hes1-protein--human and Breast-Neoplasms

Chemoresistance remains the primary reason threatening the prognosis of breast cancer (BC) patients. Extracellular vesicles (EVs) contribute to chemoresistance by carrying microRNAs (miRNAs). This study investigated the mechanism of miR-887-3p mediated by EVs in BC cell drug resistance.. MDA-MB-231-derived EVs were extracted and identified. BC cells were treated with different concentrations of doxorubicin, cisplatin, and fulvestrant, and the cell survival was evaluated. PKH26-labeled EVs were cocultured with BC cells, and the uptake of EVs was observed. miR-887-3p expression in BC cells and EVs was detected. After silencing miR-887-3p in MDA-MB-231 cells, BC cells were treated with EV-inhi to observe drug resistance. The target gene of miR-887-3p was predicted and verified. The levels of downstream Notch1/Hes1 pathway were detected. Xenograft experiment was conducted to evaluate the effect of EVs on the growth and drug resistance. MDA-MB-231-derived EVs enhanced the drug resistance of BC cells. EVs carried miR-887-3p into BC cells. miR-887-3p expression was elevated in BC cells and EVs. miR-887-3p inhibition reduced the drug resistance of BC cells. miR-887-3p targeted BTBD7. Overexpression of BTBD7 partially reversed the drug resistance of BC cells caused by EV treatment. EV treatment increased the level of Notch1/Hes1, and overexpression of BTBD7 decreased the level of Notch1/Hes1.. EVs carrying miR-887-3p could target BTBD7 and activate the Notch1/Hes1 signaling pathway, thereby promoting BC cell drug resistance. This study may offer novel insights into BC treatment.

Topics: Adaptor Proteins, Signal Transducing; Breast Neoplasms; Drug Resistance, Neoplasm; Extracellular Vesicles; Female; Humans; MicroRNAs; Receptor, Notch1; Signal Transduction; Transcription Factor HES-1

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

Loss of BRCA1 p220 function often results in basal-like breast cancer (BLBC), but the underlying disease mechanism is largely opaque. In mammary epithelial cells (MECs), BRCA1 interacts with multiple proteins, including NUMB and HES1, to form complexes that participate in interstrand crosslink (ICL) DNA repair and MEC differentiation control. Unrepaired ICL damage results in aberrant transdifferentiation to a mesenchymal state of cultured, human basal-like MECs and to a basal/mesenchymal state in primary mouse luminal MECs. Loss of BRCA1, NUMB, or HES1 or chemically induced ICL damage in primary murine luminal MECs results in persistent DNA damage that triggers luminal to basal/mesenchymal transdifferentiation. In vivo single-cell analysis revealed a time-dependent evolution from normal luminal MECs to luminal progenitor-like tumor cells with basal/mesenchymal transdifferentiation during murine BRCA1 BLBC development. Growing DNA damage accompanied this malignant transformation.

Topics: Animals; BRCA1 Protein; Breast Neoplasms; Cell Differentiation; Cell Transdifferentiation; Cell Transformation, Neoplastic; Disease Models, Animal; DNA Damage; DNA Repair; Epithelial Cells; Female; HEK293 Cells; Humans; Mammary Glands, Animal; MCF-7 Cells; Membrane Proteins; Mice; Mice, Transgenic; Nerve Tissue Proteins; Transcription Factor HES-1; Transfection

Breast cancer is one of the most frequently occurring malignant tumors affecting women's health. At least one million new cases are diagnosed each year. Therefore, research that aims to identify strategies that inhibit the growth of breast cancer cells has become a primary worldwide focus. Traditional Chinese medicine (TCM) is regarded as a valuable resource in China, and numerous monomer compositions extracted from TCMs have been demonstrated to exhibit antitumor effects. The present study aimed to determine the impact of paeoniflorin (PF) on breast cancer cell proliferation and invasion, and to explore the mechanisms underlying its effects. Different concentrations of PF were applied to MCF‑7 cells at various time points and the Cell Counting kit‑8 assay was used to determine cell proliferation, a transwell invasion assay was employed to determine cell invasion, reverse transcription‑polymerase chain reaction was used to determine notch homolog‑1 (NOTCH‑1) and Hes family basic helix‑loop helix transcription factor (HES)‑1 mRNA expression levels, and western blotting was used to determine NOTCH‑1 and HES‑1 protein expression levels. The results demonstrated that PF inhibited the proliferation of MCF‑7 cells in a dose‑ and time‑dependent manner. Following treatment with different concentrations of PF, the total number of cells present in the PF‑treated groups was significantly lower when compared with the untreated control group (P<0.05). With increasing doses of PF, the rate of cell invasion significantly decreased, indicating a dose‑dependent association. NOTCH‑1 and HES‑1 mRNA expression levels were reduced when compared with the untreated control group, which reached a statistical significance following treatment with 15 and 30 µM PF (P<0.05). NOTCH‑1 and HES‑1 protein levels demonstrated a similar trend to the mRNA levels, whereby an increase in the concentration of PF was associated with a decrease in NOTCH‑1 and HES‑1 protein expression levels. The results of the present study therefore suggest that PF may inhibit the proliferation and invasiveness of breast cancer cells via inhibition of the NOTCH‑1 signaling pathway.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Drug Screening Assays, Antitumor; Female; Gene Expression; Glucosides; Humans; MCF-7 Cells; Monoterpenes; Receptor, Notch1; Signal Transduction; Transcription Factor HES-1

