy; Lactulose; Mannitol; Middle Aged; Peptides; Permeability; Prospective Studies; Protein Precursors; Stomatitis; Time Factors; Treatment Outcome

Recent evidence has suggested a possible role for progesterone receptor (PR) in the progression of non-small cell lung cancer (NSCLC). However, little is known concerning roles of PR in NSCLC. PR contains a polyproline domain (PPD), which directly binds to the SH3 domain of signaling molecules. Because PPD-SH3 interactions are essential for EGFR signaling, we hypothesized that the presence of PR-PPD interfered with EGFR-mediated signaling and cell proliferation. We examined the role of PR-PPD in cell proliferation and signaling by stably expressing PR-B, or PR-B with disrupting mutations in the PPD (PR-BΔSH3), from a tetracycline-regulated promoter in A549 NSCLC cells. PR-B dose-dependently inhibited cell growth in the absence of ligand, and progestin (R5020) treatment further suppressed the growth. Treatment with RU486 abolished PR-B- and R5020-mediated inhibition of cell proliferation. Expression of PR-BΔSH3 and treatment with R5020 or RU486 had no effect on cell proliferation. Furthermore, PR-B expression but not PR-BΔSH3 expression reduced EGF-induced A549 proliferation and activation of ERK1/2, in the absence of ligand. Taken together, our data demonstrated the significance of PR extranuclear signaling through PPD interactions in EGFR-mediated proliferation and signaling in NSCLC.

Topics: Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Non-Small-Cell Lung; Cell Growth Processes; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Female; HEK293 Cells; Humans; Lung Neoplasms; Proline-Rich Protein Domains; Receptors, Progesterone; Signal Transduction

Myoferlin is a member of the ferlin family of proteins that participate in plasma membrane fusion, repair, and endocytosis. While some reports have implicated myoferlin in cancer, the extent of its expression in and contributions to cancer are not well established. In this study, we show that myoferlin is overexpressed in human breast cancers and that it has a critical role in controlling degradation of the epidermal growth factor (EGF) receptor (EGFR) after its activation and internalization in breast cancer cells. Myoferlin depletion blocked EGF-induced cell migration and epithelial-to-mesenchymal transition. Both effects were induced as a result of impaired degradation of phosphorylated EGFR via dysfunctional plasma membrane caveolae and alteration of caveolin homo-oligomerization. In parallel, myoferlin depletion reduced tumor development in a chicken chorioallantoic membrane xenograft model of human breast cancer. Considering the therapeutic significance of EGFR targeting, our findings identify myoferlin as a novel candidate function to target for future drug development.

Topics: Adenocarcinoma; Animals; Apoptosis; Breast Neoplasms; Calcium-Binding Proteins; Carcinoma, Ductal, Breast; Carcinoma, Lobular; Caveolae; Cell Movement; Cell Proliferation; Chick Embryo; Chorioallantoic Membrane; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; ErbB Receptors; Female; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; Glycolysis; Humans; Immunoenzyme Techniques; Membrane Proteins; Muscle Proteins; Neoplasm Invasiveness; Proteomics; RNA, Small Interfering; Tumor Cells, Cultured

Ductal carcinoma in situ (DCIS) is an early stage noninvasive breast cancer that originates in the epithelial lining of the milk ducts, but it can evolve into comedo DCIS and ultimately, into the most common type of breast cancer, invasive ductal carcinoma. Understanding the progression and how to effectively intervene in it presents a major scientific challenge. The extracellular matrix (ECM) surrounding a duct contains several types of cells and several types of growth factors that are known to individually affect tumor growth, but at present the complex biochemical and mechanical interactions of these stromal cells and growth factors with tumor cells is poorly understood. Here we develop a mathematical model that incorporates the cross-talk between stromal and tumor cells, which can predict how perturbations of the local biochemical and mechanical state influence tumor evolution. We focus on the EGF and TGF-β signaling pathways and show how up- or down-regulation of components in these pathways affects cell growth and proliferation. We then study a hybrid model for the interaction of cells with the tumor microenvironment (TME), in which epithelial cells (ECs) are modeled individually while the ECM is treated as a continuum, and show how these interactions affect the early development of tumors. Finally, we incorporate breakdown of the epithelium into the model and predict the early stages of tumor invasion into the stroma. Our results shed light on the interactions between growth factors, mechanical properties of the ECM, and feedback signaling loops between stromal and tumor cells, and suggest how epigenetic changes in transformed cells affect tumor progression.

