7392 Vol. 10, 7392–7401, November 1, 2004 Clinical Cancer Research Androgen Receptor Is Targeted to Distinct Subcellular Compartments in Response to Different Therapeutic Antiandrogens Hayley C. Whitaker,1 Sarah Hanrahan,3 informed sequential treatment regime may benefit prostate Nick Totty,3 Simon C. Gamble,1 cancer patients. The observed subnuclear and subcytoplas- Jonathan Waxman,1 Andrew C. B. Cato,4 mic associations of the AR suggest new areas of study to 2 1 investigate the role of the AR in the response and resistance Helen C. Hurst, and Charlotte L. Bevan of prostate cancer to antiandrogen therapy. 1Prostate Cancer Research Group and 2Cancer Research UK Molecular Oncology Unit, Department of Cancer Medicine, Faculty of Medicine, Imperial College London, London, United Kingdom; INTRODUCTION 3Protein Analysis, Cancer Research UK, London Research Institute, Prostate cancer is the most commonly diagnosed male London, United Kingdom; and 4Forschungszentrum Karlsruhe, cancer in the United States and the second leading cause of male Institute of Toxicology and Genetics, Karlsruhe, Germany cancer death (1). Prostate growth is initially androgen depend- ent; thus, treatment involves reducing circulating androgen lev- ABSTRACT els using leuteinizing hormone-releasing hormone analogs and opposing androgen action using antiandrogens. Little is known Purpose: Antiandrogens are routinely used in the treat- about how antiandrogens elicit their effects, although they act at ment of prostate cancer. Although they are known to pre- the level of the androgen receptor (AR), which mediates the vent activation of the androgen receptor (AR), little is effects of all androgens including the major circulating androgen known about the mechanisms involved. This report repre- testosterone and the more potent dihydrotestosterone (DHT). sents the first study of the localization of wild-type AR The AR is a ligand-dependent transcription factor, closely following expression at physiologic relevant levels in pros- related to the other steroid receptors, whose cellular localization tate cells and treatment with androgen and antiandrogens. patterns are diverse. Unliganded progesterone receptor is mainly Experimental Design: We have characterized a cellular cytoplasmic, whereas unliganded estrogen receptor is predom- model for prostate cancer using in situ cellular fractionation, inantly nuclear, but both shuttle between the cytoplasm and proteomics, and confocal microscopy and investigated the nucleus (2, 3). Conversely, unliganded glucocorticoid receptor effect of antiandrogens in clinical use on the subcellular resides entirely in the cytoplasm (4). There is controversy con- localization of the AR. cerning the localization of unliganded AR: different groups have Results: Different antiandrogens have diverse effects on reported predominantly cytoplasmic or nuclear localization (5– the subcellular localization of the AR. Treatment with an- 9). The current consensus is that unliganded AR resides largely drogen results in translocation from the cytoplasm to the in the cytoplasm, complexed with heat shock proteins (10). On nucleoplasm, whereas the antiandrogens hydroxyflutamide binding androgen, the AR is believed to dissociate from heat and bicalutamide lead to reversible association with the shock proteins, dimerize, translocate into the nucleus, and bind nuclear matrix. In contrast, treatment with the antiandro- to androgen response elements, from which it promotes the gen cyproterone acetate results in AR association with cy- transcription of androgen-regulated genes. toplasmic membranes and irreversible retention within the Antiandrogens bind to the AR but do not promote andro- cytoplasm. In addition, we demonstrate that AR transloca- gen-dependent gene transcription. It is unclear which steps in tion requires ATP and the cytoskeleton, regardless of ligand. the AR signaling pathway are blocked by antiandrogens, and it Conclusions: These results reveal that not all antian- was long thought that they simply competed with the ligand for drogens work via the same mechanism and suggest that an binding to the AR or prevented nuclear import of the receptor. Although these factors may contribute to the overall effect of the drugs, they do not account for all their actions. In some systems antiandrogens have been shown to promote AR nuclear import Received 2/27/04; revised 7/27/04; accepted 8/11/04. and even DNA binding while still inhibiting androgen-respon- Grant support: The Prostate Cancer Charity and Cancer Research sive gene transcription (5, 6, 11, 12). Using reporter assays, United Kingdom. several groups have shown partial AR activation in the presence The costs of publication of this article were defrayed in part by the of the therapeutic antiandrogens hydroxyflutamide (OHF) and payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to cyproterone acetate (CPA), thus these are termed partial antag- indicate this fact. onists. Bicalutamide (BIC), beginning to be widely used in Requests for reprints: Charlotte L. Bevan, Prostate