Molecular and Cellular Pathobiology FoxA 1 Speci fi es Unique Androgen and Glucocorticoid Receptor Binding Events in Prostate Cancer Cells

The forkhead protein FoxA1 has functions other than a pioneer factor, in that its depletion brings about a significant redistribution in the androgen receptor (AR) and glucocorticoid receptor (GR) cistromes. In this study, we found a novel function for FoxA1 in defining the cell-type specificity of ARandGR-binding events in a distinct fashion, namely, for AR in LNCaP-1F5 cells and for GR in VCaP cells. We also found different, cell-type and receptor-specific compilations of cis-elements enriched adjacent to the ARand GR-binding sites. The AR pathway is central in prostate cancer biology, but the role of GR is poorly known. We find that AR and GR cistromes and transcription programs exhibit significant overlap, and GR regulates a large number of genes considered to be AR pathway-specific. This raises questions about the role of GR in maintaining the AR pathway under androgen-deprived conditions in castration-resistant prostate cancer patients. However, in the presence of androgen, ligand-occupied GR acts as a partial antiandrogen and attenuates the AR-dependent transcription program. Cancer Res; 73(5); 1570–80. 2012 AACR. Introduction The majority of prostate cancers are initially androgendependent, and the first-line treatment is androgen deprivation (1, 2). Administration of nonsteroidal antiandrogens, such as bicalutamideandflutamide, is commonlyused inprostatecancer therapy (3). Bicalutamide was thought to be a pure antiandrogen (4) but has androgen agonistic activity in the presence of high cellular androgen receptor (AR) content (5). Cyproterone acetate (CPA)wasoneof thefirst antiandrogensused forprostate cancer treatment (6) but also has androgen-agonistic actions (7). Mifepristone (RU486) is an antiprogestin and antiglucocorticoid, but has androgenic/antiandrogenic properties as well (8, 9). All these compounds antagonize AR function by competing with testosterone (T) or dihydrotestosterone (DHT) for binding to the ligand-binding domain (LBD) of AR. Recent reports have delineated genome-wide AR-binding sites (ARB) and androgen target genes in LNCaP cells, or clones derived from it, using chromatin immunoprecipitation (ChIP) combined with microarray (ChIP-on-chip) or massively parallel sequencing (ChIP-seq; refs. 10–15). Only DHT or the synthetic androgen R1881 has been used as ligands, and information on other compounds such as those with androgen agonistic/antagonistic properties is lacking. Likewise, there is paucity of information on the mechanisms that underlie celltype specificity in AR cistromes and androgen-regulated transcription programs among prostate cancer cells. The glucocorticoid receptor (GR) plays an important role in inflammation and cancer progression; dexamethasone (Dex) has been used in treatment of castration-resistant prostate cancer (16); and GR signaling exhibits tumor suppressor activity in prostate cancer cells (17, 18). AR and GR can bind to the same androgen/glucocorticoid response element (ARE/ GRE) when examined by using naked DNA in vitro or transient expression conditions (19), or even on chromatin (12, 20), but there is very little information on the factors that ensure receptor specificity in a genuine chromatin environment. Here, we have addressed several outstanding issues in androgen action biology by examining AR and GR cistromes and transcription programs in two androgen-responsive prostate cancer cell lines, the LNCaP-1F5 and VCaP cells. The specific questions included the role of the ligand in guiding AR binding to chromatin sites, mechanisms that define ARbinding events in a cell-type specific fashion, and the interplay and cross-talk between ARand GR-binding events and transcription programs in prostate cancer cells. Materials and Methods Cell culture LNCaP-1F5 cells, engineered to express rat GR (21), were obtained from Dr. Jan Trapman (Erasmus Medical Center, Rotterdam, the Netherlands) and grown in RPMI-1640 supplemented with 10% FBSwith antibiotics. The cells were tested on a regular basis for AR and GR levels by immunoblotting as well as expression of androgen-regulated transcripts, such as PSA Authors' Affiliations: Institute of Biomedicine and Research Programs Unit, Genome-Scale Biology, Biomedicum Helsinki, University of Helsinki; and Department of Clinical Chemistry, Helsinki University Central Hospital, Helsinki, Finland Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). CorrespondingAuthor:Olli A. J€ anne, InstituteofBiomedicine, Physiology, Biomedicum Helsinki, University of Helsinki, P.O. Box 63, FI-00014 Helsinki, Finland. Phone: 358-9-19125040; Fax: 358-9-19125047 E-mail: olli.janne@helsinki.fi doi: 10.1158/0008-5472.CAN-12-235