Published online 24 October 2013 Nucleic Acids Research, 2014, Vol. 42, No. 2 999–1015 doi:10.1093/nar/gkt921 REST mediates androgen receptor actions on gene repression and predicts early recurrence of prostate cancer Charlotte Svensson1, Jens Ceder2, Diego Iglesias-Gato1, Yin-Choy Chuan1, See Tong Pang3, Anders Bjartell2, Roxana Merino Martinez4, Laura Bott5, Leszek Helczynski6, David Ulmert2,7, Yuzhuo Wang8, Yuanjie Niu9, Colin Collins8 and Amilcar Flores-Morales1,* 1Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, Downloaded from DK-2200 Copenhagen, Denmark, 2Division of Urological Cancers, Department of Clinical Sciences, Ska˚ ne University Hospital, Lund University, 20502 Malmo¨ , Sweden, 3Department of Urology, Chang Gung Memorial Hospital, Tao-Yuan 33305, Taiwan, R.O.C., 4Department of Epidemiology, Karolinska Institutet, 171 77 Stockholm, Sweden, 5Department of Cell and Molecular Biology, Karolinska Institute, 171 77 Stockholm, Sweden, 6Regional Laboratories Region Ska˚ ne, Clinical Pathology, 205 80 Malmo¨ , Sweden, 7Department of Surgery (Urology), Memorial Sloan-Kettering Cancer Center, New York, NY 100 65, USA, 8Vancouver Prostate http://nar.oxfordjournals.org/ Centre and The Department of Urologic Sciences, University of British Columbia, Vancouver, BC Canada V6H 3Z6 and 9Tianjin Institute of Urology, Tianjin Medical University, Tianjin 300 211, China Received December 19, 2012; Accepted September 20, 2013 ABSTRACT that has previously been implicated in the growth at University of British Columbia on August 12, 2014 The androgen receptor (AR) is a key regulator of NE-like castration-resistant tumors. The of prostate tumorgenesis through actions that are analysis of prostate cancer tissue microarrays not fully understood. We identified the repressor revealed that tumors with reduced expression of element (RE)-1 silencing transcription factor REST have higher probability of early recurrence, (REST) as a mediator of AR actions on gene repres- independently of their Gleason score. The demon- sion. Chromatin immunoprecipitation showed stration that REST modulates AR actions in prostate that AR binds chromatin regions containing well- epithelia and that REST expression is negatively characterized cis-elements known to mediate correlated with disease recurrence after prostatec- REST transcriptional repression, while cell imaging tomy, invite a deeper characterization of its role in studies confirmed that REST and AR closely co- prostate carcinogenesis. localize in vivo. Androgen-induced gene repression also involves modulation of REST protein turnover INTRODUCTION through actions on the ubiquitin ligase b-TRCP. Androgen deprivation or AR blockage with inhibitor The androgen receptor (AR) plays a key role in prostate cancer (PCa) development at all stages (1–3). The import- MDV3100 (Enzalutamide) leads to neuroendocrine ance of the AR in PCa progression seems at odds with its (NE) differentiation, a phenomenon that is function in controlling differentiation of the normal mimicked by REST inactivation. Gene expression prostate epithelial cells and the fact that PCa incidence profiling revealed that REST not only acts to increases with aging, when the levels of circulating andro- repress neuronal genes but also genes involved in gens are reduced (4,5). In the normal prostate gland, the cell cycle progression, including Aurora Kinase A, luminal epithelial cells express high AR levels. These AR+ *To whom correspondence should be addressed. Tel: +45 35325021; Fax: +45 35325001; Email: amilcar.fl[email protected] Present address: Amilcar Flores-Morales, Department of Disease of Biology, Novo Nordisk Foundation Center for Protein Research, Blegdamsvej 3B, 2200 Copenhagen, Denmark. ß The Author(s) 2013. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] 1000 Nucleic Acids Research, 2014, Vol. 42, No. 2 cells are terminally differentiated and quiescent (6,7). University Hospital, Malmo¨, Sweden, were used to con- However, there is a population of AR+cancer-initiating struct a tissue microarray (TMA), with duplicate tissue cells in the prostate, suggesting a role for AR in cancer cores (1.0 mm in diameter), as previously described (17). development that is supported by the effectiveness of A senior National Board–certified pathologist castration therapy (8). (L. Helczynski) examined the hematoxylin- and eosin- The AR is a ligand-dependent transcription factor stained sections for Gleason grading. The study was belonging to the nuclear hormone receptor superfamily. approved by the ethics committee of Lund University, The