An intratumoral hypoxic microenvironment is frequently observed in solid tumors, including breast cancer. Lutein, a plant-derived compound and non-vitamin A carotenoid, has been demonstrated to possess multiple protective properties including anti-inflammation, anti-oxidative stress and antitumor effects. The main objective of the present research was to elucidate the involvement of lutein in the production of reactive oxygen species (ROS) under hypoxia, the activation of hairy and enhancer of split 1 (HES1), and the proliferation, invasion and migration of breast cancer cells. The human breast cancer cell lines MDA‑MB‑157 and MCF‑7 were exposed to hypoxic conditions and various concentrations of lutein. An MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was performed to examine cell proliferation, and Annexin V-fluorescein isothiocyanate/propidium iodide staining was performed to analyze the apoptosis ratio. The levels of hypoxia inducible factor-1α (HIF‑1α), NOTCH signaling molecules, HES1 and epithelial-mesenchymal transition (EMT)-associated factors were examined by reverse transcription-quantitative polymerase chain reaction and western blot analysis. Wound healing and Transwell invasion assays were used to detect the invasion and migration of breast cancer cells. Intracellular ROS levels were examined using 2,7-dichlorodihydrofluorescein-diacetate and flow cytometry. The results revealed that cell proliferation was inhibited by lutein in a dose-dependent manner, and the apoptosis ratio gradually increased with lutein treatment under hypoxia as evident from flow cytometry-based analysis. Exposure to lutein inhibited hypoxia-mediated activation of HIF‑1α, NOTCH signaling and HES1 expression, and suppressed the hypoxia-induced expression of EMT-associated factors. Lutein markedly inhibited the invasion and migration of breast cancer cells under hypoxia. Hypoxia-induced production of ROS was also decreased by lutein. Furthermore, the ROS scavenger N‑acetylcysteine also suppressed hypoxia inducible factor 1α and HES1 expression in breast cancer cells during hypoxia, but hydrogen peroxide (H2O2) levels were increased. Taken together, the results of the present study suggested that lutein may be a novel candidate for the chemoprevention of breast cancer. Furthermore, HES1 may be crucial in mediating the involvement of lutein in the suppression of hypoxia-driven ROS-induced breast cancer progression.

Topics: Antineoplastic Agents, Phytogenic; Breast Neoplasms; Cell Hypoxia; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Down-Regulation; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Lutein; MCF-7 Cells; Neoplasm Invasiveness; Reactive Oxygen Species; Signal Transduction; Transcription Factor HES-1

MicroRNAs (miRNAs) play a key role in the regulation of gene expression. In this study, we aimed to identify the clinical values of miR-1179 and to investigate the potential molecular mechanisms in breast cancer (BC).. RT-PCR was used to detect the expression levels of miR-1179 in both BC tissues and cell lines. We analyzed the association between the miR-1179 levels and clinicopathological factors and patient prognosis. The proliferation ability of miR-1179 on BC cells was assessed by MTT and colony formation assay. The role of miR-1179 in BC cells migration and invasion was measured by transwell assays. Western blot analysis was used to quantify the expression of the molecular biomarkers of the Notch signaling pathway.. Our results showed that miR-1179 expression was frequently downregulated in BC tissues and cell lines. Clinicopathologic analysis revealed that low miR-1179 expression is correlated with lymph node metastasis, advanced clinical stage and shorter overall survival. Multivariable Cox proportional hazards regression analysis suggested that increased miR-1179 expression was an independent prognostic factor of overall survival in BC patients. Gain-of-function assay indicated that the overexpression of miR-1179 significantly suppressed BC cells proliferation, migration and invasion. Mechanistically, miR-1179 up-regulation inhibited the expression of Notch 1, Notch 4 and Hes1, indicating that miR-1179 could suppress the activation of the Notch signaling pathway.. We showed that miR-1179 was a tumor suppressor that may serve as a novel potential prognostic biomarker or molecular therapeutic target for BC.

