14-3-3η Amplifies Androgen Receptor Actions in Prostate Cancer

Purpose: Androgen receptor abundance and androgen receptor–regulated gene exAuth Repr and L Com Medi Rosw Buffa Dep Atlan Rece 12/8/ Gran HD-1 State 009 a throu Coop ww pression in castration-recurrent prostate cancer are indicative of androgen receptor activation in the absence of testicular androgen. Androgen receptor transactivation of target genes in castration-recurrent prostate cancer occurs in part through mitogen signaling that amplifies the actions of androgen receptor and its coregulators. Herein we report on the role of 14-3-3η in androgen receptor action. Experimental Design and Results: Androgen receptor and 14-3-3η colocalized in COS cell nuclei with and without androgen, and 14-3-3η promoted androgen receptor nuclear localization in the absence of androgen. 14-3-3η interacted with androgen receptor in cell-free binding and coimmunoprecipitation assays. In the recurrent human prostate cancer cell line, CWR-R1, native endogenous androgen receptor transcriptional activation was stimulated by 14-3-3η at low dihydrotestosterone concentrations and was increased by epidermal growth factor. Moreover, the dihydrotestosteroneand epidermal growth factor–dependent increase in androgen receptor transactivation was inhibited by a dominant negative 14-3-3η. In the CWR22 prostate cancer xenograft model, 14-3-3η expression was increased by androgen, suggesting a feed-forward mechanism that potentiates both 14-3-3η and androgen receptor actions. 14-3-3η mRNA and protein decreased following castration of tumor-bearing mice and increased in tumors of castrate mice after treatment with testosterone. CWR22 tumors that recurred 5 months after castration contained 14-3-3η levels similar to the androgenstimulated tumors removed before castration. In a human prostate tissue microarray of clinical specimens, 14-3-3η localized with androgen receptor in nuclei, and the similar amounts expressed in castration-recurrent prostate cancer, androgen-stimulated prostate cancer, and benign prostatic hyperplasia were consistent with androgen receptor activation in recurrent prostate cancer. Conclusion: 14-3-3η enhances androgenand mitogen-induced androgen receptor transcriptional activity in castration-recurrent prostate cancer. (Clin Cancer Res 2009;15(24):7571–81) The androgenic steroid hormones, testosterone and its more active metabolite dihydrotestosterone, maintain and stimulate the growth of prostate cancer cells. Removal of the testicular source of circulating androgens by medical or surgical castraors' Affiliations: Departments of Pediatrics (Laboratories for oductive Biology), Surgery (Division of Urology) and Pathology aboratory Medicine, Biochemistry and Biophysics, and Lineberger prehensive Cancer Center, University of North Carolina School of cine, Chapel Hill, North Carolina; Department of Urologic Oncology, ell Park Cancer Institute, and Department of Urology, University at lo School of Medicine and Biotechnology, Buffalo, New York; and artment of Pharmacology, Emory University School of Medicine, ta, Georgia ived 8/20/08; revised 3/19/09; accepted 4/13/09; published OnlineFirst 09. t support: NIH Grants HD-07315 (to M.A. Titus), HD-04466 (F.S. French), 6910 (E.M.Wilson), CA-77739 (F.S. French, J.L.Mohler, E.M.Wilson);United s Army Medical Research and Material Command Grants DAMD17-00-1nd DAMD17-02-1-0110 (C.W. Gregory, E.M. Wilson) and NICHD/NIH gh cooperative agreement U54-HD35041 as part of the Specialized erative Centers Program in Reproduction and Infertility Research. 7571 w.aacrjournals.org Cancer Re on O clincancerres.aacrjournals.org Downloaded from tion causes regression of androgen-dependent prostate cancer (1). However, androgen deprivation therapy is only palliative because prostate cancer recurs (referred to as castrationrecurrent prostate cancer) and almost always causes death. