Androgen Receptor Promotes Ligand-Independent Prostate Cancer Progression through c-Myc Upregulation

Androgen Receptor Promotes Ligand-Independent Prostate Cancer Progression through c-Myc Upregulation Lina Gao1, Jacob Schwartzman1, Angela Gibbs1, Robert Lisac1, Richard Kleinschmidt1, Beth Wilmot2,3, Daniel Bottomly2,3, Ilsa Coleman4, Peter Nelson4, Shannon McWeeney2,3, Joshi Alumkal1* 1 Division of Hematology/Oncology, Knight Cancer Institute, Oregon Health and Science University, Portland, Oregon, United States of America, 2 Oregon Clinical and Translational Research Institute, Portland, Oregon, United States of America, 3 Department of Medical Informatics and Clinical Epidemiology; Division of Bioinformatics and Computational Biology; Oregon Health and Science University, Portland, Oregon, United States of America, 4 Fred Hutchinson Cancer Research Center, University of Washington, Seattle, Washington, United States of America Abstract The androgen receptor (AR) is the principal therapeutic target in prostate cancer. For the past 70 years, androgen deprivation therapy (ADT) has been the major therapeutic focus. However, some patients do not benefit, and those tumors that do initially respond to ADT eventually progress. One recently described mechanism of such an effect is growth and survival-promoting effects of the AR that are exerted independently of the AR ligands, testosterone and dihydrotestosterone. However, specific ligand-independent AR target genes that account for this effect were not well characterized. We show here that c-Myc, which is a key mediator of ligand-independent prostate cancer growth, is a key ligand-independent AR target gene. Using microarray analysis, we found that c-Myc and AR expression levels strongly correlated with each other in tumors from patients with castration-resistant prostate cancer (CRPC) progressing despite ADT. We confirmed that AR directly regulates c-Myc transcription in a ligand-independent manner, that AR and c-Myc suppression reduces ligand-independent prostate cancer cell growth, and that ectopic expression of c-Myc attenuates the anti-growth effects of AR suppression. Importantly, treatment with the bromodomain inhibitor JQ1 suppressed c-Myc function and suppressed ligand-independent prostate cancer cell survival. Our results define a new link between two critical proteins in prostate cancer – AR and c-Myc – and demonstrate the potential of AR and c-Myc-directed therapies to improve prostate cancer control. Citation: Gao L, Schwartzman J, Gibbs A, Lisac R, Kleinschmidt R, et al. (2013) Androgen Receptor Promotes Ligand-Independent Prostate Cancer Progression through c-Myc Upregulation. PLoS ONE 8(5): e63563. doi:10.1371/journal.pone.0063563 Editor: Natasha Kyprianou, University of Kentucky College of Medicine, United States of America Received October 30, 2012; Accepted April 2, 2013; Published May 21, 2013 Copyright: ß 2013 Gao 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 publication was made possible with support from the Oregon Clinical and Translational Research Institute, grant number KL2 RR024141 from the National Center for Research Resources and the National Center for Advancing Translational Sciences of the National Institutes of Health (JA). This work was also supported by the Pacific Northwest Prostate Cancer SPORE/National Cancer Institute (P50CA097186) (JA, PN, IC), the Department of Defense (PC093509) (PSN), a Flight Attendant Medical Research Institute Young Clinical Scientist Award (JA), a Wayne D. Kuni & Joan E. Kuni Foundation Kuni Scholar Award (JA), and a Prostate Cancer Foundation Young Investigator Award (JA). With special thanks to Platt Electric, Bruce Burns, and The Burns Family Fund of the Oregon Community Foundation for their philanthropic support of this work. 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: There are several possible explanations for AR-dependent mechanisms of progression despite the suppression or interference with androgen ligands. These include intratumoral androgen synthesis, the generation of constitutively active AR transcript variants, AR gene amplification, activating AR mutations, or activation of the AR by growth factors [7–16]. It is also now clear that the AR protein can promote the activation of AR ligandindependent pathways distinct from AR’s canonical ligandactivated pathways in CRPC [17]. However, critical downstream AR target genes of this type that account for AR dependent, ligand-independent prostate cancer cell survival have not been fully clarified. The study of such AR target genes and mechanisms by which AR regulates their expression will improve our understanding of castration-resistance and lead to the identification of key AR dependent proteins whose activity may control growth and survival of CRPC cells. Such targets and pathways would naturally become high priorities for drug development. Introduction Prostate cancer is the most common cancer in men in the United States with 241,740 new cases anticipated this year [1]. Despite screening and early treatment, prostate cancer commonly recurs, and 28,170 men are predicted to die from prostate cancer this year [1]. Nearly all of these prostate cancer deaths are attributable to metastatic, castration-resistant prostate cancer (CRPC) that has progressed despite androgen deprivation therapy (ADT) – the most common treatment for patients with recurrent or advanced prostate cancer. ADT works by lowering levels of the potent AR ligands testosterone and dihydrotestosterone (DHT) or interfering with binding of androgen ligands to the androgen receptor (AR) protein, the principal therapeutic target in prostate cancer [2]. Despite ADT, including novel and more potent treatments, all prostate cancers eventually progress [3,4]. At progression, the AR is ubiquitously expressed [5,6]. PLOS ONE | www.plosone.org 1 May 2013 | Volume 8 | Issue 5 | e63563 AR and c-Myc Promote Prostate Cancer Progression To understand genes that might account for that effect, we focused on c-Myc. This is because: 1) c-Myc overexpression promotes prostate cancer development [18]; 2) c-Myc is upregulated in androgen ligand-dependent prostate cancer and further upregulated in CRPC [19,20]; and 3) prior reports have demonstrated that c-Myc, like AR, contributes to ligand-independent prostate cancer cell growth [21]. Our review of prior data that localized AR binding sites throughout the genome by chromatin immunoprecipitation (ChIP) showed that the AR localizes to an enhancer element of the c-Myc gene [17]. However, it was unclear if c-Myc was a direct AR target gene and whether androgen ligands were necessary for AR regulation of c-Myc expression. We de (...truncated)