Topics: Breast Neoplasms; Cell Movement; Female; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; MicroRNAs; Middle Aged; Neoplasm Metastasis; Neoplasm Staging; Receptor, Notch1; Receptor, Notch4; Receptors, Notch; Signal Transduction; Transcription Factor HES-1; Up-Regulation

Resistance to the HER2-targeted antibody trastuzumab remains to be a major clinical challenge in the treatment of HER2-positive breast cancer. Hyper-activation of STAT3 is proposed to be a predictive biomarker of trastuzumab resistance. However, the precise mechanism(s) remains poorly defined. Evidence is emerging that HIF-1α, a central downstream element of STAT3 pathway, serves a pivotal role in the complex signaling network with subsequent diverse cellular events.. We have established trastuzumab resistant SKBR3 cells (SKBR3-TR). The cell viability, apoptosis as well as western blot, siRNA transfection and co-immunoprecipitation assays were performed to evaluate the involvement of STAT3/HIF-1α in modulation of trastuzumab resistance.. We found that in SKBR3-TR cells and conditioned medium-treated parental cells, constitutive phosphorylated STAT3 coincided with prominent up-regulation of HIF-1α which was accompanied with PTEN attenuation. Moreover, the inhibition of STAT3 activation by Stattic and/or genetically STAT3 knocking down decreased HIF-1α level in SKBR3-TR cells. Additionally, treatment with Stattic and/or STAT3 siRNA engendered the up-regulation of PTEN protein in STAT3-inhibited resistant cells. Restoration of PTEN was also observed following siRNA-mediated silencing of HIF-1α expression. Moreover, down-regulation of HIF-1α caused a reduction in the HES-1 content. Further study with HES-1 specific siRNA revealed the elevation of PTEN expression in HES-1 knock-down trastuzumab resistant cells.. The impairment of STAT3-HIF-1α-HES-1 pathway restored trastuzumab sensitivity through up-regulation of PTEN protein.. These findings highlighted the signal integrator role of HIF-1α in STAT3-mediated trastuzumab resistance induction which would be valuable in designing more efficient chemosensitization strategies.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Down-Regulation; Drug Resistance, Neoplasm; Female; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; PTEN Phosphohydrolase; Receptor, ErbB-2; STAT3 Transcription Factor; Transcription Factor HES-1; Trastuzumab

Trastuzumab (Herceptin) monoclonal antibody directed against HER2 receptor has been administered as a treatment for metastatic HER2 positive breast cancer. The problematic issue in treatment of HER2 positive breast cancer cells is commonly the induction of resistance to trastuzumab which might be due to modulation of some vital signaling elements such as Notch1 and Pin1. In this study, we were aimed to investigate whether the cross talk between pin1 and Notch1 has a role in this event. Our results indicated that the expression level of Pin1 in resistant SKBR3 cells increased by about twofold relative to sensitive SKBR3 cells. Besides, Pin1 inhibition via juglone reduced the extent of proliferation, colony formation and migration capacity of resistant SKBR3 cells. In addition, despite a feed forward loop between Notch1 and Pin1 in sensitive SKBR3 cells, inhibition of Notch1 cleavage in resistant SKBR3 cells did not affect pin1 level whereas pin1 inhibition by juglone reduced the level of Hes1, p-Akt and increased the cellular content of Numb. Therefore, we concluded that pin1 inhibition could be considered as a promising sensitizing strategy to weaken trastuzumab resistance.

Topics: Breast Neoplasms; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Humans; Membrane Proteins; Naphthoquinones; Nerve Tissue Proteins; NIMA-Interacting Peptidylprolyl Isomerase; Proto-Oncogene Proteins c-akt; Receptor, ErbB-2; Receptor, Notch1; Signal Transduction; Transcription Factor HES-1; Trastuzumab

Trastuzumab is a humanized monoclonal antibody against the human epidermal growth factor receptor 2 (HER2) that is overexpressed in about 25 % of breast cancer patients. However, primary and/or acquired resistance to trastuzumab develops in most affected persons. In this study, we explored the functional role of miR-182 inhibition with aiming the sensitization of SKBR3 cells to trastuzumab. Cell viability, apoptosis, colony formation, and migration capacities of SKBR3(S) (sensitive) and SKBR3(R) (resistant) cells were assessed to determine the anti-proliferative effects of PNA-antimiR-182. In addition, the expression levels of miR-182, mRNA of FOXO1, and Bim as well as the protein levels of HER2 and Notch1 signaling factors were evaluated by stem-loop RT-qPCR, RT-qPCR, and Western blot, respectively. The results indicated that miR-182 might play a causal role in the mechanism of trastuzumab. In line with that, PNA-antimiR-182 inhibited synergistically the viability of both the sensitive and resistant cell groups. Furthermore, the inhibitory effect of PNA-anitmiR-182 on migration in SKBR3 cells was more than the induction of apoptosis. In addition, PNA-antimiR-182 reduced the levels of NICD, Hes1, HIF-1α, and p-Akt in both cell groups, while it augmented the intracellular content of FOXO1 and Numb suppressor proteins. In other words, PNA-antimiR-182-mediated upregulation of Numb was associated with downregulation of HIF-1α and Hes1. Consequently, downregulation of miR-182 might find therapeutical value for overcoming trastuzumab resistance. Graphical Abstract The crosstalk between HER2 and Notch1 signaling pathway is mediated by miR-182.