Topics: Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Intraductal, Noninfiltrating; Epidermal Growth Factor; Female; Humans; Mathematical Concepts; Models, Biological; Neoplasm Invasiveness; Signal Transduction; Stromal Cells; Transforming Growth Factor beta; Tumor Microenvironment

Mesenchymal stem cells (MSCs) play a critical role in promoting cancer progression. However, it is not clear whether MSCs are located in breast cancer tissues and correlated with tumor proliferation. The aim of this study was to investigate the presence of MSCs in breast cancer tissues and evaluate their interactions with cancer cells. We successfully isolated and identified MSCs from primary breast cancer tissues. Breast cancer-associated MSCs (BC-MSCs) showed homogenous immunophenotype, and possessed tri-lineage differentiation potential (osteoblast, adipocyte, and chondrocyte). When co-transplanted with cancer cells in a xenograft model in vivo, BC-MSCs significantly increased the volume and weight of tumors. We observed that BC-MSCs stimulated mammosphere formation in the transwell co-culture system in vitro. This effect was significantly suppressed by the EGF receptor inhibitor. We verified that BC-MSCs could secrete EGF and activate cancer cell's EGF receptors. Furthermore, our data showed that EGF derived from BC-MSCs could promote mammosphere formation via the PI3K/Akt signaling pathway. Our results confirmed the presence of MSC in primary breast cancer tissues, and they could provide a favorable microenvironment for tumor cell growth in vivo, partially enhance mammosphere formation via the EGF/EGFR/Akt pathway.

Topics: Animals; Antigens, Differentiation; Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Differentiation; Cell Proliferation; Cell Shape; Coculture Techniques; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Mesenchymal Stem Cells; Mice; Mice, Nude; Neoplasm Transplantation; Neoplastic Stem Cells; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Spheroids, Cellular; Tumor Burden; Tumor Cells, Cultured

Patients with invasive breast ductal carcinoma (IBDC) with metastasis have a very poor prognosis. Little is known about the synergistic action of growth and inflammatory factors in IBDC metastases.. The expression of activated extracellular signal-regulated kinase1/2 (phosphorylated or p-ERK1/2) was analyzed by immunohistochemistry in IBDC tissue samples from 80 cases. BT474 IBDC cell migration and invasion were quantified using the Transwell assay. Matrix metalloproteinase (MMP)-9 expression and activity were analyzed by RT-PCR, Western blotting and zymography. Activator protein (AP)-1 activity was measured with a luciferase reporter gene assay. The Wilcoxon signed-rank test, Chi-square test, the partition of Chi-square test, independent t-test, and Spearman's method were used for the statistical analysis.. Phosphorylated ERK1/2 was detected in 58/80 (72.5%) IBDC tissues, and was associated with higher TNM stage and lymph node metastasis, but not patient age or tumor size. Individually, epidermal growth factor (EGF), and interleukin (IL)-1β activated ERK1/2, increased cell migration and invasion, MMP-9 expression and activity, AP-1 activation in vitro and the expression of p-ERK1/2 was positively correlated with EGF expression levels, as well as IL-1β, MMP-9 and c-fos in IBDC tissue samples. Co-stimulation with EGF and IL-1β synergistically increased ERK1/2 and AP-1 activation, cell migration and invasion, and MMP-9 expression and activity. Inhibition of ERK1/2 using U0126 or siRNA abolished EGF and/or IL-1β-induced cell migration and invasion in a dose-dependent manner.. Activated ERK1/2 was associated with higher TNM stage and lymph node metastasis in IBDC. Both in vitro and in vivo studies indicated that ERK-1/2 activation may increase the metastatic ability of IBDC cells. Growth and inflammatory factors synergistically induced IBDC cell migration and invasion via ERK1/2 signaling, AP-1 activation and MMP-9 upregulation.

