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Molecular Endocrinology, doi:10.1210/me.2003-0189
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Molecular Endocrinology 18 (1): 70-85
Copyright © 2004 by The Endocrine Society

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

Address all correspondence and requests for reprints to: Edward P. Gelmann, Department of Oncology, Lombardi Cancer Center, Georgetown University, 3800 Reservoir Road, Northwest, Washington, D.C. 20057. E-mail: gelmanne{at}georgetown.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 transcriptional intermediary factor-2 or ß-catenin but could inhibit the R1881-induced binding of AR to transcriptional intermediary factor-2 and ß-catenin. Similarly, mifepristone could inhibit the R1881-induced 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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE 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 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, 6, 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 receptors, such as the thyroid receptor. More recently, NCoR and SMRT were found to interact with antagonist-bound estrogen receptor (8, 9), glucocorticoid receptor (10), and progesterone receptor (11, 12). Although the function of NCoR and SMRT in the regulation of transrepression of several members of nuclear receptors has been well documented, the role of these two corepressors in regulating AR-mediated transcriptional regulation of target genes is unknown. More recently, several papers have shown that SMRT and NCoR might play important roles in AR-mediated action by functional and physical interaction with AR (13, 14).

Clinically, androgen ablation is the most effective systemic therapy for disseminated prostate cancer. Over 80% of men with disseminated prostate cancer will show some degree of clinical response to chemical or surgical castration. Although the median duration of response to hormonal ablation among patients with metastatic prostate cancer is less than 3 yr, response durations range from a few months to many years. Androgen responsiveness in prostate cancer does not correlate with either the presence or the levels of AR in cancer tissues (15, 16, 17, 18, 19, 20). Furthermore, AR expression persists after the patient no longer enjoys clinical remission induced by androgen deprivation. Once prostate cancer progresses after initial androgen ablation up to 20% of patients will have a transient response to second-line hormonal therapy that involves either an antiandrogen (21) or adrenal steroidogenesis (22).

Mifepristone is a derivative of norethindrone, a synthetic 19-nor-steroid. Mifepristone strongly binds to progesterone as well as glucocorticoid receptors and thus acts as an antagonist to progestational and glucocorticoid functions (23). Mifepristone has been tested in phase II studies for ovarian and breast cancer (24, 25, 26). Mifepristone has not yet been tested in human prostate cancer. Preclinical studies have suggested that mifepristone may induce prostate cancer cell death and inhibit growth of prostate cancer cells in tumor model systems (27, 28, 29, 30). Mifepristone has been shown to interact with the AR in vitro and to mediate the binding of AR to an androgen response element (ARE), in contrast to the effects of flutamide, that inhibited the AR/ARE interaction (31, 32, 33, 34). In this study, we demonstrate that mifepristone functions as a potent antiandrogen with minor agonist activity that depends on the cellular milieu. The molecular basis of the antagonist effect of mifepristone on AR-mediated action is addressed in comparison with other known antiandrogens.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mifepristone Inhibits Transcriptional Activation of the Androgen-Responsive Promoters
To study the effects of mifepristone on AR action, CV-1 cells were cotransfected with wild-type, full-length AR and the murine mammary tumor virus long terminal repeat (LTR)-luciferase (MMTV-Luc) reporter. As shown in Fig. 1AGo, 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. 1Go, B and C). Thus mifepristone antagonized AR-mediated transactivation.



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  		Fig. 1. Effect of Mifepristone on AR-Mediated Transcription

A, The effect of mifepristone alone or in the presence of 0.1 nM R1881 was examined in an MMTV-Luc reporter gene assay in CV-1 cells expressing wild-type AR. CV-1 cells were transiently transfected with 10 ng of AR, 100 ng of MMTV-Luc, and 10 ng of Renilla. After 16 h incubation, cells were treated for 24 h with 0.1 nM R1881 and/or the indicated concentrations of mifepristone. Luciferase activities from reporter and Renilla plasmids were measured as described in Materials and Methods. The Luciferase values were normalized for Renilla expression. B, The effects of mifepristone and bicalutamide were compared in CV-1 cells transfected with probasin-Luc and wild-type AR. C, Similar experiment as in panel B with PSA/PSE-Luc.