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Current address for C.W. Gregory: Clinsys Clinical Research, 5840 Colonnade Center Drive, Suite 501, Raleigh, NC 27615. Current address for R.R. Subramanian: Pfizer Target Biology, Pfizer Research Technology Center, Cambridge MA 02139. Requests for reprints: Frank S. French, Department of Pediatrics (Laboratories for Reproductive Biology), CB#7500, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7500. Phone: 919-966-0930; Fax: 919-966-2203; E-mail: [email protected] and Mark A. Titus, Department of Urologic Oncology, Roswell Park Cancer Institute; Buffalo, NY 14263. Phone: 716-845-4876; Fax: 716-845-4165; E-mail:[email protected]. F 2009 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-08-1976 Clin Cancer Res 2009;15(24) December 15, 2009 search. ctober 16, 2017. © 2009 American Association for Translational Relevance We show that 14-3-3η is an androgen receptor coactivator in postcastration recurrent prostate cancer (CR-CaP) through its direct interaction with androgen receptor and by augmenting mitogen effects on androgen receptor transcriptional activity. The use of androgen receptor siRNA technology in the experimental setting as well as in recent phase I clinical trials with a CYP17 inhibitor of androgen production provides strong evidence that androgen receptor action is central to the growth of CR-CaP. Our studies identify a new potential target to inhibit CR-CaP growth. We show that 14-3-3η causes androgen receptor to be nuclear in the absence of androgen, whereas without 14-3-3η androgen receptor is cytoplasmic. We also show that androgen receptor signaling increases the production of 14-3-3η through the synthesis of new mRNA and that 143-3η levels remain elevated together with androgen receptor in CR-CaP. Studies described in this report provide a potential new target for combination therapy to block the androgen receptor–driven growth of CR-CaP. Human Cancer Biology Published OnlineFirst December 8, 2009; DOI: 10.1158/1078-0432.CCR-08-1976 The expression of androgen receptor (2–6) and androgenregulated genes (7, 8) in castration-recurrent cancer implicates androgen receptor signaling in recurrent growth despite low levels of circulating androgen. Potential mechanisms include androgen receptor gene amplification (9), gain of function mutations in the androgen receptor gene resulting in promiscuous ligand binding (refs. 6, 10, see also www.androgendb.mcgill. ca), coactivator overexpression (11), intracrine production of testosterone and dihydrotestosterone (12, 13), and enhanced activation of androgen receptor through mitogen signaling (14–17). The 14-3-3 family of homodimeric or heterodimeric α-helical proteins interact with a variety of signaling proteins including kinases, phosphatases, transcription factors, and nonkinase receptors (18). 14-3-3 proteins were the first to be identified as phosphoserine/threonine motif binding proteins. Three recognition motifs for 14-3-3 have been identified, but 14-3-3 target proteins do not always contain sequences that conform to these motifs or require phosphorylation for interaction with 14-3-3 (18, 19). Possible modes of action of 14-3-3 on target proteins include directed conformational change, modification of nuclear/ cytoplasmic localization, physical occlusion of sequencespecific interactive regions, and scaffolding (20, 21). 14-3-3 binding often leads to client protein stabilization or altered subcellular localization that in many cases alters protein function. Some 14-3-3 isoforms have been directly associated with tumorigenesis and may serve as targets for cancer therapy (22). CWR22 is an androgen-dependent human prostate cancer xenograft model propagated in male nude mice (23). After castration of the tumor-bearing male host, prostate-specific antigen protein and mRNA decrease rapidly and CWR22 xenograft tumors regress in size (7, 24) but within 5 months undergo recurrent growth in the absence of testicular androgens, a progression pattern that resembles the recurrence of prostate cancer in 7572 Clin Cancer Res 2009;15(24) December 15, 2009 Cancer Rese on Oc clincancerres.aacrjournals.org Downloaded from humans but in a shorter timeframe (23). Castration-recurrent CWR22 tumors remain androgen responsive, and an androgen-sensitive cell line, CWR-R1 (25), was developed from a recurrent CWR22 xenograft tumor. Herein we show that 14-3-3η is a human androgen receptor– binding protein in CWR-R1 cells (25) and that 14-3-3η binding to androgen receptor is associated with increased androgen receptor transcriptional activity that is enhanced by epidermal growth factor (EGF) and dihydrotestosterone. We found that the expression of 14-3-3η was androgen dependent in the androgen-dependent CWR22 xenograft tumor and decreased after castration but reappeared in the castration-recurrent CWR22 tumor despite the absence of circulating testicular androgen. In addition, clinical samples of castration-recurrent cancer revealed amounts of 14-3-3η similar to the amounts expressed in androgen-stimulated benign prostate and androgenstimulated prostate cancer. The results indicate that androgen receptor regulates the expression of 14-3-3η and that androgen receptor regulation is active in castration-recurrent prostate cancer. 