Topics: Carcinoma, Ductal, Breast; Cell Line, Tumor; Cell Movement; Drug Synergism; Enzyme Activation; Epidermal Growth Factor; Female; Humans; Interleukin-1beta; Matrix Metalloproteinase 9; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phosphorylation; RNA Interference; Transcription Factor AP-1

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

Basal-like carcinomas and human epidermal growth factor receptor 2 (HER-2/neu) overexpression carcinomas are the subgroups of breast cancers that have the most aggressive clinical behavior. Phosphorylation/activation of c-Jun NH2-terminal kinase is characterized as a stress-activated protein kinase, which regulates apoptosis after cellular stress. The aim of this study was to evaluate the association of phosphorylated c-Jun NH2-terminal kinase expression with phenotypes and clinicopathologic parameters of breast cancer. Phosphorylated c-Jun NH2-terminal kinase was immunohistochemically measured in a cohort of 160 patients with invasive breast cancer treated with therapeutic surgery followed by anthracycline or docetaxel-based chemotherapy. These results were further correlated with the phenotypes and clinicopathologic characteristics of breast cancers. Increased phosphorylated c-Jun NH2-terminal kinase expression was significantly associated with lack of estrogen receptor expression (P < .0001), positivity for cytokeratins 5/6 (P = .029), epidermal growth factor receptor (P = .035), basal-like phenotype (P = .015), and "triple-negative" phenotype (P = .01). Furthermore, the positive expression of phosphorylated c-Jun NH2-terminal kinase was positively correlated with p-glycoprotein (r = 0.54, P < .0001) and multidrug resistance-associated protein 1(r = 0.38, P < .0001) but not with lung resistance protein (r = -0.02, P = .78). Our results indicate that the activation of phosphorylated c-Jun NH2-terminal kinase may play a role in the carcinogenesis of basal-like and triple-negative breast carcinoma.

Topics: Adenocarcinoma, Mucinous; ATP Binding Cassette Transporter, Subfamily B, Member 1; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Lobular; Carcinoma, Medullary; Chi-Square Distribution; Epidermal Growth Factor; Estrogen Receptor alpha; Female; Humans; Immunohistochemistry; JNK Mitogen-Activated Protein Kinases; Keratins; Multidrug Resistance-Associated Proteins; Neoplasm Staging; Patient Selection; Phosphorylation; Up-Regulation; Vault Ribonucleoprotein Particles

This study was undertaken to investigate epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER-2)/neu expression in a cohort of apocrine carcinomas of the breast with emphasis on the classification of the breast tumors with apocrine morphology. In total, 55 breast carcinomas morphologically diagnosed as apocrine were evaluated for the steroid receptor expression profile characteristic of normal apocrine epithelium (androgen receptor positive/estrogen receptor (ER) negative/progesterone receptor (PR) negative), and for the expression of EGFR and Her-2/neu proteins, and the copy number ratios of the genes EGFR/CEP7 and HER-2/CEP17. On the basis of the results of steroid receptors expression, 38 (69%) cases were classified as pure apocrine carcinoma (androgen receptor positive/ER negative/PR negative), whereas 17 (31%) were re-classified as apocrine-like carcinomas because they did not have the characteristic steroid receptor expression profile. Her-2/neu overexpression was observed in 54% of the cases (57% pure apocrine carcinomas vs 47% apocrine-like carcinomas). HER-2/neu gene amplification was demonstrated in 52% of all cases (54% pure apocrine carcinomas vs 46% apocrine-like carcinomas). EGFR protein (scores 1 to 3+) was detected in 62% of all cases and was expressed in a higher proportion of pure apocrine carcinomas than in the apocrine-like carcinomas group (76 vs 29%, P=0.006). In the pure apocrine carcinoma group, Her-2/neu and EGFR protein expression were inversely correlated (P=0.006, r=-0.499). EGFR gene amplification was observed in two pure apocrine carcinomas and one apocrine-like carcinoma. Polysomy 7 was commonly present in pure apocrine carcinomas (61 vs 27% of apocrine-like carcinomas; P=0.083) and showed a weak positive correlation with EGFR protein expression (P=0.025, r=0.326). Our study showed that apocrine breast carcinomas are molecularly diverse group of carcinomas. Strictly defined pure apocrine carcinomas are either HER-2-overexpressing breast carcinomas or triple-negative breast carcinomas, whereas apocrine-like carcinomas predominantly belong to the luminal phenotype. Pure apocrine carcinomas show consistent overexpression of either EGFR or HER-2/neu, which could have significant therapeutic implications.