 
The effect of mifepristone on transcription from an androgen-dependent reporter construct was compared with other ligands. As shown in Fig. 2AGo, 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. 1AGo 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 2BGo shows the MMTV-LTR-driven reporter assay in LNCaP cells treated with different ligands including mifepristone. Note that the agonism of E2 and CPA was enhanced markedly by the T877A mutation compared with wild-type AR (Fig. 2AGo). Hydroxyflutamide was an agonist for this mutant receptor. But the response of AR(T877A) to bicalutamide and mifepristone was similar to the response of the wild-type receptor in agreement with previously published findings (34).



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  		Fig. 2. Comparison of Mifepristone with Other Antiandrogens

A and B, MMTV-Luc reporter assay performed (A) in CV-1 cells expressing wild-type AR or (B) in LNCaP cells that express endogenous AR(T877A). Concentrations of ligands used were as follows: 0.1 nM R1881, 1 µM E2, 1 µM CPA, 1 µM OH-flutamide, 10 µM bicalutamide, 1 µM mifepristone. C and D, Studies of interaction between antiandrogens. MMTV-Luc reporter gene assays in CV-1 cells expressing wild-type AR and treated with 0.1 nM R1881. C, Cultures were exposed to 0.1 nM R1881 and treated with increasing concentrations of bicalutamide with (+) or without (-) 10 nM mifepristone. D, Cultures were exposed to 0.1 nM R881 and treated with increasing concentrations of mifepristone with (+) or without (-)1 µM bicalutamide. No synergy was observed. In C and D, the control (R1881 treated only) is the second bar only, all bars show data with either R1881 plus increasing concentration of bicalutamide (C) or mifepristone (D), or R1881 + bicalutamide + 10 nM mifepristone (C) or R1881 + mifepristone + 1 µM bicalutamide (D).

 
Because both mifepristone and bicalutamide were highly antagonistic to AR activation, we determined whether there was any interaction between the two or whether they behaved as independent ligands that bound to the same receptor site. As shown in Fig. 2Go, C and D, there was no significant additive or synergistic effect of mifepristone and bicalutamide on R1881-AR-mediated reporter gene expression. Similar results were obtained when reporters under control of the rat probasin promoter and human prostate-specific antigen promoter and enhancer were used (data not shown). These results suggest that these two antagonists act in concert most consistent with their binding to the same site on the AR.

Mifepristone Binding to AR
To study the relative binding affinities of mifepristone and other ligands, a whole cell competition-binding assay was performed in COS-7 cells transfected with wild-type AR. The binding assay was performed using [3H]-R1881 as the tracer and unlabeled antiandrogens at 0.1-, 1-, 10-, 100-, and 1000-fold molar excess as competitors. The displacement curves of the antiandrogens compared with the reference agonist R1881 are shown in Fig. 3Go. The relative binding affinities of these antiandrogens for AR ranked as follows: R1881 100% > CPA 5.64% = mifepristone 5.64% > E2 1.65% > hydroxyflutamide 0.44% >> bicalutamide (<<0.16). The relative binding affinities of RU-486 to CPA and OH-flutamide are similar to the values reported by Kemppainen et al. (31), published before the general availability of bicalutamide.



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  		Fig. 3. Competitive Radioligand Binding Assay Showing the Retention of [3H]-R1881 Bound to Wild-Type AR by Increasing Concentrations of Competitor Ligands

For whole cell, radioligand binding assay, COS-7 cells were transiently transfected with wild-type AR (pCMVhAR, 500 ng/well in 24-well plates). Cells were cultured in phenol red-free medium containing 5% CCS for 24 h before binding assays. Radioligand binding assay was performed as described in Materials and Methods, and the total binding was expressed as 100%. Relative binding affinity values were calculated from the constructed competitive binding curves and expressed as the ratio of concentration of R1881 over concentration of competitor, each of which produces a 50% decrease in specific [3H]-R1881 binding x 100.

