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

Abstract

ABSTRACTExpression levels of retinoic acid receptor gamma (NR1B3/RARG, encodes RARγ), are commonly reduced in prostate cancer (PCa). Therefore we sought to establish the cellular and gene regulatory consequences of reduced RARγ expression, and determine RARγ regulatory mechanisms.RARGshRNA approaches in non-malignant (RWPE-1 and HPr1-AR) and malignant (LNCaP) prostate models revealed that reducing RARγ levels, rather than adding exogenous retinoid ligand, had the greatest impact on prostate cell viability and gene expression. ChIP-Seq defined the RARγ cistrome which was significantly enriched at active enhancers associated with AR binding sites. Reflecting a significant genomic role for RARγ to regulate androgen signaling, RARγ knockdown in HPr1-AR cells significantly regulated the magnitude of the AR transcriptome. RARγ down-regulation was explained by increased miR-96 in PCa cell and mouse models, and TCGA PCa cohorts. Biochemical approaches confirmed that miR-96 directly regulated RARγ expression and function Capture of the miR-96 targetome by biotin-miR96 identified that RARγ and a number of RARγ interacting co-factors includingTACC1were 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 ofRARGandTACC1and upper quartile miR-96, compared to the reverse, identified a gene network including several RARγ 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 RARγ network to govern AR signaling, PCa progression and disease outcome.Conflict of interest:The authors certify that they have NO affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript.FUNDINGLESCacknowledges support, in part, of Roswell Park Comprehensive Cancer Center-University of Pittsburg Cancer Institute Ovarian Cancer Specialized Program of Research Excellence National Institutes of Health [P50CA159981-01A1].MDLacknowledges support of Molecular Pharmacology and Experimental Therapeutics NRSA T32 program [T32CA009072] held at Roswell Park Comprehensive Cancer Center.MJCandDJSacknowledges support in part from the Prostate program of the Department of Defense Congressionally Directed Medical Research Programs [W81XWH-14-1-0608, W81XWH-11-2-0033] and the National Cancer Institute (NCI) grant P30CA016056 involving the use of Roswell Park Comprehensive Cancer Center’s Genomic Shared Resource.MJC, GL, AR, HWandPvdBacknowledges support from the European Union-United States Atlantis Program [P116J090011].MJCandLESCacknowledge support from the National Cancer Institute (NCI) grant P30CA016056 involving the use of OSUCCC The James, CCSG P30CA016058