Topics: Breast Neoplasms; Carcinoma, Ductal, Breast; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; In Situ Hybridization, Fluorescence; Polymorphism, Single Nucleotide; Receptor, ErbB-2; Receptors, Steroid; RNA, Messenger; Statistics, Nonparametric

We explored the crosstalk between cell survival (phosphatidylinositol 3-kinase (PI3K)/Akt) and mitogenic (Ras/Raf/MEK/extracellular signal-regulated kinase (ERK)) signaling pathways activated by an epidermal growth factor (EGF) and analyzed their sensitivity to small molecule inhibitors in the PI3K-mutant estrogen receptor (ER)-positive MCF7 and T47D breast cancer cells. In contrast to MCF7 cells, ERK phosphorylation in T47D cells displayed resistance to MEK inhibition by several structurally different compounds, such as U0126, PD 098059 and PD 198306, MEK suppression by small interfering RNA (siRNA) and was also less sensitive to PI3K inhibition by wortmannin. Similar effect was observed in PI3K-wild type ER-positive BT-474 cells, albeit to a much lesser extent. MEK-independent ERK activation was induced only by ErbB receptor ligands and was resistant to inhibition of several kinases and phosphatases that are known to participate in the regulation of Ras/mitogen-activated protein kinase (MAPK) cascade. Although single agents against PDK1 or Akt did not affect EGF-induced ERK phosphorylation, a combination of PI3K/Akt and MEK inhibitors synergistically suppressed ERK activation and cellular growth. siRNA-mediated silencing of class I PI3K or Akt1/2 genes also significantly decreased U0126-resistant ERK phosphorylation. Our data suggest that in T47D cells ErbB family ligands induce a dynamic, PI3K/Akt-sensitive and MEK-independent compensatory ERK activation circuit that is absent in MCF7 cells. We discuss candidate proteins that can be involved in this activation circuitry and suggest that PDZ-Binding Kinase/T-LAK Cell-Originated Protein Kinase (PBK/TOPK) may play a role in mediating MEK-independent ERK activation.

Topics: Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Line, Tumor; Epidermal Growth Factor; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Intercellular Signaling Peptides and Proteins; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase Kinases; Mutation; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Receptors, Estrogen

Epidermal growth factor (EGF) mediates breast cancer cell chemotaxis and metastasis through mechanisms that involve the growth-regulatory mammalian target of rapamycin (mTOR) complex mTORC2, but the mechanisms involved remain obscure. Here, we report that the rapamycin-insensitive mTORC2 component protein Rictor is a critical mediator of metastasis in breast cancer cells. In patients with ductal carcinoma, Rictor expression was associated with increased lymph node metastasis. EGF induced translocation and colocalization of Rictor with protein kinase Cζ (PKCζ), a pivotal molecule in chemotaxis signaling. Further, Rictor coimmunoprecipitated with PKCζ in the absence of the mTORC2 complex. Small interfering RNA-mediated knockdown of Rictor inhibited EGF-induced PKCζ phosphorylation and translocation along with phosphorylation of the key F-actin binding protein cofilin. In parallel, Rictor knockdown reduced cellular chemotactic capacity and ablated pulmonary metastasis in a xenograft mouse model of breast cancer. Our findings identify Rictor as an important mediator of chemotaxis and metastasis in breast cancer cells.

Topics: Animals; Breast Neoplasms; Carcinoma, Ductal, Breast; Carrier Proteins; Cell Line, Tumor; Cell Movement; Chemotaxis; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Immunohistochemistry; Lung Neoplasms; Lymphatic Metastasis; Mammary Neoplasms, Experimental; Mice; Mice, SCID; Microscopy, Confocal; Phosphorylation; Protein Binding; Protein Kinase C; Rapamycin-Insensitive Companion of mTOR Protein; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Transplantation, Heterologous

Epithelial-mesenchymal transition (EMT) is a process occurring during both embryogenesis and early stages of invasive cancer. Epithelial cells that undergo EMT become more migratory and invasive with a mesenchymal morphology. Herein we assess EMT induction in a mouse mammary epithelial cell line driven by Msx2, a homeobox-containing transcription factor important during mammary gland development. NMuMG cells, a normal mouse mammary epithelial cell line, stably transfected with a Msx2 cDNA showed downregulation of an epithelial marker E-cadherin and upregulation of the mesenchymal markers vimentin and N-cadherin. Furthermore, overexpression of Cripto-1, a member of the epidermal growth factor-CFC protein family already known to be involved in EMT, was detected in Msx2-transfected cells. The expression of Cripto-1 was accompanied by activation of the tyrosine kinase c-Src pathway and an increase in the invasive ability of the cells. Functional assays also demonstrated inhibition of the invasive behavior of the Msx2-transfected cells by a c-Src specific inhibitor. Moreover, immunohistochemistry of human infiltrating breast carcinomas showed positive staining for Msx2 only in the infiltrating tumor cells while the non-infiltrating tumor cells were negative. These results suggest that Msx2 may play a significant role in promoting EMT in epithelial cells that acquire properties involved in tumor invasion. J. Cell. Physiol. 219: 659-666, 2009. Published 2009 Wiley-Liss, Inc.