 
Mifepristone Induces DNA Binding of AR
Transcriptional activation of target genes by AR involves a multistep process to assemble a transcriptional complex (38). To ask if mifepristone binding could induce DNA binding of AR to its target response elements, the herpes virus viral protein 16 (VP16)/AR fusion protein was used. The VP16 transactivation domain was fused to the N terminus of wild-type, full-length AR. With this construct, the nuclear translocation and DNA binding activity were separated from the transactivation function of the DNA-bound receptor within the nucleus, because after nuclear translocation VP16/AR constructs transactivate the reporter gene much more efficiently (39). VP16/AR, both wild type and T877A, were inactive in the absence of ligands. However, in the presence of either R1881, E2, CPA or mifepristone, the reporter was stimulated, indicating that the VP16/AR fusion proteins (wild type and T877A) must have been translocated to the nucleus and bound to DNA, a result that is consistent with immunohistochemical nuclear localization data of Kemppainen et al. (31) (Fig. 4Go). In contrast, hydroxyflutamide could only induce a minimal binding of the wild-type AR but a more efficient binding of the AR T877A to the reporter gene promoters. Bicalutamide, on the other hand, could only induce minimal binding of both the wild-type and T877A mutant AR to the reporter genes promoters. Further evidence that ligands mediated binding of this fusion protein to DNA was provided by the agonistic interaction of hydroxyflutamide with VP16/AR(T877A). Because mifepristone was able to activate binding of AR to DNA, we next investigated the effects of the drug on intra- and intermolecular interactions of the AR.



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  		Fig. 4. Ligand Mediation of AR Interaction with the AREs in MMTV LTR

CV-1 cells were transiently transfected with 10 ng of the VP16/AR (wild-type, WT) or T877A mutant, 100 ng of MMTV-Luc, and 10 ng of Renilla. Cells were treated with either 1 nM R1881 or the indicated ligands. The final concentrations of bicalutamide was 10 µM, all others were 1 µM. Transcription in this assay is driven by VP16 in the fusion proteins, and therefore the reporter read-out is an indicator of receptor binding to the AREs of the MMTV promoter.

 
Mifepristone Inhibits the AR N/C-Terminal Interaction
Agonist-induced interaction between the AR N and C termini play a major role in AR transcriptional activity (1, 40). To test whether mifepristone could affect the AR N/C-terminal interaction, a mammalian two-hybrid assay was used. CV-1 cells were cotransfected with AR(507–919) that contains the DNA binding domain and LBD and AR(1–503) in conjunction with an MMTV-Luc reporter. As shown in Fig. 5AGo, there was no interaction between these two fusion proteins in the absence of ligand, but R1881 induced a strong interaction and E2 mediated a weaker interaction. As reported before, CPA, hydroxyflutamide and bicalutamide could not induce any detectable interaction of the wild-type AR(LBD) with the N terminus (13). Hydroxyflutamide and CPA induced interaction with the mutant AR(LBD) (T877A). Mifepristone did not induce any N/C-terminal interaction of either the wild-type or mutant LBD. Furthermore, using GAL4/AR(LBD), both wild type and T877A, and VP16/AR(1–660) fusion constructs in a pure binding assay we showed that CPA and hydroxyflutamide could induce an N/C-terminal interaction with the mutant AR(LBD) (T877A), but bicalutamide and mifepristone had no effect (Fig. 5BGo). Similar results were also obtained using a GAL4 fusion construct that contained the first FXXLF motif in the AR NH2-terminal domain (23FQNLF27) (Fig. 5CGo) (1). R1881 induced a strong interaction between GAL4/FQNLF and VP16/AR(LBD), whereas all antiandrogens tested did not. However, when cells were cotransfected with GAL4/FQNLF and VP16/AR(LBD) (T877A), hydroxyflutamide, CPA, and E2-induced moderate interactions, whereas bicalutamide and mifepristone were inactive. Therefore, even though mifepristone binding to AR was permissive for DNA binding, it was not permissive for the N/C-terminal interaction. To test if mifepristone could inhibit AR N/C-terminal interaction, CV-1 cells were cotransfected with GAL4/AR(LBD) and VP16/AR(1–660) in conjunction with a FR-Luc reporter. As shown in Fig. 5DGo, mifepristone could inhibit the R1881-induced N/C-terminal interaction in a dose-dependent manner.



