Abstract 3101: RET, an estrogen-dependent proto-oncogene, is required for the proliferation of ER-positive breast cancer cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: RET is a member of the cadherin superfamily, which encodes a receptor tyrosine kinase. RET is a transforming proto-oncogene associated with many endocrine carcinomas. Regulation of RET expression is very complex. The RET promoter can be regulated by many transcription factors, including retinoic acid receptor, Sp1, Nkx2-1, and others. Using a transcriptional profiling assay, our laboratory previously identified RET as an estrogen-induced gene in breast cancer cells. In this study, we investigated the molecular mechanism by which estrogen regulates RET expression in breast cancer, and assessed the function of RET proto-oncogene in breast cancer. Methods: RET expression in breast cancer cell lines was analyzed by Q-RT-PCR, and RET expression in tissue specimens was retrieved from the oncomine database. Transcriptional regulation of RET promoter and distal enhancer elements was analyzed by luciferase assay and chromatin immunoprecipitation (ChIP) after treatment with vehicle or estrogen. Cell proliferation was measured by MTS assay and cell counting. Results: RET was highly expressed in estrogen receptor (ER)-positive breast cancer cell lines and breast cancer tissue specimens compared to ER-negative ones. Expression of RET was induced robustly by estrogen, and this induction was dependent on ER via a primary transcriptional regulatory mechanism, as determined by pretreatment of cycloheximide, ER siRNA, or the pure ER-antagonist, fulvestrant. The proximal promoter of RET gene was not responsive to estrogen treatment and ER did not bind to the proximal promoter. ChIP analysis showed that of nine potential ER binding sites in the RET gene locus, only three bind ER with different efficiency. A 1kb DNA fragment, which is located about 50kb upstream of the transcriptional start site, had an extremely high affinity to ER. When inserted in front of the promoter-luciferase cassette, only this 1kb fragment, but not other eight fragments, enhanced estrogen inducibility and therefore functions as an enhancer acting in cis. This enhancer region has two half estrogen responsive elements (EREs) and a full ERE, which are critical for estrogen-induced RET expression. In a functional assay, siRNA knockdown of RET inhibited estrogen-stimulated cell proliferation in ER-positive breast cancer cells, but not in ER-negative ones. Conclusion: The RET proto-oncogene is induced by estrogen treatment and is required for the proliferation of ER-positive breast cancers. Estrogen-induced RET expression is facilitated by ER binding to a distal enhancer containing novel EREs. This pathway of estrogen regulation of RET gene expression and cell proliferation may be targeted in the future for better treatment of breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3101. doi:10.1158/1538-7445.AM2011-3101