ETS1 regulates NKX3.1 5' 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 GST-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.

Androgen regulation of the prostatic tumour suppressor NKX3.1 is mediated by its 3' untranslated region.

The homeodomain transcription factor NKX3.1 is a prostate-specific tumour suppressor, expression of which is reduced or undetectable in the majority of metastatic prostate tumours. In the normal prostate and in prostate cancer cells, NKX3.1 expression is under tight androgenic control that we have shown to be mediated by its ~2.5 kb 3'UTR (3' untranslated region). Reporter deletion analysis of the NKX3.1 3'UTR identified three regions that were transactivated by DHT (5alpha-dihydrotestosterone) in the AR (androgen receptor)-expressing prostate cancer cell line LNCaP. Reversal of DHT effects by the anti-androgen bicalutamide supported an AR-mediated mechanism, and bioinformatic analysis of the NKX3.1 3'UTR identified canonical AREs (androgen-response elements) in each of the androgen-responsive regions. EMSAs (electrophoretic mobility-shift assays) indicated binding of the AR DNA-binding domain to two of the AREs, a proximal ARE at +2378-2392 from the transcription start site, and a more distal ARE at +3098-3112. ChIP (chromatin immunoprecipitation) analysis provided further evidence of ligand-dependent recruitment of endogenous AR to sequence encompassing each of the two elements, and site-directed mutagenesis and deletion analysis confirmed the contribution of each of the AREs in reporter assays. The present studies have therefore demonstrated that the NKX3.1 3'UTR functions as an androgen-responsive enhancer, with the proximal ARE contributing the majority and the distal ARE providing a smaller, but significant, proportion of the androgen responsiveness of the NKX3.1 3'UTR. Characterization of androgen-responsive regions of the NKX3.1 gene will assist in the identification of transcriptional regulatory mechanisms that lead to the deregulation of NKX3.1 expression in advanced prostate cancers.