Topics: Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Membrane Proteins; MicroRNAs; Nerve Tissue Proteins; Receptor, ErbB-2; RNA, Messenger; Transcription Factor HES-1; Trastuzumab

MicroRNAs (miRNAs or miRs) regulate gene expression by negatively modulating the stability or translational efficiency of their target genes by targeting the 3'-untranslated region (3'-UTR). Aberrant miRNA expression has been reported in various types of cancer; miRNAs can function as either oncogenes or tumor suppressor genes in cancer. In this study, we examined the expression level of miR‑101 in breast cancer tissues and cell lines by RT-qPCR, and found that miR‑101 expression was downregulated in breast cancer tissues and cell lines; indeed, in 6 of the 28 tissue samples, miR‑101 could not be detected. Furthermore, miR‑101, when transfected into SKBR3 cells, inhibited cell proliferation and promoted apoptosis, while miR‑101 inhibitor had the opposite effect. A dual-luciferase reporter assay revealed that miR‑101 targeted the 3'-UTR of eyes absent homolog 1 (Drosophila) (EYA1). Western blot analysis demonstrated a significantly decreased protein level of EYA1 in the SKBR3 cells transfected with miR‑101 mimic, whereas transfection with miR‑101 inhibitor led to an increased level of EYA1. Moreover, an increased expression of EYA1 was also found in breast cancer tissues and cell lines. The silencing of EYA1 using siRNA targeting EYA1 (EYA1‑siRNA) significantly inhibited SKBR3 cell proliferation and promoted apoptosis, and also suppressed the increased proliferation induced by transfection with miR‑101 inhibitor. The protein expression levels of Notch signaling components (jagged1, Hes1 and Hey1) were significantly decreased by transfection with miR‑101 mimic and EYA1-siRNA, and were increased by transfection with miR‑101 inhibitor. Furthermore, the elevated protein expression levels of jagged1, Hes1 and Hey1 induced by transfection with miR‑101 inhibitor in the SKBR3 cells were significantly decreased by transfection with EYA1-siRNA. Taken together, these results suggest that miR‑101 is down-regulated in breast cancer, and can inhibit cell proliferation and promote apoptosis by targeting EYA1 through the Notch signaling pathway.

Topics: 3' Untranslated Regions; Apoptosis; Base Sequence; Basic Helix-Loop-Helix Transcription Factors; Binding Sites; Breast Neoplasms; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Humans; Intracellular Signaling Peptides and Proteins; Jagged-1 Protein; MicroRNAs; Nuclear Proteins; Oligonucleotides, Antisense; Protein Tyrosine Phosphatases; RNA, Small Interfering; Signal Transduction; Transcription Factor HES-1

Tumor-initiating cells (also known as cancer stem cells) are the subpopulation of cells shown to be responsible for tumor initiation, maintenance and recurrence. In breast cancer, CD44(+)/CD24(-/low) cells were identified as tumor-initiating cells. We previously reported that a Gemini vitamin D analog, 1,25-dihydroxy-20R-21(3-hydroxy-3-deuteromethyl-4,4,4-trideuterobutyl)-23-yne-26,27-hexafluoro-cholecalciferol (BXL0124), reduced CD44(+)/CD24(-/low) cells in MCF10DCIS basal-like breast cancer cells. Since Notch has been identified as one of the key signaling pathways involved in breast cancer stem cells, the effect of BXL0124 on the Notch signaling pathway was investigated in breast cancer. The CD44(+)/CD24(-/low) subpopulation of MCF10DCIS cells showed elevated Notch1 signaling and increased cell proliferation compared to the CD44(+)/CD24(high) subpopulation. Treatment with the Gemini vitamin D analog BXL0124 decreased the level of activated Notch1 receptor. In addition, mRNA and protein levels of the Notch ligands, Jagged-1, Jagged-2 and DLL1, were significantly reduced by treatment with BXL0124, which was followed by repression of c-Myc, a key downstream target of Notch signaling. Interestingly, HES1, a known downstream target of Notch signaling, was rapidly induced by treatment with BXL0124. The inhibitory effect of BXL0124 on Notch signaling was reversed by knockdown of HES1. Overexpression of HES1 inhibited Notch1 signaling and reduced the CD44(+)/CD24(-/low) subpopulation, confirming a role of HES1 in Notch1 signaling. In conclusion, the Gemini vitamin D analog, BXL0124, represses the tumor-initiating subpopulation by HES1-mediated inhibition of Notch1 signaling. The present study demonstrates BXL0124 as a potent inhibitor of Notch signaling to target tumor-initiating cells in basal-like breast cancer. This article is part of a Special Issue entitled "17th Vitamin D Workshop".

