Antiandrogen Effects of Mifepristone on Coactivator and Corepressor Interactions with the Androgen Receptor

0888-8809/04/$15.00/0 Printed in U.S.A. Molecular Endocrinology 18(1):70–85 Copyright © 2004 by The Endocrine Society doi: 10.1210/me.2003-0189 Antiandrogen Effects of Mifepristone on Coactivator and Corepressor Interactions with the Androgen Receptor LIANG-NIAN SONG, MEGHAN COGHLAN, AND EDWARD P. GELMANN Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, D.C. 20057 transcriptional intermediary factor-2 or ␤-catenin but could inhibit the R1881-induced binding of AR to transcriptional intermediary factor-2 and ␤-catenin. Similarly, mifepristone could inhibit the R1881induced N/C-terminal interaction in a dose-dependent manner even though mifepristone alone has no effect on the N/C-terminal interaction of AR. We found that mifepristone could induce a strong interaction between AR and corepressors nuclear receptor corepressor and silencing mediator for retinoid and thyroid hormone receptors in both transactivation and two-hybrid assays to a greater degree than hydroxyflutamide, cyproterone acetate, and bicalutamide. The AR-corepressor interaction was also seen in coimmunoprecipitation assays. Finally, mifepristone at high concentrations induced a low level of prostate-specific antigen expression in LNCaP and antagonized prostate-specific antigen expression induced by R1881. Mifepristone also antagonized R1881 action on the growth of LNCaP prostate cancer cells. (Molecular Endocrinology 18: 70–85, 2004) T the ligand-dependent transcriptional AF-2 domain. The N terminus has been shown to interact directly with the C terminus in a ligand-dependent manner. The N/C interaction is found to be required for full transcription potential of the AR. Two LXXLL-related sequences in the N terminus have been found responsible for mediating the N/C-terminal interaction (1). The transcriptional activity of AR is modulated by proteins known as coactivators and corepressors that bind to the receptor. Coactivators, such as the p160 family of coactivators steroid receptor coactivator-1 (2), transcriptional intermediary factor-2 (TIF2)/glucocorticoid receptor-interacting protein 1 (3, 4) were originally defined as factors that increase the total amount of induced gene product with saturating concentrations of hormone. X-ray crystallographic studies indicate that the AR(LBD) adopts a similar structural fold as other members of steroid/nuclear receptor super family, suggesting that members of this super family share a common regulatory mechanism (5–7). The nuclear receptor corepressor (NCoR) and the related silencing mediator for retinoid and thyroid hormone receptors (SMRT) were initially discovered on the basis of their ability to bind to ligand-free nuclear HE ANDROGEN RECEPTOR (AR) is one member of the steroid/nuclear receptor super family of ligand-dependent transcription factors. AR plays a critical role in normal male development as well as prostate cancer development and progression. As a member of steroid/nuclear receptor super family, AR contains a central DNA binding domain, which separates the receptor amino (N) terminus from the carboxy (C) terminus. The N terminus contains an activation function (AF)-1 transactivation domain and the C terminus harbors the ligand binding domain (LBD) and Abbreviations: AF, Activation function; AR, androgen receptor; ARE, androgen response element; CCS, charcoalstripped fetal calf serum; CPA, cyproterone acetate; DHT, dihydrotesterone; E2, estradiol; LBD, ligand binding domain; LTR, long terminal repeat; MMTV, murine mammary tumor virus; NTD, N-terminal domain; NCoR, nuclear receptor corepressor; PSA, prostate-specific antigen; SMRT, silencing mediator for retinoid and thyroid hormone receptor; TIF2, transcriptional intermediary factor-2; VP16, herpes simplex viral protein 16. Molecular Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the endocrine community. 70 Mifepristone is a potent antagonist of steroid hormone receptors such as glucocorticoid and progesterone receptors. We investigated the potential for mifepristone to act as an antiandrogen and compared it with partial androgen receptor (AR) agonists and antagonists, in particular bicalutamide. Mifepristone was an effective antiandrogen in vitro that inhibited transcription from three androgen-responsive promoters and blocked the agonist R1881 in a dose-dependent manner. Like bicalutamide, mifepristone also antagonized the action of androgen receptor with a (T877A) mutation. Mifepristone competed effectively with R1881 with a relative binding affinity comparable to that of cyproterone acetate, and much higher than that of hydroxyflutamide and bicalutamide in a binding assay. Mifepristone could effectively induce the binding of the herpes simplex viral protein 16/AR fusion protein to the hormone response elements in the murine mammary tumor virus-luciferase reporter. With either wild-type or T877A mutant AR, mifepristone alone was unable to induce any detectable interaction with coactivators Song et al. • Antiandrogen Effects of Mifepristone RESULTS Mifepristone Inhibits Transcriptional Activation of the Androgen-Responsive Promoters 71 terminal repeat (LTR)-luciferase (MMTV-Luc) reporter. As shown in Fig. 1A, mifepristone alone induced minimal activation of AR reporter expression at high concentrations (⬎100 nM). When cells were treated with both R1881 (0.1 nM) and increasing concentrations of mifepristone, the R1881-induced reporter expression was markedly inhibited by mifepristone. To examine the effect of mifepristone on other androgen-responsive promoters, we used reporters under control of the rat probasin promoter and human prostate-specific antigen promoter and enhancer. Mifepristone could significantly inhibit the R1881-induced reporter expression in a dose-dependent manner to a greater degree than bicalutamide (Fig. 1, B and C). Thus mifepristone antagonized AR-mediated transactivation. The effect of mifepristone on transcription from an androgen-dependent reporter construct was compared with other ligands. As shown in Fig. 2A, estradiol (E2) and cyproterone acetate (CPA) stimulated transcription less than half the level induced by R1881. Hydroxyflutamide and bicalutamide, two nonsteroidal antiandrogens, had minimal, if any, measurable effects. Mifepristone induced minimal, but reproducible, activation of AR similar to the result shown in Fig. 1A and reminiscent of previously published findings (31). In prostate cancer cells mutations in the AR(LBD) have been shown to broaden ligand specificity and alter the response of mutant AR to antiandrogens so that they act as agonists (35). In LNCaP prostate cancer cells the T877A mutation in the AR(LBD) causes AR antagonists CPA and hydroxyflutamide to act as agonists (36, 37). Figure 2B shows the MMTV-LTR-driven reporter assay in LNCaP cells treated with different ligands including mifepristone. Note (...truncated)