The miR-96 and RAR gamma signaling axis governs androgen signaling and prostate cancer progression

Expression levels of retinoic acid receptor gamma (NR1B3/RARG, encodes RAR gamma) are commonly reduced in prostate cancer (PCa). Therefore, we sought to establish the cellular and gene regulatory consequences of reduced RAR gamma expression, and determine RAR gamma regulatory mechanisms. RARG shRNA approaches in non-malignant (RWPE-1 and HPr1-AR) and malignant (LNCaP) prostate models revealed that reducing RAR gamma levels, rather than adding exogenous retinoid ligand, had the greatest impact on prostate cell viability and gene expression. ChIP-Seq defined the RAR gamma cistrome, which was significantly enriched at active enhancers associated with AR binding sites. Reflecting a significant genomic role for RAR gamma to regulate androgen signaling, RAR gamma knockdown in HPr1-AR cells significantly regulated the magnitude of the AR transcriptome. RAR gamma downregulation was explained by increased miR-96 in PCa cell and mouse models, and TCGA PCa cohorts. Biochemical approaches confirmed that miR-96 directly regulated RAR gamma expression and function. Capture of the miR-96 targetome by biotin-miR-96 identified that RAR gamma and a number of RAR gamma interacting co-factors including TACC1 were all targeted by miR-96, and expression of these genes were prominently altered, positively and negatively, in the TCGA-PRAD cohort. Differential gene expression analyses between tumors in the TCGA-PRAD cohort with lower quartile expression levels of RARG and TACC1 and upper quartile miR-96, compared to the reverse, identified a gene network including several RAR gamma target genes (e.g., SOX15) that significantly associated with worse disease-free survival (hazard ratio 2.23, 95% CI 1.58 to 2.88, p = 0.015). In summary, miR-96 targets a RAR gamma network to govern AR signaling, PCa progression and disease outcome.