Topics: Animals; Base Sequence; Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Line; CSK Tyrosine-Protein Kinase; DNA Primers; Epidermal Growth Factor; Epithelial Cells; Female; Homeodomain Proteins; Humans; Mammary Glands, Animal; Membrane Glycoproteins; Mesoderm; Mice; Neoplasm Invasiveness; Neoplasm Proteins; Protein-Tyrosine Kinases; Recombinant Proteins; RNA, Small Interfering; Signal Transduction; src-Family Kinases; Transfection; Up-Regulation

Growth factors are important mediators of proliferation. Deregulation in growth factor mechanisms as well as in its receptors will contribute to cancer development. One of the most important is the epidermal growth factor (EGF), which is encoded by EGF gene. A functional polymorphism at position 61 (A/G) is associated with increased expression of EGF. Thus, we proposed to assess genotype frequencies in a case-control study and appraise their association to breast cancer risk. Using the polymerase chain reaction technique combined with restriction enzyme fragment length polymorphism (PCR-RFLP) we analyzed DNA from 883 women (500 controls and 383 breast cancer patients). Our results suggested that carriers of G homozygous genotype had a lower risk for developing breast cancer (odds ratio = 0.68; 95% confidence intervals, 0.46-1.01). Further, we showed that the waiting time for onset of breast cancer in G homozygous patients for EGF genotypes (55 years) was significantly lower in comparison to A-allele carriers (59 years) (log-rank test: p = 0.038). EGF is produced in mammary tissue and acts in the mammalian development. A lower risk for breast cancer in GG carriers might be explained through EGF receptor internalization promoted by EGF.

Topics: Adenocarcinoma, Mucinous; Adult; Age of Onset; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Lobular; Carcinoma, Papillary; Case-Control Studies; Epidermal Growth Factor; ErbB Receptors; Female; Genotype; Humans; Middle Aged; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Polymorphism, Single Nucleotide; Prognosis

The aim of this study is to provide an expression profile of ErbB/HER ligands in breast cancer. We analysed the relationships with their receptors, the bio-pathological features and prognosis.. Epidermal growth factor (EGF), transforming growth factor-alpha (TGFalpha), amphiregulin (AREG), betacellulin (BTC), heparin-binding EGF-like growth factor (HB-EGF), epiregulin (EREG) and neuregulins1-4 (NRG1-4) were quantified in 363 tumours by real-time reverse transcription-polymerase chain reaction using TaqMan probes.. Ligands were detected in 80%-96% of the cases, except NRG3 (42%) and EREG (45.5%). At least one ligand was expressed in 304 cases (cut-off: upper quartile). Almost all combinations of receptor and ligand co-expressions were observed, but TGFalpha is preferentially expressed in tumours co-expressing EGFR/HER3, NRG3 in those co-expressing EGFR/HER4, AREG and EREG in those co-expressing HER2/HER4. EGF and AREG were associated with estradiol receptors, small tumour size, low histoprognostic grading, high HER4 levels. TGFalpha, HB-EGF and NRG2 were negatively related to these parameters. In Cox univariate analyses, EGF was a prognostic factor.. Our study demonstrates that (i) ErbB/HER ligands, including BTC and EREG, are expressed in most breast cancers; and (ii) TGFalpha, HB-EGF and NRG2 high expressions are related to the biological aggressiveness of the tumours.

