Published OnlineFirst February 15, 2011; DOI: 10.1158/0008-5472.CAN-10-2512 Cancer Tumor and Stem Cell Biology Research Identification of a Clinically Relevant Androgen-Dependent Gene Signature in Prostate Cancer Hannelore V. Heemers1, Lucy J. Schmidt3, Zhifu Sun4, Kevin M. Regan3, S. Keith Anderson5, Kelly Duncan1, Dan Wang2, Song Liu2, Karla V. Ballman5, and Donald J. Tindall3 Abstract The androgen receptor (AR) is the principal target for treatment of non–organ-confined prostate cancer (PCa). Androgen deprivation therapies (ADT) directed against the AR ligand–binding domain do not fully inhibit androgen-dependent signaling critical for PCa progression. Thus, information that could direct the development of more effective ADTs is desired. Systems and bioinformatics approaches suggest that considerable variation exists in the mechanisms by which AR regulates expression of effector genes, pointing to a role for secondary transcription factors. A combination of microarray and in silico analyses led us to identify a 158-gene signature that relies on AR along with the transcription factor SRF (serum response factor), representing less than 6% of androgen-dependent genes. This AR-SRF signature is sufficient to distinguish microdissected benign and malignant prostate samples, and it correlates with the presence of aggressive disease and poor outcome. The AR- SRF signature described here associates more strongly with biochemical failure than other AR target gene signatures of similar size. Furthermore, it is enriched in malignant versus benign prostate tissues, compared with other signatures. To our knowledge, this profile represents the first demonstration of a distinct mechanism of androgen action with clinical relevance in PCa, offering a possible rationale to develop novel and more effective forms of ADT. Cancer Res; 71(5); 1–11. Ó2011 AACR. Introduction ADT prevents tumor growth and leads to a favorable clinical response. Unfortunately, ADT does not eradicate disease and Prostate cancer (PCa) remains the most frequently diag- eventually PCa recurs as castration-recurrent PCa (CRPC), nosed cancer of an internal organ and the second leading which is invariably lethal. Intriguingly, the emergence of cause of cancer-related death in men (1). One in 6 American CRPC is due, at least in part, to inappropriate activation of men will be confronted with PCa, which makes this disease a the AR (4–7). Recent therapeutic approaches that are tai- significant health problem. lored specifically to target aberrant AR action in CRPC lead Localized PCa is treated with surgical or radiation thera- to antitumor activity in a substantial subset of patients (8– pies that have a curative intent (2). For patients with locally 10). However, these effects are partial and temporary, which advanced PCa, those with metastatic disease, or whose indicates that AR activity is not inhibited fully by current cancer recurs after initial treatment, treatment options ADTs (11, 12). Developing more effective means to interfere are limited to prevent disease progression. As PCa progres- with AR signaling requires an in-depth understanding of the sion depends on androgen signaling, the androgen receptor molecular mechanism(s) by which AR governs clinically (AR) is the principal target for treating non–organ-confined relevant events in PCa. disease. Traditional androgen deprivation therapy (ADT) The AR is a ligand-dependent transcription factor belong- interferes with the systemic production of androgens and/ ing to the nuclear receptor superfamily. On androgen binding, or involves administration of antiandrogens (3). Initially, the AR translocates from the cytoplasm to the nucleus, binds as a dimer to "androgen response elements" (ARE) in the regulatory regions of target genes, and recruits a productive Authors' Affiliations: Departments of 1Urology and 2Biostatistics, Roswell transcriptional complex (reviewed in ref. 13). Considerable Park Cancer Institute, Buffalo, New York and 3Department of Urology efforts have been directed toward identifying AR-dependent Research, 4Division of Biomedical Statistics and Informatics, and 5Cancer genes which contribute to PCa progression. Large-scale gene Center Statistics, Mayo Clinic, Rochester, Minnesota expression profiling studies in model systems have identified Note: Supplementary data for this article are available at Cancer Research dozens of androgen-regulated genes (14–19). Many of these Online (http://cancerres.aacrjournals.org/). have been proposed to play a role in differentiated cell Corresponding Author: Hannelore V. Heemers, Roswell Park Cancer Institute, Department of Urology, C&V Annex Room 142, Elm & Carlton function, cell proliferation, and cell survival (20). Nonetheless, Streets, Buffalo, NY 