Identification of novel genes that regulate androgen receptor signaling and growth of androgen-deprived prostate cancer cells

// Elina Levina 1, 2 , Hao Ji 1 , Mengqiang Chen 1 , Mirza Baig 5 , David Oliver 1 , Patrice Ohouo 5 , Chang-uk Lim 1 , Garry Schools 1 , Steven Carmack 3 , Ye Ding 3 , Eugenia V. Broude 1 , Igor B. Roninson 1 , Ralph Buttyan 4, * , Michael Shtutman 1, * 1 Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, Columbia, SC, USA 2 Department of Biological Sciences, University of South Carolina, Columbia, SC, USA 3 Wadsworth Center, NY State Department of Health, Albany, NY, USA 4 The Vancouver Prostate Centre, Vancouver, BC, Canada 5 Cancer Center, Ordway Research Institute, Albany, NY, USA * These authors have contributed equally to this work Correspondence to: Ralph Buttyan, e-mail: rbuttyan@prostatecentre.com Michael Shtutman, e-mail: shtutmanm@sccp.sc.edu Keywords: prostate cancer, androgen receptor, shRNA, IGSF8, tumor progression Received: February 02, 2015 Accepted: April 10, 2015 Published: April 23, 2015 ABSTRACT Prostate cancer progression to castration refractory disease is associated with anomalous transcriptional activity of the androgen receptor (AR) in an androgen-depleted milieu. To identify novel gene products whose downregulation transactivates AR in prostate cancer cells, we performed a screen of enzymatically-generated shRNA lenti-libraries selecting for transduced LNCaP cells with elevated expression of a fluorescent reporter gene under the control of an AR-responsive promoter. The shRNAs present in selected populations were analyzed using high-throughput sequencing to identify target genes. Highly enriched gene targets were then validated with siRNAs against selected genes, testing first for increased expression of luciferase from an AR-responsive promoter and then for altered expression of endogenous androgen-regulated genes in LNCaP cells. We identified 20 human genes whose silencing affected the expression of exogenous and endogenous androgen-responsive genes in prostate cancer cells grown in androgen-depleted medium. Knockdown of four of these genes upregulated the expression of endogenous AR targets and siRNAs targeting two of these genes (IGSF8 and RTN1) enabled androgen-independent proliferation of androgen-dependent cells. The effects of IGSF8 appear to be mediated through its interaction with a tetraspanin protein, CD9, previously implicated in prostate cancer progression. Remarkably, homozygous deletions of IGSF8 are found almost exclusively in prostate cancers but not in other cancer types. Our study shows that androgen independence can be achieved through the inhibition of specific genes and reveals a novel set of genes that regulate AR signaling in prostate cancers.