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  		Fig. 5. N/C-Terminal Interactions of AR in the Presence of Different Ligands

A, MMTV-Luc reporter assay. CV-1 cells were transiently transfected with 10 ng of either wild type or T877A mutant AR507–919, 100 ng of MMTV-Luc, 50 ng of AR(1–503), and 10 ng of Renilla. Cells were treated for 24 h with 1 nM R1881 or indicated antiandrogens. The concentrations of all the antiandrogens used were 1 µM for hydroxyflutamide, CPA, E2, mifepristone, and 10 mM for bicalutamide. B, Mammalian two-hybrid assay. CV-1 cells were cotransfected with 10 ng of GAL4/AR(LBD) (wild type or T877A), 100 ng of FR-Luc reporter, 50 ng of VP16/AR(1–660) and 10 ng of Renilla. Cells were treated for 24 h with the indicated ligands (concentrations were the same as panel A). C, Mammalian two-hybrid assay showing activation of the GAL4-responsive FR-Luc reporter after interaction of GAL4/FQNLF and VP16/AR(LBD) in the presence of different ligands. FQNLF is the N-terminal AR pentapeptide that mediates binding with the AR(LBD). Concentrations of ligands used were as follows: 1 nM R1881, 1 µM E2, 1 µM CPA, 1 µM OH-flutamide, 10 µM bicalutamide, 1 µM mifepristone. D, Mifepristone inhibits the N/C interaction. Mammalian two-hybrid assay was done as described in panel B and cells were treated with 0.1 nM R1881 and increasing concentrations of mifepristone as indicated.

 
Mifepristone Inhibits the Interaction between AR and Coactivators
Coactivators play a pivotal role in amplifying the transcriptional signal initiated by the interaction of ligand-bound AR with DNA. Coactivators bind to the C terminus of AR in a region of the molecule termed AF-2 (41). To determine whether mifepristone-bound AR was able to form complexes with coactivators we used a mammalian two-hybrid assay. In this assay R1881 induces a strong interaction between GAL4/AR(LBD) and coactivators TIF2 or VP16/ß-catenin (Fig. 6AGo). In contrast, mifepristone alone did not induce any detectable interaction between the AR(LBD) and coactivators. Mifepristone could inhibit the binding of AR to both TIF2 and VP16/ß-catenin in a dose-dependent manner. When 1 µM mifepristone was used, the interaction was completely blocked. We also examined the effects of different antiandrogens on the interaction between AR(T877A) and coactivators. To this end, GAL4/AR(LBD) (T877A) was used in the mammalian two-hybrid assay. We found that like R1881, dihydrotestosterone (DHT), CPA, hydroxyflutamide, and E2 induced a strong interaction between AR(T877A) and TIF2, but bicalutamide and mifepristone had no effect (Fig. 6BGo). Interaction between AR(LBD) (T877A) and VP16/ß-catenin was supported by R1881, hydroxyflutamide, and E2, but in this case not CPA, bicalutamide or mifepristone (Fig. 6BGo).



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  		Fig. 6. Interaction of AR with TIF2 and ß-Catenin

A, Antiandrogens inhibit the interaction between AR and coactivators. Mammalian two-hybrid assay in CV-1 cells with the FR-Luc GAL4-responsive reporter and GAL4/AR(LBD) and either TIF2 or VP16/ß-catenin as previously described (39 ). Cells were treated with R1881, hydroxyflutamide (HF), CPA, mifepristone individually or R1881 plus each of these antiandrogens at the indicated concentrations. B, Antiandrogens-induced interaction between AR T877A and coactivators. Experiments were done as described in panel A except that GAL4/AR(LBD) T877A was used. Cells were treated with 1 nM R1881 and DHT, 1 µM hydroxyflutamide, CPA, mifepristone (RU-486), and E2, and 10 µM bicalutamide (BIC). C, Reporter gene assay in CV-1 cells with MMTV-Luc, AR(507–919) T877A and either TIF2 or VP16/ß-catenin. The concentration of each ligand is the same as those used in panel B.

