ETS1 Regulates NKX3.15′ Promoter Activity and Expression in Prostate Cancer Cells

BACKGROUND. NKX3.1 controls the differentiation and proliferation of prostatic epithelial cells both during development and in the adult, while its expression is frequently downregulated in prostate cancers. Transcriptional control of NKX3.1 expression and in particular, factors that function via the NKX3.1 5' proximal promoter are poorly characterized. METHODS. Deletion reporter analyses, bioinformatics, electromobility shift assays (EMSA), chromatin immunoprecipitation (ChIP) and Western blotting were performed to identify and functionally characterize sites of transcription factor binding within the initial 2,062 bp of the NKX3.1 5' promoter. RESULTS. Deletion reporter studies of the 2,062 bp NKX3.1 5' promoter sequence localized positive transcriptional activity between -1069 and -993. Bioinformatic analyses identified the presence of two overlapping ETS1 binding sites within this region, designated EBS1 and EBS2, which exhibited 82% and 74% homology, respectively, to the ETS consensus binding sequence. EMSA and supershift assays indicated binding of both endogenous ETS1 and a recombinant CST-ETS1 protein solely to EBS1, a result that was confirmed in vivo by ChIP analysis. ETS1 overexpression transactivated NKX3.1 promoter reporter activity and upregulated endogenous NKX3.1 mRNA and protein levels in the LNCaP prostate cancer cell line, demonstrating a functional role for ETS1 in the regulation of NKX3.1 expression. CONCLUSIONS. ETS1 upregulation of NKX3.1 expression in LNCaP cells is mediated in part via its interaction with an EBS located in the NKX3.1 5' proximal promoter. ETS1 may regulate NKX3.1 during prostate development, with the aberrant ETS1 expression and cellular localization frequently observed in human prostate tumors potentially contributing to the abnormal expression of NKX3.1. Prostate 71: 403-414, 2011. (C) 2010 Wiley-Liss, Inc.