Identification of Novel Androgen-Regulated Pathways and mRNA Isoforms through Genome-Wide Exon- Specific Profiling of the LNCaP Transcriptome Prabhakar Rajan1,2,3., Caroline Dalgliesh1., Phillippa J. Carling 1 , Thomas Buist1, Chaolin Zhang4¤a, Sushma N. Grellscheid1, Kelly Armstrong5, Jacqueline Stockley5, Cedric Simillion6, Luke Gaughan 5 , Gabriela Kalna2, Michael Q. Zhang4¤b, Craig N. Robson 5 , Hing Y. Leung2,3, David J. Elliott1* 1 Institute of Human Genetics, Newcastle University, Newcastle-upon-Tyne, United Kingdom, 2 Beatson Institute for Cancer Research, Glasgow, United Kingdom, 3 Institute of Cancer Sciences, University of Glasgow, Glasgow, United Kingdom, 4 Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, United States of America, 5 Northern Institute for Cancer Research, Newcastle University, Newcastle-upon-Tyne, United Kingdom, 6 Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle-upon-Tyne, United Kingdom Abstract Androgens drive the onset and progression of prostate cancer (PCa) by modulating androgen receptor (AR) transcriptional activity. Although several microarray-based studies have identified androgen-regulated genes, here we identify in-parallel global androgen-dependent changes in both gene and alternative mRNA isoform expression by exon-level analyses of the LNCaP transcriptome. While genome-wide gene expression changes correlated well with previously-published studies, we additionally uncovered a subset of 226 novel androgen-regulated genes. Gene expression pathway analysis of this subset revealed gene clusters associated with, and including the tyrosine kinase LYN, as well as components of the mTOR (mammalian target of rapamycin) pathway, which is commonly dysregulated in cancer. We also identified 1279 putative androgen-regulated alternative events, of which 325 (,25%) mapped to known alternative splicing events or alternative first/last exons. We selected 30 androgen-dependent alternative events for RT-PCR validation, including mRNAs derived from genes encoding tumour suppressors and cell cycle regulators. Of seven positively-validating events (,23%), five events involved transcripts derived from alternative promoters of known AR gene targets. In particular, we found a novel androgen-dependent mRNA isoform derived from an alternative internal promoter within the TSC2 tumour suppressor gene, which is predicted to encode a protein lacking an interaction domain required for mTOR inhibition. We confirmed that expression of this alternative TSC2 mRNA isoform was directly regulated by androgens, and chromatin immunoprecipitation indicated recruitment of AR to the alternative promoter region at early timepoints following androgen stimulation, which correlated with expression of alternative transcripts. Together, our data suggest that alternative mRNA isoform expression might mediate the cellular response to androgens, and may have roles in clinical PCa. Citation: Rajan P, Dalgliesh C, Carling PJ, Buist T, Zhang C, et al. (2011) Identification of Novel Androgen-Regulated Pathways and mRNA Isoforms through Genome-Wide Exon-Specific Profiling of the LNCaP Transcriptome. PLoS ONE 6(12): e29088. doi:10.1371/journal.pone.0029088 Editor: Franky L. Chan, The Chinese University of Hong Kong, Hong Kong Received March 12, 2011; Accepted November 21, 2011; Published December 14, 2011 Copyright: 2011 Rajan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by an Association for International Cancer Research project grant (06-705 to DJE and HYL), a Medical Research Council Clinical Research Training Fellowship (G0500482 to PR), a Royal College of Surgeons of England Surgical Research Fellowship (to PR), an Academy of Medical Sciences/ Wellcome Trust Starter Grant for Clinical Lecturers (to PR), a Wellcome Trust Project grant (080388 to DJE) and a National Institutes for Health grant (GM074688 to MQZ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: david.elliott@ncl.ac.uk . These authors contributed equally to this work. ¤a Current address: Howard Hughes Medical Institute, Laboratory of Molecular Neuro-Oncology, The Rockefeller University, New York, New York, United States of America ¤b Current address: Center for Systems Biology, The University of Texas at Dallas, Richardson, Texas, United States of America Introduction The growth, development and function of the prostate gland are dependent on androgens, which affect gene expression via the androgen receptor (AR), a steroid hormone receptor transcription factor. AR transcriptional activity also drives the onset and progression of prostate cancer (PCa) [1], the second leading cause of male cancer-related deaths with ,899,000 men diagnosed worldwide each year [2]. Cognate ligand-activated AR is thought to exert its transcriptional effects via direct or indirect binding to DNA sequences termed androgen response elements (AREs) [3,4]. Effective first and second line therapies for advanced PCa inhibit endogenous androgen production and AR function [5], highlight- ing the critical importance of AR signalling and downstream gene targets in PCa biology. Alternative mRNA isoforms with different exon contents can be generated through variable alternative splicing patterns, and selection of alternative transcriptional initiation and termination sites. Using these mechanisms, mRNAs with different exon combinations are transcribed from most (,70 to 90%) human genes [6,7,8], and can encode proteins with different sub-cellular localisations and functions. In addition to transcriptional effects, PLoS ONE | www.plosone.org 1 December 2011 | Volume 6 | Issue 12 | e29088