Transcription profiling of erB2-dependent response over time to EGF and HRG stimulation of human mammary luminal epithelial cells
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ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Worthington J
PROVIDER: S-ECPF-TABM-106 | biostudies-other | 2010
REPOSITORIES: biostudies-other
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Project description:Our aim was to identify time-dependent changes in gene expression associated with Epidermal Growth Factor (EGF) and Hergulin B1 (HRG) stimulation of normal human mammary luminal epithelial cells (HB4a) and a derivative cell line (C3.6)overexpressing the receptor tyrosine kinase ErbB2/HER2. Serum-starved HB4a parental cells and ErbB2-overexpressing C3.6 cells were stimulated with EGF or HRG for 4 h, 18 h and 24 h or left unstimulated (0 h) prior to microarray analysis of mRNA levels of 9,932 probes, representing ~6,000 genes. Four replicates of each sample were co-hybridized to chips with a common RNA reference allowing direct comparison of gene expression across all experimental conditions: time, growth factor and cell line.
2006-08-15 | E-TABM-106 | biostudies-arrayexpress
Project description:BACKGROUND: The AP2 transcription factor family is a set of developmentally regulated, retinoic acid (RA) inducible genes, which regulate expression of estrogen receptor-alpha (ERalpha) in breast carcinoma. We hypothesized that AP2 factors regulate a set of genes characteristic of the hormone responsive breast cancer phenotype. To better understand the role of AP2 factors in hormone responsive breast cancer, we sought to identify AP2-target genes in breast epithelial cells. MATERIALS AND METHODS: Overexpression of AP2 factors was achieved in human mammary epithelial cells (HMECs) using adenoviral vectors. AP2 target genes were identified by comparative hybridization to cDNA microarrays containing 30,000 human genes. Expression patterns were confirmed by Northern and Western blot and by elimination of AP2 using siRNA. Potential regulatory elements in promoters of target genes were identified by DNase I hypersensitive site mapping. RESULTS: Comparative cDNA microarray hybridization identified a set of genes induced by overexpression of AP2alpha and AP2gamma in HMECs. The up-regulation of cellular retinoic acid-binding protein 2 (CRABPII), EST-1, and ECM1 was induced by overexpression of AP2alpha, AP2gamma, or a chimeric AP2 factor in which the activation domain of AP2alpha was replaced by the activation domain of herpesvirus VP16. Interestingly, hormone unresponsive MDA-MB-231 cells were resistant to CRABPII induction by any of the AP2 factors. Elimination of AP2gamma in MCF7 cells resulted in a significant reduction in CRABPII expression. AP2alpha induced DNase I hypersensitive sites in the promoter of the CRABPII gene at -5000 bp, which corresponds to the site of action of RAR/RXR factors. CONCLUSIONS: AP2 factors regulate CRABPII expression in HMECs and breast cancer cells and accounts for the associated expression of ERalpha and CRABPII in hormone responsive breast cancer. Because CRABPII mediates growth suppressive effects of RA in breast cancer, the data suggest that AP2 factors have the ability to mediate RA responsiveness through the regulation of CRABP II expression.
| S-ECPF-SMDB-4003 | biostudies-other
Project description:p130Cas adaptor protein regulates basic processes such as cell cycle control, survival and migration. p130Cas over-expression has been related to mammary gland transformation, however the in vivo consequences of p130Cas over-expression during mammary gland morphogenesis are not known. In ex vivo mammary explants from MMTV-p130Cas transgenic mice, we show that p130Cas impairs the functional interplay between Epidermal Growth Factor Receptor (EGFR) and Estrogen Receptor (ER) during mammary gland development. Indeed, we demonstrate that p130Cas over-expression upon the concomitant stimulation with EGF and estrogen (E2) severely impairs mammary morphogenesis giving rise to enlarged multicellular spherical structures with altered architecture and absence of the central lumen. These filled acinar structures are characterized by increased cell survival and proliferation and by a strong activation of Erk1/2 MAPKs and Akt. Interestingly, antagonizing the ER activity is sufficient to re-establish branching morphogenesis and normal Erk1/2 MAPK activity. Overall, these results indicate that high levels of p130Cas expression profoundly affect mammary morphogenesis by altering epithelial architecture, survival and unbalancing Erk1/2 MAPKs activation in response to growth factors and hormones. These results suggest that alteration of morphogenetic pathways due to p130Cas over-expression might prime mammary epithelium to tumorigenesis.