Topics: Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Blotting, Western; Breast Neoplasms; Calcitriol; Carcinoma, Basal Cell; CD24 Antigen; Cell Proliferation; Female; Flow Cytometry; Homeodomain Proteins; Humans; Hyaluronan Receptors; Microscopy, Fluorescence; Neoplastic Stem Cells; Real-Time Polymerase Chain Reaction; Receptor, Notch1; Receptors, Calcitriol; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Transcription Factor HES-1; Tumor Cells, Cultured

Breast cancers (BCs) typically express estrogen receptors (ERs) but frequently exhibit de novo or acquired resistance to hormonal therapies. Here, we show that short-term treatment with the anti-estrogens tamoxifen or fulvestrant decrease cell proliferation but increase BC stem cell (BCSC) activity through JAG1-NOTCH4 receptor activation both in patient-derived samples and xenograft (PDX) tumors. In support of this mechanism, we demonstrate that high ALDH1 predicts resistance in women treated with tamoxifen and that a NOTCH4/HES/HEY gene signature predicts for a poor response/prognosis in 2 ER+ patient cohorts. Targeting of NOTCH4 reverses the increase in Notch and BCSC activity induced by anti-estrogens. Importantly, in PDX tumors with acquired tamoxifen resistance, NOTCH4 inhibition reduced BCSC activity. Thus, we establish that BCSC and NOTCH4 activities predict both de novo and acquired tamoxifen resistance and that combining endocrine therapy with targeting JAG1-NOTCH4 overcomes resistance in human breast cancers.

Topics: Aldehyde Dehydrogenase 1 Family; Animals; Antineoplastic Agents, Hormonal; Basic Helix-Loop-Helix Transcription Factors; Benzazepines; Breast Neoplasms; Calcium-Binding Proteins; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Drug Resistance, Neoplasm; Estradiol; Estrogen Receptor Antagonists; Female; Fulvestrant; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Intercellular Signaling Peptides and Proteins; Isoenzymes; Jagged-1 Protein; Membrane Proteins; Mice; Neoplastic Stem Cells; p-Aminoazobenzene; Proto-Oncogene Proteins; Receptor, Notch4; Receptors, Estrogen; Receptors, Notch; Retinal Dehydrogenase; Serrate-Jagged Proteins; Signal Transduction; Survival Analysis; Tamoxifen; Transcription Factor HES-1; Xenograft Model Antitumor Assays

Dysregulation of epigenetic controls is associated with tumorigenesis in response to microenvironmental stimuli; however, the regulatory pathways involved in epigenetic dysfunction are largely unclear. We have determined that a critical epigenetic regulator, microRNA-205 (miR-205), is repressed by the ligand jagged1, which is secreted from the tumor stroma to promote a cancer-associated stem cell phenotype. Knockdown of miR-205 in mammary epithelial cells promoted epithelial-mesenchymal transition (EMT), disrupted epithelial cell polarity, and enhanced symmetric division to expand the stem cell population. Furthermore, miR-205-deficient mice spontaneously developed mammary lesions, while activation of miR-205 markedly diminished breast cancer stemness. These data provide evidence that links tumor microenvironment and microRNA-dependent regulation to disruption of epithelial polarity and aberrant mammary stem cell division, which in turn leads to an expansion of stem cell population and tumorigenesis. This study elucidates an important role for miR-205 in the regulation of mammary stem cell fate, suggesting a potential therapeutic target for limiting breast cancer genesis.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Breast Neoplasms; Calcium-Binding Proteins; Carcinogenesis; Cell Line, Tumor; Cell Polarity; Cell Proliferation; Epigenesis, Genetic; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Homeodomain Proteins; Humans; Intercellular Signaling Peptides and Proteins; Jagged-1 Protein; Kruppel-Like Transcription Factors; Mammary Neoplasms, Experimental; Membrane Proteins; Mice; MicroRNAs; Neoplastic Stem Cells; Receptor, Notch2; RNA, Neoplasm; Serrate-Jagged Proteins; Signal Transduction; Transcription Factor HES-1; Transcription Factors; Tumor Microenvironment; Zinc Finger E-box-Binding Homeobox 1

