title: Expression of the RET proto-oncogene is regulated by TFAP2C in breast cancer independent of the estrogen receptor.
abstract: 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 < 0.001). Knockdown of TFAP2C in ER-negative MDA-MB-453 cells also led to a significant reduction in Ret mRNA compared to NT siRNA (0.16 vs. 1.0, P < 0.001). In MCF-7 cells, knockdown of TFAP2C abrogated Ret protein expression (0.02 vs. 1.0, P < 0.001) before reduction in ER.
  
  
  CONCLUSIONS
  TFAP2C regulates expression of the RET proto-oncogene through five AP-2 regulatory sites in the RET promoter. Regulation of Ret by TFAP2C occurs independently of ER expression in breast carcinoma.
authors.affiliation: Department of Surgery, University of Iowa, Iowa City, IA, USA.
authors.forename: Philip M
authors.initials: PM
authors.lastname: Spanheimer
authors.forename: George W
authors.initials: GW
authors.lastname: Woodfield
authors.forename: Anthony R
authors.initials: AR
authors.lastname: Cyr
authors.forename: Mikhail V
authors.initials: MV
authors.lastname: Kulak
authors.forename: Lola S
authors.initials: LS
authors.lastname: White-Baer
authors.forename: Thomas B
authors.initials: TB
authors.lastname: Bair
authors.forename: Ronald J
authors.initials: RJ
authors.lastname: Weigel
mesh_terms: D001665:Binding Sites; D015536:Down-Regulation / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D055785:Gene Knockdown Techniques; D006801:Humans; D061986:MCF-7 Cells; D011401:Promoter Regions, Genetic; D000090063:Proto-Oncogene Mas; D051096:Proto-Oncogene Proteins c-ret / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011960:Receptors, Estrogen / Q000378:metabolism*; D011980:Receptors, Progesterone / Q000378:metabolism; D050656:Transcription Factor AP-2 / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation / Q000235:genetics
chemical_list: C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D012333:RNA, Messenger; D011960:Receptors, Estrogen; D011980:Receptors, Progesterone; C493679:TFAP2C protein, human; D050656:Transcription Factor AP-2; D051096:Proto-Oncogene Proteins c-ret