Abstract

Abstract The Raf kinase inhibitory protein (RKIP) acts as an activator of apoptosis and inhibitor of metastasis in prostate cancer. Diminished expression of RKIP has been reported in several cancers including prostate carcinomas; however, the underlying mechanism of RKIP regulation remains largely unknown. RKIP transcription is inhibited by the EMT inducer Snail which also represses the expression of the metastasis suppressor E-cadherin. E-cadherin and RKIP are co-expressed in many tumors. E-cadherin transcription is also suppressed by the polycomp repressive complex (PRP) transcription repressor, enhancer of zeste homolog 2 (EZH2), which mediates an H3 lysine 27 trimethylation in target promoters. EZH2 is highly expressed in several metastatic tumors including prostate cancer and its levels are inversely correlated with E-cadherin and RKIP expressions. The zinc-finger transcription factor Yin-Yang 1 (YY1) is also overexpressed in prostate tumors and has been associated with tumor progression. Silencing of YY1 by siRNA reverses the EMT phenotype of metastatic prostate cancer cell lines by induction of E-cadherin and RKIP. Here, we explored the direct roles of YY1 and EZH2 in the transcriptional regulation of RKIP in prostate cancer. We used as experimental models the metastatic and non-metastatic prostate cancer cell lines DU145 and LNCaP, respectively, which differ in the expression levels of EZH2, YY1 and RKIP. DU145 cells express low RKIP and high EZH2 and YY1 levels, whereas LNCaP cells express the opposite. Putative binding sites were identified for both YY1 and EZH2 (proximal E-boxes) within the RKIP promoter. CHiP analyses showed that both factors can physically associate with the RKIP promoter. Loss and gain of function reporter approaches showed that ectopic expression of EZH2 in LNCaP cells inhibits the wild type, but not the E-box mutant RKIP basal promoter activity. Similarly, a mutated EZH2 vector that lacks efficient binding to the target promoter couldn't inhibit the RKIP promoter activity. EZH2-mediated inhibition of RKIP promoter activity could also be inhibited by HDAC inhibitors, suggesting that HDAC recruitment and deacetylation of histone H3 is perquisite for EZH2 suppressive activity. Silencing of EZH2 or YY1 in DU145 also resulted in induction of RKIP promoter activity. The negative effect of YY1 on RKIP promoter activity was corroborated by inhibition of RKIP mRNA and protein levels. The above observations were further validated in prostate cancer tissue microarrays whereby YY1 and RKIP protein expressions were found to be inversely correlated. The present findings support the roles of EZH2 and YY1 as transcription repressors of RKIP in prostate cancer and suggest that their overexpression may be associated, in part, with the high cell metastatic potential through downregulation of RKIP. We propose that EZH2 and YY1 might be potential prognostic markers of prostate cancer progression and targets for therapeutic intervention. 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 2162. doi:10.1158/1538-7445.AM2011-2162

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