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Transcription profiling of human mammary epithelial cells over-expressing AP2

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

SUBMITTER: McPherson LA

PROVIDER: S-ECPF-SMDB-4003 | biostudies-other | 2007 Mar

REPOSITORIES: biostudies-other

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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.

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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Transcription profiling of human mammary epithelial cells over-expressing AP2

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