| S-EPMC3519769 | biostudies-literature
Project description:Sharing common ErbB/HER receptor signaling pathway, heregulin (HRG) induces differentiation of MCF-7 breast cancer cells while epidermal growth factor (EGF) elicits proliferation. Although the cell fate led by those two ligands was totally different, the gene expression profile in early transcription was unexpectedly qualitatively similar, suggesting that the gene expression in late transcription, not early transcription, may reflect a respect of ligand specificity. In this study, based on the data from time-course microarray of all human genes, we predicted and determined a series of transcription factors which may control HRG-specific timed-late transcription and cellular differentiation of MCF-7 cells. Validation analyses showed that one of activator protein 1 (AP-1) families appeared just after c-Fos expression, another AP-1 family partner, induced expression of another transcription factor through activation of AP-1 complex. Furthermore, expression of this transcription factors caused suppression of extracellular signal-regulated kinase (ERK) phosphorylation which is sustainedly regulated by HRG-initiated ErbB signaling. Overall, our analysis indicated an importance of formation of timed-transcriptional regulatory network and its function to control upstream signaling pathway through negative feedback for cellular differentiation. Experiment Overall Design: MCF7 human breast cancer cells were stimulated by the growth hormone (epidermal growth factor (EGF) or heregulin (HRG)). Control was set as non-treated cells.
| S-ECPF-GEOD-13009 | biostudies-other
Project description:To investigate changes in gene expression associated with ERBB2, expression profiling of immortalised human mammary luminal epithelial cells and variants expressing a moderate and high level of ERBB2 has been carried out using cDNA microarrays corresponding to approximately 6,000 unique genes/ESTs. Sixty-three (63) significantly up- or down-regulated (>2.0 fold) genes were identified and further validated by RT-PCR and Northern blot analysis as well as microarray comparisons with a spontaneously ERBB2- overexpressing breast cancer cell line and ERBB2-positive primary breast tumours. Proteins predicted to be associated ERBB2 overexpression in breast cancers were analysed together with their clinical relevance by antibody screening using a tissue array. Differentially regulated genes include those involved in cell matrix interactions including P4HA2 (proline 4-hydroxylase), galectin 1 (LGALS1) and galectin 3 (LGALS3), FN1 (fibronectin 1), and CDH3 (p-cadherin) and cell proliferation (CRIP1, IGFBP3) and transformation (S100P, S100A4). A number of genes associated with MYC signalling were also differentially expressed, including NDRG1, USF2 and the epithelial membrane proteins 1 and 3 (EMP1, EMP3). These data represent profile of the transcriptional changes associated with ERBB2-related pathways in the breast, and identify novel and potentially useful targets for prognosis and therapy.
| S-ECPF-SNGR-8 | biostudies-other
Project description:We analyzed gene expression in 184 (finite life span) and HMT3522 S1 (immortal non-malignant) HMECs on successive days (3, 5, and 7) post-seeding in a laminin-rich extracellular matrix assay. Both HMECs underwent growth arrest in G0/G1 and differentiated into polarized acini between days 5 and 7.
| S-ECPF-MEXP-1006 | biostudies-other
Project description:To understand how integration of multiple data types can help decipher cellular responses at the systems level, we analyzed the mitogenic response of human mammary epithelial cells to epidermal growth factor (EGF) using whole genome microarrays, mass spectrometry-based proteomics and large-scale western blots with over 1000 antibodies. A time course analysis revealed significant differences in the expression of 3172 genes and 596 proteins, including protein phosphorylation changes measured by western blot. Integration of these disparate data types showed that each contributed qualitatively different components to the observed cell response to EGF and that varying degrees of concordance in gene expression and protein abundance measurements could be linked to specific biological processes. Networks inferred from individual data types were relatively limited, whereas networks derived from the integrated data recapitulated the known major cellular responses to EGF and exhibited more highly connected signaling nodes than networks derived from any individual dataset. While cell cycle regulatory pathways were altered as anticipated, we found the most robust response to mitogenic concentrations of EGF was induction of matrix metalloprotease cascades, highlighting the importance of the EGFR system as a regulator of the extracellular environment. These results demonstrate the value of integrating multiple levels of biological information to more accurately reconstruct networks of cellular response. Keywords: time-course analysis
2010-04-14 | GSE15668 | GEO
Project description:BACKGROUND: Members of the ErbB family of growth factor receptors are intricately linked with epithelial cell biology, development and tumourigenesis; however, the mechanisms involved in their downstream signalling are poorly understood. Indeed, it is unclear how signal specificity is achieved and the relative contribution each receptor has to specific gene expression. METHODS: Gene expression profiling of a human mammary luminal epithelial cell model of ErbB2-overexpression was carried out using cDNA microarrays with a common RNA reference approach to examine long-term overlapping and differential responses to EGF and heregulin beta1 treatment in the context of ErbB2 overexpression. Altered gene expression was validated using quantitative real time PCR and/or immunoblotting. One gene of interest was targeted for further characterisation, where the effects of siRNA-mediated silencing on IGF1-dependent signalling and cellular phenotype were examined and compared to the effects of loss of ErbB2 expression. RESULTS: 775 genes were differentially expressed and clustered in terms of their growth factor responsiveness. As well as identifying uncharacterized genes as novel targets of ErbB2-dependent signalling, ErbB2 overexpression augmented the induction of multiple genes involved in proliferation (e.g. MYC, MAP2K1, MAP2K3), autocrine growth factor signalling (VEGF, PDGF) and adhesion/cytoskeletal regulation (ZYX, THBS1, VCL, CNN3, ITGA2, ITGA3, NEDD9, TAGLN), linking them to the hyper-poliferative and altered adhesive phenotype of the ErbB2-overexpressing cells. We also report ErbB2-dependent down-regulation of multiple interferon-stimulated genes that may permit ErbB2-overexpressing cells to resist the anti-proliferative action of interferons. Finally, IGFBP3 was unique in its pattern of regulation and we further investigated a possible role for IGFBP3 down-regulation in ErbB2-dependent transformation through suppressed IGF1 signalling. We show that IGF1-dependent signalling and proliferation were enhanced in ErbB2-overexpressing cells, whilst loss of ErbB2 expression by siRNA silencing reduced IGF1 signalling. Furthermore, IGFBP3 knockdown resulted in basal ERK and Akt activation in luminal epithelial cells and increased invasiveness and anchorage-independent colony formation in SKBR3 cells. CONCLUSIONS: These data show IGFBP3 as a negative regulator of transformation and that its down-regulation enhances IGF1-dependent signalling. They also show that ErbB2 can up-regulate IGF1-dependent signalling, possibly via the regulated expression of IGFBP3.
