[CANCER RESEARCH 63, 4903– 4913, August 15, 2003] Specificity of Cyclin D1 for Androgen Receptor Regulation 1 Christin E. Petre-Draviam, Stephen L. Cook, Craig J. Burd, Thomas W. Marshall, Yelena B. Wetherill, and Karen E. Knudsen 2 Department of Cell Biology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0521 ABSTRACT Androgen receptor (AR) activity is required for prostate growth, dif- ferentiation, and secretion. Deregulation of AR activity results in inap- propriate mitogenic signaling and is thought to contribute both to the initiation and progression of prostate cancers. Cyclin D1 functions as a strong AR corepressor by directly interacting with and inhibiting receptor activity. However, the extent to which cyclin D1 functions to inhibit AR activity under conditions associated with cancer progression has not been determined. We now demonstrate that cyclin D1 action is conserved in multiple tumor cell backgrounds, inhibiting AR-dependent gene activa- tion in breast, bladder, and androgen-independent prostatic adenocarci- noma cell lines. In androgen-dependent prostatic adenocarcinomas, cyclin D1 effectively muted androgen-stimulated target gene expression in a manner analogous to dominant negative ARs. The ability of cyclin D1 to inhibit AR activity was conserved with regard to target promoter, repress- ing transactivation from mouse mammary tumor virus, probasin, and prostate-specific antigen promoters. Inappropriate, nonligand AR activa- tion, postulated to act through regulation of receptor phosphorylation, was also sensitive to cyclin D1 regulation. Moreover, we show that several phosphorylation site mutants of the AR were equally inhibited by cyclin D1 as compared with the wild-type receptor. Given these data establishing the potency of cyclin D1-mediated repression, we evaluated the ability of cyclin D1 to inhibit tumor-derived AR alleles and polymorphisms associ- ated with tumor progression and increased prostate cancer risk. We demonstrate that the AR alleles and polymorphisms tested respond com- pletely to cyclin D1 corepressor activity. In addition, activation of a common tumor-derived AR allele by 17-estradiol and progesterone was inhibited through ectopic expression of cyclin D1. Taken together, these data establish the potency of cyclin D1 as an AR corepressor and provide support for additional studies examining the efficacy of developing novel prostate cancer therapies for both androgen-dependent and -independent tumors. INTRODUCTION Treatment of nonorgan confined prostate cancer relies on its unique requirement for androgen (1–3). The objective of prostate cancer therapy is to eliminate androgen action through bilateral orchiectomy and/or through administration of antiandrogens. Such androgen abla- tion results in cell cycle arrest and cell death in prostatic adenocarci- nomas and is highly effective because 80% of patients respond favorably to treatment (4). Unfortunately, median time to the forma- tion of recurrent tumors is only 24 –36 months with relapse occurring in virtually 100% of treated patients (2). Cells of the recurrent tumors proliferate in the absence of androgen, and no effective treatment currently exists for androgen-independent prostate cancer. Given the importance of androgen in prostate cancer formation and treatment, much emphasis has been placed on understanding the molecular mechanisms of androgen action. Prostatic epithelial and adenocarcinoma cells sense androgen through the AR, 3 a member of the steroid hormone receptor super- family of transcription factors (5, 6). The AR contains three functional domains, classified based upon their homology to other known nu- clear receptors: a NH 2 -terminal transactivation domain; a highly con- served DNA binding region; and a COOH-terminal ligand binding pocket (5, 7). The AR differs from other nuclear receptors in that its NH 2 -terminal domain is the site of its major transactivation function, AF-1 (7). In addition, interaction between the NH 2 and COOH- terminal regions of the AR is necessary for complete receptor activity (8). Binding of androgens such as DHT to the AR causes the disso- ciation of heat shock proteins from the receptor and allows for its dimerization and translocation into the nucleus (9, 10). Within the nucleus, the AR binds to AREs located on target genes such as PSA, which is used clinically to monitor prostate cancer progression (11– 13). The gene expression profile initiated by the AR results in a diverse set of biological outcomes, including secretion, differentia- tion, growth, and survival (11). The specificity of such biological outcomes is hypothesized to hinge upon the cellular environment and availability of AR cofactors. Nevertheless, the precise gene targets involved in these diverse functions remain largely undefined. Intriguingly, in recurrent androgen independent prostate cancer, the AR is expressed and inappropriately activated (i.e., in the absence of ligand; Ref. 2). This activation event is known to occur through multiple mechanisms, including AR amplification (up to 30% of recurrent tumors) and mutations within the AR itself, which allow alternative steroids (e.g., 17-estradiol, progesterone) to serve as ligands (2). Also thought to contribute to the androgen-independent phenotype is indirect stimulation of the AR by growth factors and signal transduction pathways (reviewed in Ref. 14). Specifically, EGF, IGF-I, KGF, and IL-6 were previously demonstrated to induce AR activity in the absence of ligand and may synergize with low-level DHT to enhance AR action (15, 16). It has been hypothesized that activation of signal transduction pathways in response to cytokines and growth factors results in phosphorylation of the AR, thus provid- ing a potential mechanism by which receptor activity is modulated (17). It is through these disparate pathways that the AR is thought to be inappropriately activated, facilitating proliferation and tumor pro- gression in the absence of canonical ligand. Thus, inhibition of AR activity is a major goal of therapies used to treat both early and late stage prostate cancers. We and others have previously shown that cyclin D1 is a potent inhibitor of AR activity (18, 19). Although well characterized for its role in cell cycle transitions, cyclin D1 has been shown to harbor multiple transcriptional functions independent of the cell cycle. Through an LxxLL motif in its COOH terminus and independent of CDK association, cyclin D1 forms a trimeric complex with ER and the steroid receptor coactivator, SRC-1, to enhance estrogen-respon- sive transcription (20, 21). Association of cyclin D1 with the AR Received 12/4/02; revised 6/6/03; accepted 6/9/03. 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. 1 This work was supported by NIH Grant R01CA099996 (to K. E. K.) and the Department of Defense Grant DAMD17-02-1-0037 (to K. E. K.). C. E. P-D. and C. J. B. are supported by the University Distinguished Graduate Fellowship (University of Cin- cinnati) and the Albert J. Ryan Foundation. 2 To whom requests for reprints should be addressed, at Phone: (513) 558-7371; Fax: (513) 558-4454; E-mail: Karen.Knudsen@uc.edu. 3 The abbreviations used are: AR, androgen receptor; DHT, dihydrotestosterone; ARE, androgen-responsive element; PSA, prostate-specific antigen; EGF, epidermal growth factor; IGF, insulin-like growth factor; KGF, keratinocyte growth factor; IL, interleukin; CDK, cyclin-dependent kinase; ER, estrogen receptor; MMTV, mouse mammary tumor virus; CDT, charcoal dextran treated; GFP, green fluorescent protein; GST, glutathione S-transferase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. 4903 Research. on July 15, 2015. © 2003 American Association for Cancer cancerres.aacrjournals.org Downloaded from