Treating metastasis has been challenging due to tumors complexity and heterogeneity. This complexity is partly related to the crosstalk between tumor and its microenvironment. Endothelial cells -the building blocks of tumor vasculature- have been shown to have additional roles in cancer progression than angiogenesis and supplying oxygen and nutrients. Here, we show an alternative role for endothelial cells in supporting breast cancer growth and spreading independent of their vascular functions. Using endothelial cells and breast cancer cell lines MDA-MB231 and MCF-7, we developed co-culture systems to study the influence of tumor endothelium on breast tumor development by both in vitro and in vivo approaches. Our results demonstrated that endothelial cells conferred survival advantage to tumor cells under complete starvation and enriched the CD44HighCD24Low/- stem cell population in tumor cells. Moreover, endothelial cells enhanced the pro-metastatic potential of breast cancer cells. The in vitro and in vivo results concordantly confirmed a role for endothelial Jagged1 to promote breast tumor through notch activation. Here, we propose a role for endothelial cells in enhancing breast cancer progression, stemness, and pro-metastatic traits through a perfusion-independent manner. Our findings may be beneficial in developing novel therapeutic approaches.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Breast Neoplasms; Calcium-Binding Proteins; CD24 Antigen; Cell Line, Tumor; Cell Survival; Cells, Cultured; Cellular Microenvironment; Coculture Techniques; Endothelial Cells; Homeodomain Proteins; Humans; Hyaluronan Receptors; Intercellular Signaling Peptides and Proteins; Jagged-1 Protein; MCF-7 Cells; Membrane Proteins; Mice, Inbred NOD; Mice, Knockout; Mice, SCID; Microscopy, Confocal; Neoplasm Metastasis; Neoplastic Stem Cells; Receptors, Notch; Reverse Transcriptase Polymerase Chain Reaction; Serrate-Jagged Proteins; Transcription Factor HES-1; Transplantation, Heterologous

Benzyl isothiocyanate (BITC) is a promising anticancer constituent of edible cruciferous vegetables with in vivo efficacy against chemically induced as well as oncogene-driven breast cancer in experimental rodents. However, the mechanism underlying anticancer effect of BITC is not fully understood. This study was undertaken to determine the role of Notch signaling in anticancer responses to BITC as this pathway is often hyperactive in human breast cancer. Exposure of MCF-7, MDA-MB-231, and SUM159 human breast cancer cells to pharmacologic concentrations of BITC (2.5 and 5 μM) resulted in cleavage (activation) of Notch1, Notch2, and Notch4, which was accompanied by induction of γ-secretase complex components Presenilin1 and/or Nicastrin. The BITC-mediated cleavage of Notch was associated with its transcriptional activation as revealed by RBP-Jk and Hes-1A/B luciferase reporter assays. Inhibition of cell migration or cell viability resulting from BITC exposure was not influenced by pharmacological suppression of Notch1 using a γ-secretase inhibitor or RNA interference of Notch1 as well as Notch4. On the other hand, the BITC-mediated inhibition of cell migration, but not cell viability, was significantly augmented by siRNA and shRNA knockdown of Notch2 protein. Furthermore, the BITC-mediated inhibition of MDA-MB-231 xenograft growth in vivo was associated with a significant increase in nuclear levels of cleaved Notch2 and Hes-1 proteins. In conclusion, the results of this study indicate that (a) BITC treatment activates Notch2 in cultured and xenografted human breast cancer cells, and (b) Notch2 activation impedes inhibitory effect of BITC on cell migration.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Female; Homeodomain Proteins; Humans; Isothiocyanates; Mice; Mice, Nude; Proteolysis; Receptor, Notch1; Receptor, Notch2; Receptors, Notch; RNA Interference; Transcription Factor HES-1; Transcription, Genetic; Xenograft Model Antitumor Assays

We have previously shown that the anti-proliferative effect of retinoic acid in human breast cancer cell line MCF-7 is dependent on HES-1 expression. Here we show that retinoic acid induces HES-1 expression via upregulation of transcription factor SOX9. By expressing a dominant negative form of SOX9, disrupting endogenous SOX9 activity, the retinoic acid-induced HES-1 mRNA expression was inhibited. We found an enhancer regulating HES-1 expression: two SOX9 binding sites upstream of the HES-1 gene that were capable of binding SOX9 in vitro. By performing chromatin immunoprecipitation, we showed that SOX9 binding to the HES-1 enhancer was induced by retinoic acid in vivo. In reporter assays, transfection of a SOX9 expression plasmid increased the activity of the HES-1 enhancer. The enhancer responded to retinoic acid; furthermore, the expression of a dominant negative SOX9 abolished this response. Taken together, we present here a novel transcriptional mechanism in regulating hormone-dependent cancer cell proliferation.