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Project description:"Deficiencies in the ATM gene are the underlying cause for ataxia telangiectasia, a congenital syndrome characterized by neurological, motor and immunological defects, as well as a predisposition to cancer risks. MicroRNAs (miRNAs) are small regulators of post-transcriptional gene expression and a useful tool for cancer diagnosis, staging, and prediction of therapeutic responses to clinical regimens. In particular, miRNAs have been used to develop signatures for breast cancer profiling. We are interested in the consequences of ATM deficiency on miRNA expression in breast epithelial cells and the potential contribution to cancer predisposition. In this study we investigate the effects of ATM loss on the miRNA expression and related gene expression changes in normal human mammary epithelial cells (HME-CC). We have identified 81 significantly differently expressed miRNAs in the ATM-deficient HME-CCs using small RNA sequencing. Many of these differentially expressed miRNAs have been described and implicated in tumorigenesis and proliferation. These changes include down-regulation of tumor suppressor miRNAs, such as hsa-miR-29c and hsa-miR-16, as well as the over-expression of pro-oncogenic miRNAs hsa-miR-93 and hsa-mir-221. All 81 miRNAs were combined with genome wide gene expression profiles to investigate possible targets of miRNA regulation. We identified messenger RNA (mRNA) targets of these miRNAs that were also significantly regulated after the depletion of ATM. Predicted targets included many genes implicated in cancer formation and progression, including SOCS1 and the proto-oncogene MAF. Integrated analysis of miRNA and mRNA expression allows us to build a more complete understanding of the pathways and networks involved in the breast cancer predisposition observed in individuals deficient in ATM. This study highlights miRNA and predicted mRNA target expression changes in ATM-deficient HME-CCs and suggests a mechanism for the breast cancer-prone phenotype seen in ATM deficient cells and patients. Additionally, this study provides preliminary data for defining miRNA profiles that may be used prognostic biomarkers for breast cancer predisposition. Examination of small RNA population in human mammary epithelial cell lines. Each condition was preformed in triplicate. This submission represents the transcriptome component of study."
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Project description:Molecular subtypes of breast cancer are characterized by distinct patterns of gene expression that are predictive of outcome and response to therapy. The luminal breast cancer subtypes are defined by the expression of estrogen receptor-alpha (ERα)-associated genes, many of which are directly responsive to the transcription factor activator protein 2C (TFAP2C). TFAP2C participates in a gene regulatory network controlling cell growth and differentiation during ectodermal development and regulating ESR1/ERα and other luminal cell-associated genes in breast cancer. TFAP2C has been established as a prognostic factor in human breast cancer, however, its role in the establishment and maintenance of the luminal cell phenotype during carcinogenesis and mammary gland development have remained elusive. Herein, we demonstrate a critical role for TFAP2C in maintaining the luminal phenotype in human breast cancer and in influencing the luminal cell phenotype during normal mammary development. Knockdown of TFAP2C in luminal breast carcinoma cells induced epithelial-mesenchymal transition with morphological and phenotypic changes characterized by a loss of luminal-associated gene expression and a concomitant gain of basal-associated gene expression. Conditional knockout of the mouse homolog of TFAP2C, Tcfap2c, in mouse mammary epithelium driven by MMTV-Cre promoted aberrant growth of the mammary tree leading to a reduction in the CD24(hi)/CD49f(mid) luminal cell population and concomitant gain of the CD24(mid)/CD49f(hi) basal cell population at maturity. Our results establish TFAP2C as a key transcriptional regulator for maintaining the luminal phenotype in human breast carcinoma. Furthermore, Tcfap2c influences development of the luminal cell type during mammary development. The data suggest that TFAP2C has an important role in regulated luminal-specific genes and may be a viable therapeutic target in breast cancer.
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