Abstract LB-293: MicroRNA-96 targets RARγ and associates with prostate cancer progression
Authors: Long Mark D. , Singh Prashant K. , Campbell Moray J. 
Published: August 2015

Abstract
Nuclear receptors (NRs) integrate dietary and steroidal signals to control cell fate and are strong chemoprevention and chemotherapy targets, for example in prostate cancer (PCa). We mined TCGA and MSKCC PCa cohorts using bootstrap approaches revealing that NRs mRNA was significantly down-regulated more than predicted by chance (p = 7.1e-4). Loss of expression did not reflect somatic mutations or CNVs suggesting that epigenetic mechanisms drive this expression. Using prediction algorithms we identified NR targeting miRNA and revealed a number were significantly elevated, associated with significant negative correlations with specific NR. For example, RARG, which encodes retinoic acid receptor gamma (RARγ), was amongst the most frequently reduced NR, reflecting the prostatic metaplasia in RARγ knockout mice reported by others. Amongst the most significantly and commonly elevated miRNA was miR-96 which is predicted to target RARG and associated with worse disease free survival (p = 1.3e-4). Mining of published microarray and ChIP-Seq data revealed that miR-96 was downstream of androgen receptor (AR) signaling and supported a negative feed-forward relationship between AR and RARγ. Expression analyses revealed inverse correlations of RARG and targeting miR-96 in 14 different PCa cell lines of increasing aggressiveness and along the time course of tumor development in TRAMP and Pten(-/-) murine models of PCa. In RWPE-1 non-malignant prostate cells and in LNCaP PCa cells we validated miR-96 regulation of RARγ expression through miRNA mimic transfection and luciferase assays with miR-96 target sites from the RARG-3′UTR. Functional analyses revealed that modulating miR-96 levels affected the cellular response to the natural ligand for RARγ, all-trans retinoic acid. To capture all genes targeted by miR-96 in RWPE-1 and LNCaP cells we undertook transfection with biotin-labeled miR-96 followed by streptavidin pull-down and microarray analyses. 389 genes were significantly enriched in LNCaP cells and subsequent in silico analyses revealed significant enrichment for known and predicted targets for miR-96 (q-value 1.45e-25), down-regulation of enriched targets, and genes with annotated functions involved in hormone and apoptotic responses (q-value 5.1e-33). In LNCaP cells, approximately 10% of the targets were other transcription factors (e.g. CEBPA, FOXN2), co-regulators (e.g. TBLIX) or epigenetic regulators (e.g. ARID3B), many of which have established roles in the co-regulation of retinoid and androgen signaling. We propose that AR-driven miR-96 disrupts an interdependent network of RARγ and interacting transcription factors that control cell fate. Tumor or serum measurements of miR-96 have clinical potential to be exploited in targeted chemoprevention approaches to predict RARγ signaling status and progression risk.
Citation Format: Mark D. Long, Prashant K. Singh, Moray J. Campbell. MicroRNA-96 targets RARγ and associates with prostate cancer progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-293. doi:10.1158/1538-7445.AM2015-LB-293