14-3-3η localizes with androgen receptor in nuclei and provides a link between EGF signaling and androgen receptor transcriptional activation in prostate cancer. Materials and Methods Plasmid construction. Full-length 14-3-3η (18) was excised from the vector pPCR-Script Amp 14-3-3η using EcoR1 and Sac1. The excised DNA was blunt-ended with Klenow enzyme (Life Technologies, Inc.) and ligated into the blunt-ended BamH1 site of pSG5 (Stratagene). Sense and antisense constructs were verified by automatic sequencing using a Perkin-Elmer Corp. Model 377 DNA sequencer. A dominant negative double arginine mutant of human 14-3-3η cDNA (pcDNA3.1-myc-14-3-3η-R56, 60A) was obtained from Andrey S. Shaw, Washington University, St. Louis, MO. Solid-phase binding assays. Purified full-length human hexahistidinetagged 14-3-3η or 14-3-3ζ was incubated with Ni+2-charged Sepharose 6B beads (Novagen) for 1 h at 4°C. Radiolabeled androgen receptor was generated using the TNT in vitro transcription-translation system (Promega). The full-length DNA template pSG5-AR was incubated with TNT rabbit reticulocyte lysates in the presence of S-methionine. For the binding assays (26), the immobilized 14-3-3η or 14-3-3ζ protein (1 μg each) was mixed with S-labeled androgen receptor in Nonidet P-40 buffer [1% Nonidet P-40, 137 mmol/L NaCl, 1 mmol/L MgCl2, and 40 mmol/L Tris-HCl (pH 8.0)] for 1 h at 4°C with rotation. 14-33η or 14-3-3ζ complexes were washed three times with Nonidet P-40 buffer and three times with radioimmune precipitation assay wash (1% Nonidet P-40, 137 mmol/L NaCl, 0.5% sodium deoxycholate, 0.1% SDS, and 20 mmol/L Tris-HCL (pH 8.0)], and boiled 5 min in 2× SDS sample buffer before resolution by SDS-PAGE (12%). Gels were dried and autoradiography was done with BioMax film (Eastman Kodak Co.) at -80°C. Image analysis was carried out using the Personal Densitometer SI (Molecular Dynamics). Immunoprecipitation and immunoblot analysis. Protein lysates were prepared from COS cells grown on 10-cm culture dishes and cotransfected with sense or antisense 14-3-3η and human androgen receptor expression vectors as described (27). COS cells were derived from CV1 cells by transformation with replication origin defective SV40 virus that codes for large T antigen. Thus, expression vectors containing the SV40 origin of replication, when transiently transfected into COS cells, are replicated to multiple copies/cell. COS cell lysates were precleared with mouse IgG and protein A-agarose. To examine androgen receptor/ 14-3-3η complex formation in COS cell and CWR-R1 cell lysates, anti– 14-3-3 antibody that recognizes η and γ isoforms (Santa Cruz Biotechnology Inc.; sc-731) or anti-actin antibody (Research Diagnostic Inc.) www.aacrjournals.org arch. tober 16, 2017. © 2009 American Association for 14-3-3η and Androgen Receptor in Prostate Cancer Published OnlineFirst December 8, 2009; DOI: 10.1158/1078-0432.CCR-08-1976 were incubated with rotation for 1 h followed by the addition of protein A-agarose with constant rotation at 4°C overnight. Immunoblot analysis was done using androgen receptor monoclonal antibody (Biogenex) at 1:5,000 dilution and secondary antibody (horseradish peroxidase–conjugated anti-mouse, Promega Corporation) at 1:10,000 dilution. Antigen-antibody complexes were detected using enhanced chemiluminescence (DuPont, NEN Research Products). Prostate cancer specimens were pulverized in liquid nitrogen and mixed with 0.5 mL of radioimmunoprecipitation assay buffer containing Complete protease inhibitors (Roche; ref. 11). Tissue was homogenized for 30 s on ice, incubated for 45 min on ice, and centrifuged at 10,000× g for 20 min twice at 4°C to remove nuclei and insoluble material. Proteins were separated by electrophoresis on 10% acrylamide gels, electroblotted, and immunoblot analysis was done as described above using 14-3-3η antibody specific for the η isoform (Santa Cruz