Cancer Re- prostate cancer therapy, is thought to be a pure antagonist search Group, Department of Cancer Medicine, Faculty of Medicine, because no transactivation has yet been reported in its presence. Imperial College London, Du Cane Road, London W12 ONN, United Kingdom. Phone: 44-208-3833784; Fax: 44-208-3835830; E-mail: It is necessary to study how antiandrogens exert their [email protected]. effects to understand why hormone therapy for prostate cancer, ©2004 American Association for Cancer Research. while initially successful, inevitably fails. Investigations into the Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2004 American Association for Cancer Research. Clinical Cancer Research 7393 mechanism of action of antiandrogens in prostate cancer have with either 10Ϫ8 mol/L MB or 10Ϫ6 mol/L antiandrogen. Lu- been hampered by a lack of relevant cellular models for the ciferase activity was measured using the LucLite luciferase disease. Previous studies have used transient overexpression of assay kit (Packard Bioscience, San Diego, CA). Each treatment the AR (frequently tagged with other moieties, such as green was assayed in quadruplicate, and data are shown Ϯ SD. fluorescent protein) or the LNCaP cell line, which has a mutated In situ Cell Fractionation. In situ cell fractionation was AR that affects translocation in the presence of antiandrogens carried out immediately after incubation with ligand using a (13). Here, we use a PC3 prostate cancer cell line that has been method modified from Staufenbiel and Deppert (16). Briefly, stably transfected with wild-type AR (PC3wtAR) to express cells were incubated with the following buffers, and each was homogenous levels of AR protein similar to the endogenous retained for immunoblotting: (a) wash buffer, 10 mmol/L MES levels in target cells (14). Using a cell fractionation procedure, (pH 6.3), 10 mmol/L NaCl2, 1.5 mmol/L MgCl2, 10% glycerol, we have separated PC3wtAR cells into subcellular compart- and 5 ␮L/mL mammalian protease inhibitor mixture (Sigma); ments including the cytoplasm, nucleus, and nuclear matrix (b) cytoplasmic buffer, wash buffer plus 1% Nonidet P-40, 1 (NM) and demonstrate for the first time alternative subnuclear mmol/L EGTA, 5 mmol/L dithiothreitol (DTT) for 3 (C1) and and subcytoplasmic compartmentalization of wild-type AR after 27 (C2) minutes sequentially at 4°C; (c) DNase I buffer, wash treatment with androgens and antiandrogens in a prostate cancer buffer plus 50 ␮g/mL DNase I for 15 minutes at 37°C; (d) cell line. nuclear buffer, 4 mol/L NaCl2, 10 mmol/L MES (pH 6.2), 1.5 ␮ mmol/L MgCl2, 10% glycerol, and 5 L/mL mammalian pro- tease inhibitor mixture, 1 mmol/L EGTA, and 5 mmol/L DTT MATERIALS AND METHODS for 30 minutes at 4°C; (e) RNase A buffer, wash buffer plus 50 Cell Culture. PC3wtAR (14) and LNCaP cells (Ameri- ␮g/mL DNase I and 50 ␮g/mL RNase A for 30 minutes at 37°C; can Type Culture Collection, Manassas, VA) were cultured in (f) NM buffer, 50 mmol/L Tris (pH 9), KCl, 3% (v/v) Empigen RPMI 1640 (Sigma, St. Louis, MO) supplemented with 100 BB (Ellis & Everard Yorkshire, United Kingdom), 5 mmol/L units/mL penicillin, 0.1 mg/mL streptomycin, 2 mmol/L gluta- DTT, 1 mmol/L EGTA, 10% glycerol, and 5 ␮L/mL mamma- mine (Sigma), and 10% fetal bovine serum (Labtech Interna- lian protease inhibitor mixture for 1 hour at 4°C; and (g) urea tional, Sussex, United Kingdom). PC3wtAR medium was sup- buffer, 9 mol/L urea, 2% (w/v) CHAPS, 1% (w/v) DTT, 2% plemented with 4 ␮g/mL Geneticin (Life Technologies, Inc., (v/v) pharmalytes (Amersham Biosciences, Piscataway, NJ) for Rockville, MD). COS-1 cells were cultured in DMEM medium 5 minutes at room temperature with scraping. (Sigma) supplemented with penicillin, streptomycin, glutamine Immunoblot Analysis. Cell pellets were solubilized in and serum as above. Culture in androgen-free conditions was urea buffer to give whole cell lysates. For these and cellular carried out in “starvation media”: phenol red-free RPMI or fractions, protein concentration was determined using Bradford DMEM (Invitrogen, Carlsbad, CA) supplemented as above but reagent (Bio-Rad, Hercules, CA), and equal amounts of total with charcoal-stripped fetal bovine serum (Globepharm Surrey, protein were loaded. Samples were separated by SDS-PAGE United Kingdom). and transferred onto nitrocellulose membrane (Bio-Rad) before Before in situ cell fractionation, PC3wtAR and LNCaP probing for AR using either PG-21 antibody (Upstate Biotech- cells were grown to 60% confluence, washed three times in PBS nology, Lake Placid, NY) or AR441 (Dako Cytomation, Copen- (Sigma), and cultured in starvation medium for 24 hours. Cells hagen, Denmark). Control blots were probed with anti-actin were dosed with 10Ϫ8 mol/L of the DHT analog mibolerone (Sigma), anti-Hsp60 (Stressgen, San Diego, CA), anti-poly- (MB; Perkin-Elmer, Fremont, CA) or 10Ϫ6 mol/L antiandrogens (ADP-ribose) polymerase (PARP; Santa Cruz Biotechnology, [CPA (Sigma), OHF (Schering-Plough Hertfordshire, United Santa Cruz, CA), or anti-lamin B (Santa Cruz Biotechnology).