Expression of the RET Proto-oncogene Is Regulated by TFAP2C in Breast Cancer Independent of the Estrogen Receptor

Background The RET proto-oncogene is expressed as part of the estrogen receptor (ER) cluster in breast cancer. We sought to determine if TFAP2C regulates Ret expression directly or indirectly through ER. Methods Chromatin immunoprecipitation sequencing (ChIP-Seq) and gel-shift assay were used to identify TFAP2C binding sites in the RET promoter in four breast cancer cell lines. Ret mRNA and protein levels were evaluated in ER-positive and ER-negative breast cancer cell lines after knockdown of TFAP2C. Luciferase expression assay was performed to assess expression from two of the identified sites. Results ChIP-Seq identified five main binding peaks for TFAP2C in the RET promoter at −101.5 kb, −50.7 kb, −32.5 kb, +5.0 kb, and +33.6 from the RET transcriptional start site. Binding at three of the AP-2 sites was conserved across all four cell lines, whereas the RET −101.5 and RET +33.6 sites were each found to be unbound by TFAP2C in one cell line. A TFAP2C consensus element was confirmed for all five sites. Knockdown of TFAP2C by siRNA in ER-positive MCF-7 cells resulted in significant down regulation of Ret mRNA compared to nontargeting (NT) siRNA (0.09 vs. 1.0, P