Topics: Amphiregulin; Betacellulin; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Lobular; Disease-Free Survival; EGF Family of Proteins; Epidermal Growth Factor; Epiregulin; ErbB Receptors; Female; Gene Expression Profiling; Glycoproteins; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Intracellular Signaling Peptides and Proteins; Neoplasm Invasiveness; Neoplasm Proteins; Nerve Growth Factors; Nerve Tissue Proteins; Neuregulin-1; Neuregulins; Prognosis; Receptor, ErbB-2; Receptors, Estrogen; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor alpha

Heparan sulfate (HS) glycosaminoglycans are the oligosaccharide chains of heparan sulfate proteoglycans. The sulfation of HS glycosaminoglycan residues is required for its interaction with various heparin-binding growth factors to promote their biological activities to activate their high affinity receptor tyrosine kinases. We have identified HS glycosaminoglycan-6-O-endosulfatase HSulf-1 as a down-regulated gene in ovarian, breast, and several other cancer cell lines. Here we have shown that HSulf-1 inhibits autocrine activation of the EGFR-ERK (epidermal growth factor receptor-extracellular signal-regulated kinase) pathway induced by serum withdrawal in MDA-MB-468 breast cancer cells. Short hairpin RNA-mediated down-regulation of HSulf-1 in HSulf-1 clonal lines of MDA-MB-468 led to a significant increase in autocrine activation of ERK compared with vector only control. The autocrine signaling was also inhibited with neutralization antibodies against amphiregulin and HB-EGF, the heparin-binding growth factor family of the EGF superfamily. Furthermore, HSulf-1-mediated inhibition of autocrine signaling was associated with reduced cyclin D1 levels, leading to decreased S phase fraction and increased G(2)-M fraction, as well as increased cell death. Finally, evaluation of HSulf-1 expression levels in primary invasive breast tumors by RNA in situ hybridization indicated that HSulf-1 is down-regulated in the majority (60%) of tumors, with a predominant association with lobular histology. These data suggest a potential role of HSulf-1 down-regulation in mammary carcinogenesis.

Topics: Amphiregulin; Autocrine Communication; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Lobular; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D; Cyclins; Down-Regulation; EGF Family of Proteins; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Glycoproteins; Heparin-binding EGF-like Growth Factor; Humans; In Situ Hybridization; Intercellular Signaling Peptides and Proteins; Middle Aged; Neoplasm Invasiveness; Neoplasm Recurrence, Local; Retroviridae; RNA, Small Interfering; S Phase; Signal Transduction; Sulfotransferases; Tissue Array Analysis

The development of cancer prevention strategies depends on the elucidation of molecular pathways underlying oncogenesis. In a previous proteomic study of matched normal breast ducts and Ductal Carcinoma in Situ (DCIS), we identified overexpression of Rab11a in DCIS. Rab11a is not well studied in cancer, but is known to regulate the recycling of internalized cell surface proteins and receptors from the early endosome through the trans-Golgi network. Using immunohistochemistry, we confirmed our observation, noting increased Rab11a expression in 19 of 22 (86%) DCIS cases compared to matched normal breast epithelium. To study the function of Rab11a, immortal, nontumorigenic MCF10A breast cells were stimulated with ligands to the EGF receptor (EGFR) after transfection with empty vector (control), Rab11a, or a S25N dominant-negative (DN) Rab11a. Using an iodinated ligand:receptor recycling assay, transfection of Rab11a accelerated, while DN-Rab11a postponed EGFR recycling in vitro. The signaling and in vitro phenotypic consequences of Rab11a expression and function were studied. Transfection of DN-Rab11a increased Erk1/2 activation downstream of EGF, but exerted no effect on the Akt pathway. Expression of DN-Rab11a inhibited MCF10A proliferation by 50-60%, and also inhibited anchorage-dependent colonization. Notably, DN-Rab11a transfection increased motility toward EGFR ligands. The data provide a first demonstration that Rab11a modulates EGFR recycling, and promotes the proliferation but inhibits the motility of an immortal breast line, consistent with the DCIS phenotype.

Topics: Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epidermal Growth Factor; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Phosphorylation; rab GTP-Binding Proteins; Transforming Growth Factor alpha