paradigm of gene regulation by androgens indicates Sweden, and the Helsinki Declaration of Human Rights that on dihydrotestosterone (DHT) binding, the AR trans- was strictly observed. locates to the nucleus where it binds androgen responsive elements (ARE). This binding is followed by the recruit- Cell culture ment of co-regulators and the RNA polymerase II The human PCa cell lines LNCaP and CWR22RV1 were complex, which leads to chromatin modifications and obtained from the American Tissue Culture Collection altered transcription (9,10). The recent application of and grown in RPMI-1640, supplemented with L-glutamine chromatin immunoprecipitation (ChIP) combined with and 10% fetal bovine serum. For the experiments Downloaded from next-generation sequencing have altered our understand- involving androgen treatment, the cells were cultured in ing of the AR mechanisms of action (11–13). It is now clear phenol red-free medium, supplemented with 5% dextran- that most of the AR binding to enhancer or promoter coated charcoal-treated fetal bovine serum (HyClone Inc., regions does not result in an activation of transcription in Logan, UT, USA) for 48 h before exposure to the same proximal genes (14). Transcriptional induction by the AR medium containing 0.1% ethanol (control) or 10 nM DHT http://nar.oxfordjournals.org/ requires its binding to chromatin regions poised for tran- for the indicated time. scriptional activation. Directing the AR to such regions may involve interaction with other DNA-bound transcrip- Chromatin immunoprecipitation tion factors in gene enhancers (11). The fact that there are many cryptic AR binding sites in the genome that could be ChIP was performed using the anti-AR antibody (PG-21) activated if the appropriate chromatin remodeling occurs and the anti-REST antibody (07–579), obtained from has significant implications, as PCa progression is Millipore (Billerica, MA, USA) essentially as described accompanied by genetic instability and chromatin re- (14,18). Microarray analysis was performed using the Human HG18 385K RefSeq promoter tiling array accord- modeling. This may alter the AR transcriptional program at University of British Columbia on August 12, 2014 in tumor cells and help to explain the duality of the AR role ing to the protocols provided by NimbleGen (Roche, as a pro-differentiating factor in normal epithelial cells and Mannheim, Germany). Data analysis was performed a tumor promoter in cancer cells (14–16). using the NimbleGen proprietary software, as described In this study, we analyzed the sequence content of earlier (14). The Benjamini method was used to control the human gene promoter regions occupied by AR on DHT false discovery rate (FDR) at an FDR (a) level of 0.1. stimulation to identify enrichment in DNA elements cor- Regions with at least four consecutive probes passing responding to known transcription factor binding sites. A the statistical threshold were considered as a peak, and novel mechanism for AR control of transcription and then peaks were extended until the first insignificant cell differentiation, mediated by repressor element (RE)-1 probe was encountered. Overlapping peaks were merged. silencing transcription factor (REST), was identified. The AR-occupied regions (ARORs) were defined as the peaks expression of REST in localized tumors was also analyzed. present in the two independently replicated experiments (Supplementary Table S1). Polymerase chain reaction (PCR) analysis of ChIP products was performed as MATERIALS AND METHODS described (18). Materials Motif searches The anti-AR antibodies N20 (SC-816), 441 (SC-7305) To identify enriched transcription factor binding motifs and PG21 (Millipore), anti-b-actin (SC130301), anti- within the ARORs, we used the TFBS computational REST (SC-25398, HPA006079, Ab21635, 07-579), anti- framework for transcription factor binding site analysis chromogranin A (Ab-15160), b-TRCP (SC-33213), (19,20). All AROR identified by the ChIP microarray fluorescein-conjugated anti-mouse IgG and anti-rabbit analysis (average size of 500 bases) were included in the IgG were obtained from Santa Cruz Biotechnologies. analysis. A reference set was generated from a group of DHT and R1881 were obtained from Sigma-Aldrich randomly selected sequences corresponding to the (St. Louis, MO, USA). The control siRNA and those tar- genomic regions represented in the arrays matching the geting the AR, REST and b-TRCP were purchased from ARORs in average size and GC content. The GC Qiagen (Valencia, CA, USA). siRNAs with a modified content was 52.9% for the control