14263. Phone: 716-8457644; Fax: 716-8454165; E- correlation of androgen-responsive mRNA expression profiles mail: [email protected] obtained from in vitro model systems with transcriptomes doi: 10.1158/0008-5472.CAN-10-2512 derived from clinical PCa specimens has been challenging. To Ó2011 American Association for Cancer Research. date, specific genes and transcriptional programs that are www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2011 American Association for Cancer Research. Published OnlineFirst February 15, 2011; DOI: 10.1158/0008-5472.CAN-10-2512 Heemers et al. critical for PCa cell proliferation and metastasis and that are con) or a custom-made control SMARTpool targeting lucifer- regulated by androgens remain largely elusive. These difficul- ase (Luc condition) using Lipofectamine 2000 (Invitrogen) ties may reflect, in part, that attempts at classification have following the manufacturer's instructions. Forty-two hours encompassed all androgen-dependent gene expression, which after transfection, cells were treated with 5 nmol/L R1881 or assumed little or no variation in the manner by which AR ethanol vehicle. Three biological triplicates were included per controls transcription of target genes. Recent systems treatment group. Forty-eight hours later, cells were harvested approaches, however, suggest variability in the composition in TRIzol reagent (Invitrogen). of the AR transcriptional complex at regulatory sites in effector genes, indicate that not all androgen-dependent genes Cell line RNA preparation and microarray analysis are subject to direct ARE-driven mechanisms of regulation RNA was isolated from cells with TRIzol (Invitrogen), and provide evidence that AR signaling in PCa cells relies on purified on RNeasy columns (Qiagen), and checked for integ- secondary transcription factors (TF; refs. 21–25). Some TFs rity by Agilent testing. cDNA was generated and hybridized to interact directly with the AR to affect its ability to bind to AREs Human Genome U133 Plus 2.0 Arrays (Affymetrix) according and compete for coregulators or to cooperate in the tran- to the manufacturer's instructions at the Mayo Clinic scription of AR target genes (13). Other TFs mediate critical Advanced Genomics Technology Microarray Shared Resource effects on PCa cells by mechanisms that do not rely on core facility. The microarray data sets have been deposited in physical interaction with the AR but involve androgen regula- Gene Expression Omnibus under accession number tion of their activities (26, 27). These "indirect" mechanisms of GSE22606. A detailed description of the microarray data androgen action convey androgen responsiveness to target analysis can be found in Supplementary Data. genes that do not contain AREs and can induce coordinated responses of genes and/or cells. Identification of TFs whose Patient material and microarray data analysis transcriptional program contributes to the development of A detailed description of the patient materials and micro- aggressive disease may, therefore, open novel avenues to array data set analysis is included in Supplementary Data. target clinically relevant androgen signaling in PCa. We recently identified a novel indirect mechanism of Ingenuity Pathway Analysis androgen action in which effects of androgens on PCa cells Ingenuity Pathway Analysis (IPA) was done using the 158 are mediated by serum response factor (SRF; ref. 28), which is SRF-dependent androgen-responsive gene signature as a focus a MADS box-containing TF that was originally identified by gene set and Ingenuity curated knowledge base as a reference. its ability to convey the effects of serum to immediate early response genes (29). Since then, SRF has also been shown to Real-time reverse transcriptase PCR control expression of genes involved in the organization of the cDNA was prepared and real-time reverse transcriptase cytoskeleton and to be critical for embryonic development, (RT)-PCR was done as before (28). Primers targeting human experimental metastasis, and angiogenesis (30–33). Androgen FHL2, SRF, prostate-specific antigen (PSA), AR, and glycer- exposure induces expression of four-and-a-half-LIM domain aldehyde-3-phosphate dehydrogenase (GAPDH) have been protein 2 (FHL2) in a manner that is AR-dependent but described (28). Primer sequences used to analyze SRF-depen- independent of any AREs in the FHL2 gene. Instead, andro- dent gene expression are listed in Supplementary Table S1. gens stimulate expression of FHL2 through action of SRF on its consensus binding site (CArG box) in the FHL2 promoter Results (28). In view of these cellular and physiologic roles, it is tempting to speculate that androgen control over the SRF Identification of an SRF-dependent