Associations between p160 coactivator proteins and the development of resistance to endocrine treatment have been described. We hypothesized that nuclear receptor coregulatory proteins may interact with nonsteroid receptors. We investigated the mitogen-activated protein kinase-activated transcription factors, Ets, as possible interaction proteins for the coactivators SRC-1 and AIB1 and the corepressor NCoR in human breast cancer.. Expression and coexpression of Ets and the coregulatory proteins was investigated using immunohistochemistry and immunofluorescence in a cohort of breast tumor patients (N = 134). Protein expression, protein-DNA interactions and protein-protein interactions were assessed using Western blot, electromobility shift, and coimmunoprecipitation analysis, respectively.. Ets-1 and Ets-2 associated with reduced disease-free survival (P < 0.0292, P < 0.0001, respectively), whereas NCoR was a positive prognostic indicator (P < 0.0297). Up-regulation of Ets-1 protein expression in cell cultures derived from patient tumors in the presence of growth factors associated with tumor grade (P < 0.0013; n = 28). In primary breast tumor cell cultures and in the SKBR3 breast cell line, growth factors induced interaction between Ets and their DNA response element, induced recruitment of coactivators to the transcription factor-DNA complex, and up-regulated protein expression of HER2. Ets-1 and Ets-2 interacted with the coregulators under basal conditions, and growth factors up-regulated Ets-2 interaction with SRC-1 and AIB1. Coexpression of Ets-2 and SRC-1 significantly associated with the rate of recurrence and HER expression, compared with patients who expressed Ets-2 but not SRC-1 (P < 0.0001 and P < 0.0001, respectively).. These data describe associations and interactions between nonsteroid transcription factors and coregulatory proteins in human breast cancer.

Topics: Blotting, Western; Breast Neoplasms; Carcinoma, Ductal, Breast; Electrophoretic Mobility Shift Assay; Epidermal Growth Factor; Female; Fibroblast Growth Factor 2; Gene Expression Regulation, Neoplastic; Histone Acetyltransferases; Humans; Immunoenzyme Techniques; Immunoprecipitation; Neoplasm Invasiveness; Neoplasm Recurrence, Local; Nuclear Proteins; Nuclear Receptor Co-Repressor 1; Nuclear Receptor Coactivator 1; Nuclear Receptor Coactivator 3; Prognosis; Proto-Oncogene Protein c-ets-1; Proto-Oncogene Protein c-ets-2; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-ets; Receptor, ErbB-2; Receptors, Estrogen; Repressor Proteins; Response Elements; Survival Rate; Trans-Activators; Transcription Factors; Transcription, Genetic; Tumor Cells, Cultured

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents, Hormonal; Aromatase Inhibitors; Breast Neoplasms; Carcinoma, Ductal, Breast; Chemotherapy, Adjuvant; Clinical Trials as Topic; Drug Resistance, Neoplasm; Enzyme Inhibitors; Epidermal Growth Factor; Female; Gefitinib; Humans; Neoplasms, Hormone-Dependent; Phosphorylation; Quinazolines; Receptor Cross-Talk; Receptor, ErbB-2; Receptors, Estrogen; Retrospective Studies; Selective Estrogen Receptor Modulators; Tamoxifen; Trastuzumab

Previous studies showed that antagonists of bombesin (BN)/gastrin-releasing peptide (GRP) inhibit the growth of various cancers by interfering with the growth-stimulatory effects of BN-like peptides and down-regulating epidermal growth factor receptors on tumors. Because the overexpression of the human epidermal growth factor receptor-2 (ErbB-2/HER-2/neu) oncogene plays a role in the progression of many breast cancers, we investigated whether BN/GRP antagonists can affect HER-2 in mammary tumors. Female nude mice bearing orthotopic xenografts of MDA-MB-435 human estrogen-independent breast cancers were treated daily with BN/GRP antagonists RC-3095 (20 microg) or RC-3940-II (10 microg) for 6 weeks. The expression of BN/GRP receptors on tumors was analyzed by reverse transcription-PCR and immunoblotting. We also evaluated whether the mRNA expression for the c-jun and c-fos oncogenes is affected by the therapy. Both BN/GRP antagonists significantly inhibited growth of MDA-MB-435 cancers; RC-3095 reduced tumor volume by 40% and RC-3940-II by 65%. The GRP receptors (subtype 1) were detected in MDA-MB-435 tumors, showing that they mediate the inhibitory effect of the antagonists. Tumor inhibition was associated with a substantial reduction in the expression of mRNA and protein levels of the ErbB/HER receptor family as well as with a decrease in the expression of c-jun and c-fos oncogenes. BN/GRP antagonists RC-3940-II and RC-3095 could be considered for endocrine therapy of estrogen-independent breast cancers that express members of the ErbB/HER receptor family and the c-jun and c-fos oncogenes.