Topics: Antineoplastic Agents; Basic Helix-Loop-Helix Transcription Factors; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Electrophoretic Mobility Shift Assay; Enhancer Elements, Genetic; Female; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Immunoprecipitation; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; SOX9 Transcription Factor; Transcription Factor HES-1; Transfection; Tretinoin

Epithelial-to-mesenchymal transition (EMT) is associated with decreased adhesion and acquisition of metastatic potential of breast cancer cells. Epithelial-to-mesenchymal transition is mediated, in part, by two transcription repressors, Snail and Slug, that are known to be targets of the Notch signaling pathway, and JAGGED1-induced Notch activation increases EMT. However, the events that lead to increased Notch activity during EMT of breast cancer cells are unknown.. The accumulation of hypoxia inducible factors (HIFs) under hypoxia was detected by western blot analysis, and their effects on Notch signaling were measured by an in vitro Notch reporter assay. The expression of Notch target genes under hypoxia was tested by real-time PCR. The knockdown of HIF-1alpha was mediated by retroviral delivery of shRNA. The expression of Slug and Snail under hypoxia was measured by real-time PCR. Breast cancer cell migration and invasion under hypoxia were tested with cell migration and invasion kits.. Hypoxia increased the expression of Notch target genes such as HES1 and HEY1 in breast cancer cells, as was expression of Notch receptors and ligands. The mechanism is likely to involve the accumulation of HIF-1alpha and HIF-2alpha in these cells by hypoxia, which synergised with the Notch co-activator MAML1 in potentiating Notch activity. Hypoxia inducible factor-1alpha was found to bind to HES1 promoter under hypoxia. Knockdown of HIF-1alpha with shRNA inhibited both HES1 and HEY1 expression under hypoxia. Hypoxia increased the expression of Slug and Snail, and decreased the expression of E-cadherin, hallmarks of EMT. Notch pathway inhibition abrogated the hypoxia-mediated increase in Slug and Snail expression, as well as decreased breast cancer cell migration and invasion.. Hypoxia-mediated Notch signaling may have an important role in the initiation of EMT and subsequent potential for breast cancer metastasis.

Topics: Basic Helix-Loop-Helix Transcription Factors; Breast Neoplasms; Cadherins; Cell Cycle Proteins; Cell Movement; Female; Homeodomain Proteins; Humans; Hypoxia; Neoplasm Invasiveness; Receptors, Notch; Signal Transduction; Transcription Factor HES-1

Epidemiological studies have suggested that cigarette smoking is related to increased breast cancer risk. Nicotine is most likely related to the risk in cigarette smoking. However, the mechanisms by which nicotine promotes cancer development are not fully understood. It has recently been suggested that development of breast cancer are originated from cancer stem cells, which are a minor population of breast cancer. In the present study, we investigated the effects of nicotine on the population of cancer stem cells in MCF-7 human breast cancer cells, using flow cytometry with a cancer stem cell marker aldehyde dehydrogenase (ALDH). We found that nicotine increased ALDH-positive cell population in a dose-dependent manner. We further demonstrated that a PKC-Notch pathway is involved in the effect of nicotine. In addition, the effect of nicotine was blocked by treatment with the α7 subunit-selective antagonist of nicotinic acetylcholine receptors (nAChR) α-Bungarotoxin. These data suggest that nicotine increases the stem cell population via α7-nAChR and the PKC-Notch dependent pathway in MCF-7 cells. These findings reveal a relationship between nicotine and the cancer stem cells in human breast cancer.

Topics: Aldehyde Dehydrogenase; alpha7 Nicotinic Acetylcholine Receptor; Basic Helix-Loop-Helix Transcription Factors; Biomarkers; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Female; Homeodomain Proteins; Humans; Neoplastic Stem Cells; Nicotine; Receptors, Nicotinic; Receptors, Notch; Transcription Factor HES-1

Regulation of hairy and enhancer of split homologue-1 (HES-1) by estradiol and all-trans retinoic acid affects proliferation of human breast cancer cells. Here, we identify and characterize cis-regulatory elements involved in HES-1 regulation. In the distal 5' promoter of the HES-1 gene, we found a retinoic acid response element and in the distal 3' region, an estrogen receptor alpha(ER)alpha binding site. The ERalpha binding site, composed of an estrogen response element (ERE) and an ERE half-site, is important for both ERalpha binding and transcriptional regulation. Chromatin immunoprecipitation assays revealed that ERalpha is recruited to the ERE and associates with the HES-1 promoter. We also show recruitment of nuclear receptor co-regulators to the ERE in response to estradiol, followed by a decrease in histone acetylation and RNA polymerase II docking in the HES-1 promoter region. Our findings are consistent with a novel type of repressive estrogen response element in the distal 3' region of the HES-1 gene.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Binding Sites; Breast Neoplasms; Cell Line; Chlorocebus aethiops; Down-Regulation; Estrogen Receptor alpha; Estrogens; Female; Gene Deletion; Gene Expression Regulation, Neoplastic; Histones; Homeodomain Proteins; Humans; Promoter Regions, Genetic; RNA Polymerase II; Time Factors; Transcription Factor HES-1

Hes-6 is a member of the basic helix-loop-helix (bHLH) family of transcription factors, and its overexpression has been reported in metastatic cancers of different origins. Hes-6 has been described as an inhibitor of Hes-1 during neuronal development, although its function in cancer is not known. In this study, we investigated the function of Hes-6 in breast cancer and tested the hypothesis that Hes-6 enhances breast cancer cell proliferation and is regulated by estrogen.. To investigate the function of Hes-6, T47D cells stably expressing Hes-6 were generated by lentiviral transduction, and conversely, siRNA also was used to knock down Hes-6 expression in breast cancer cells. The Hes-6-expressing T47D cells were transplanted into immunodeficient mice to study effects on tumor growth.. We found that Hes-6 expression was significantly higher in the high-grade, estrogen receptor (ER)alpha-negative SKBR3 and MDA-MB-231 cells compared with the ERalpha-positive, non-metastasizing T47D and MCF-7 breast carcinoma cells. Moreover, the level of Hes-6 mRNA was 28 times higher in breast cancer samples compared with normal breast samples. In Hes-6-expressing T47D cells, Hes-6 ectopic expression was shown to stimulate cell proliferation in vitro as well as breast tumor growth in xenografts. Moreover, expression of Hes-6 resulted in induction of E2F-1, a crucial target gene for the transcriptional repressor Hes-1. Consistently, silencing of Hes-6 by siRNA resulted in downregulation of E2F-1 expression, whereas estrogen treatment caused induction of Hes-6 and downstream targets hASH-1 and E2F-1 in MCF-7 cells.. Together, the data suggest that Hes-6 is a potential oncogene overexpressed in breast cancer, with a tumor-promoting and proliferative function. Furthermore, Hes-6 is a novel estrogen-regulated gene in breast cancer cells. An understanding of the role and regulation of Hes-6 could provide insights into estrogen signaling and endocrine resistance in breast cancer and, hence, could be important for the development of novel anticancer drugs.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Breast Neoplasms; Cell Growth Processes; Cell Line, Tumor; E2F1 Transcription Factor; Estradiol; Estrogen Receptor alpha; Female; Homeodomain Proteins; Humans; Mice; Mice, SCID; Repressor Proteins; RNA, Messenger; RNA, Small Interfering; Transcription Factor HES-1; Transfection; Transplantation, Heterologous

Breast cancer is one of the leading causes of cancer death in women. The Notch family of proteins plays crucial roles in determining cell fates such as proliferation, differentiation and apoptosis. A role for Notch signaling in human breast cancer has been suggested by the development of adenocarcinomas in the murine mammary gland. However, it is not clear currently whether Notch signaling is frequently expressed and activated in breast cancers. Here we show that Notch signaling is overexpressed and highly activated in breast cancers. More significantly, the attenuation of Notch signaling by gamma-secretase inhibitor can inhibit the proliferation of breast cancer cells by both causing cell cycle arrest and apoptosis. Thus, targeting Notch signaling may be of therapeutic value in breast cancers.

Topics: Adaptor Proteins, Signal Transducing; Amyloid Precursor Protein Secretases; Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Breast; Breast Neoplasms; Calcium-Binding Proteins; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Homeodomain Proteins; Humans; Intercellular Signaling Peptides and Proteins; Membrane Proteins; Protease Inhibitors; Proto-Oncogene Proteins; Receptor, Notch1; Receptor, Notch3; Receptor, Notch4; Receptors, Notch; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Serrate-Jagged Proteins; Signal Transduction; Transcription Factor HES-1; Up-Regulation

The mechanism behind hormone dependent growth of breast cancer is presently not well understood. We show that the HES-1 protein level in the breast cancer cell lines T47D and MCF-7 is down regulated by 17beta-estradiol treatment. This regulation could be reversed by addition of the anti-estrogens 4OH tamoxifen, raloxifen and Imperial Chemical Industries (ICI) 182,780. In T47D cells with inducible exogenous HES-1 expression, induced expression of HES-1 protein prevented the proliferative effect of 17beta-estradiol and subsequent up regulation of proliferating cell nuclear antigen (PCNA). An inverse correlation between the HES-1 and PCNA protein levels respectively was found in colon cancer cell lines. These findings point to a potential role of HES-1 as a tumor suppressor in epithelial cells, and as a mediator of 17beta-estradiols proliferative effect on breast cancer cells.

Topics: Basic Helix-Loop-Helix Transcription Factors; Breast Neoplasms; Cell Division; Down-Regulation; Estradiol; Estrogen Antagonists; Homeodomain Proteins; Humans; Proliferating Cell Nuclear Antigen; Transcription Factor HES-1; Tumor Cells, Cultured