Topics: Adult; Animals; Bombesin; Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Division; Epidermal Growth Factor; Estrogens; Female; Gastrins; Gene Expression Regulation, Neoplastic; Genes, fos; Genes, jun; Humans; Mice; Mice, Nude; Neoplasm Transplantation; Peptide Fragments; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Receptor, ErbB-2; RNA, Messenger; Tumor Cells, Cultured

The effect of oncostatin M (OM) on epidermal growth factor (EGF)-mediated protein tyrosine phosphorylation in an infiltrating ductal breast carcinoma cell line, H3922, was investigated by Western blot analysis. Pretreatment of H3922 cells with OM for 72 h suppressed EGF-stimulated protein tyrosine phosphorylation signals by 77%. Interestingly, pretreatment with OM for 6 or 48 h had little effect on these signals. EGF-mediated tyrosine phosphorylation of EGF receptor (EGFR) was suppressed by 55% in 72-h OM pretreated H3922 cells. No reduction in EGFR protein expression was detected in these cells. Flow cytometric analysis verified that OM does not suppress EGFR expression. The effect of OM could not be attributed to induction of protein tyrosine phosphatases. An H3922 subclone cell line, designated H3922-8, was found to exhibit no proliferative response to treatment with EGF. However, EGF-mediated protein tyrosine phosphorylation was detected in these cells. Radioligand EGF binding studies comparing H3922 to H3922-8 cells indicated that the clonal cells apparently lack high affinity EGF receptors. The mechanism by which OM suppresses EGF-mediated tyrosine phosphorylation has not been completely characterized. However, the suppressive effect occurs regardless of whether the cells are acutely responsive (H3922) or virtually unresponsive (H3922-8) to EGF stimulation of cell growth.

Topics: Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Division; Culture Media; Cytokines; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Inflammation Mediators; Oncostatin M; Peptides; Phosphorylation; Phosphotyrosine; Protein Tyrosine Phosphatases; Time Factors; Tumor Cells, Cultured; Tyrosine

The importance of epidermal growth factor (EGF) in both normal and malignant mammary gland development are presented in these studies. Initial findings demonstrated that in the absence of ovarian hormones, EGF had a significant proliferative effect on mammary epithelial cells. To determine whether mammary epithelial cells grown with EGF, in the absence of ovarian hormones, could be transformed by N-methyl-N-nitrosourea (MNU), female ovariectomized Lewis rats were implanted with pellets containing EGF for 1 week and then treated with MNU for initiation. Two days after MNU treatment, ovaries were implanted and EGF pellets were removed from all ovariectomized groups in order to promote carcinogenesis. The mammary carcinoma incidence of the EGF-stimulated group (90%) was not significantly different from the intact group (100%). The mammary cancer morphology of EGF-treated carcinomas was either ductal carcinoma or cribriform adenocarcinoma, whereas intact animals developed mainly papillary and occasional cribriform carcinomas. Fifty-eight percent of the carcinomas from the EGF group were ovarian hormone-independent compared with 10% of carcinomas from the intact group. These results demonstrate that EGF-induced proliferation during initiation with MNU was sufficient to induce the transformation of mammary carcinomas in the absence of ovarian hormones. The hormonal dependency of these EGF-induced carcinomas were different compared with MNU-initiated mammary carcinomas in intact rats.

Topics: Adenocarcinoma; Animals; Carcinoma, Ductal, Breast; Carcinoma, Papillary; Cell Division; DNA Mutational Analysis; Epidermal Growth Factor; Estradiol; Estrogens; Female; Genes, ras; Mammary Neoplasms, Experimental; Methylnitrosourea; Neoplasm Proteins; Neoplasms, Hormone-Dependent; Ovariectomy; Ovary; Polymerase Chain Reaction; Progesterone; Rats; Rats, Inbred Lew; Rats, Sprague-Dawley; Receptors, Estrogen; Receptors, Progesterone

Epidermal growth factor receptors (EGFR) have been investigated in the tumors of 166 breast cancer patients and concentrations of their ligands (alpha-TGF and EGF) were measured in 104 tumors by radioimmunoassay. EGFR and both growth factors were simultaneously detected in 18% and EGFR and one of the ligands were found in 11%. There were trends to higher frequency of EGFR expression in advanced tumors, in ductal and lobular infiltrative cancers with high grade of malignancy and in estrogen receptor negative breast cancers. A possible role of the parameters studied in the prediction of breast cancer and in the choice of therapy is discussed.

Topics: Adult; Aged; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Lobular; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Ligands; Middle Aged; Radioimmunoassay; Transforming Growth Factors