 
To confirm the mammalian two-hybrid assay results, a transient transactivation assay was performed in CV-1 cells. In this experiment, cells were cotransfected with AR(507–919) (T877A) containing the DNA binding domain and LBD, together with either TIF2 or VP16/ß-catenin. As shown in Fig. 6CGo, in the absence of ligand, AR(507–919) (T877A) did not induce expression of the MMTV-Luc reporter. However, when cells cotransfected with AR(T877A) and TIF2, and treated with hydroxyflutamide, CPA or E2 we observed reporter gene expression nearly equivalent to the activity induced by R1881. Mifepristone and bicalutamide had no effect similar to the mammalian two-hybrid assay result. When cells were cotransfected with AR(T877A) and VP16/ß-catenin, we observed once again that, except for CPA, the ligands produced similar results as with TIF2.

Mifepristone Enhances Interaction between AR and Corepressors
AR interacts with corepressors that have an inhibitory effect on transcriptional activation by the receptor. We examined the effect of a variety of ligands on the interaction of AR with the corepressors NCoR and SMRT using a modified mammalian two-hybrid assay. In this assay, cells were cotransfected with wild-type, full-length AR and the MMTV-Luc reporter, together with either VP16 control or fusion constructs of VP16 with the C-terminal nuclear receptor interaction domain of NCoR (VP16/NCoR.ID), or SMRT (VP16/SMRT.ID). Binding of the VP16 fusion proteins to the AR induced reporter gene transcription activated by the VP16 transactivation function. Mifepristone induced a low level of activation of the reporter in the presence of VP16 not dissimilar from the low level of AR activation shown in Fig. 1AGo (Fig. 7AGo). However, in the presence of vectors that contained only the AR-interaction domains of the corepressors, either VP16/NCoR.ID or VP16/SMRT.ID fusion proteins, mifepristone induced a 4- to 5-fold activation of the reporter, suggesting an interaction between AR and the corepressor peptide domains. To address more directly the interaction between AR and corepressors in the presence of different ligands, we also performed a conventional mammalian two-hybrid assay in CV-1 cells with GAL4/NCoR.ID or GAL4/SMRT.ID and VP16/AR. Mifepristone induced a very strong interaction in this assay, whereas R1881, hydroxyflutamide, bicalutamide, CPA, and E2 had minimal, if any, effects (Fig. 7BGo). We performed a competition assay to determine whether the induction of the reporter in response to mifepristone treatment in Fig. 7BGo was due to direct interaction of the corepressors with AR, as opposed to indirect reactions of corepressors with VP16/AR that are mediated by any of a variety of possible bridging factors. Excess expression of full-length corepressors attenuated the induction of reporter gene expression. Figure 7CGo shows a significant dose-dependent increase in the ability of full-length SMRT to reduce the total activity for interaction of GAL4/SMRT.ID with mifepristone-bound VP16/AR. A similar concentration-dependent decrease in total activity, albeit not as evident as SMRT, was seen with full-length NCoR. These results strongly support a direct interaction of mifepristone-bound AR with corepressors. The physical association of AR with a corepressor was confirmed by coimmunoprecipitation of AR and Flag-tagged NCoR in the presence of different ligands. R1881 and mifepristone induced strong physical association between AR and NCoR. Bicalutamide also mediated an interaction, but to a lesser degree than mifepristone (Fig. 7DGo).



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  		Fig. 7. Interaction of AR with Corepressors NCoR and SMRT

A, Modified mammalian two-hybrid assay. CV-1 cells were cotransfected with 10 ng of pCMVhAR, 100 ng of MMTV-Luc, 50 ng of VP16, VP16/NCoR.ID, or VP16/SMRT.ID, and 10 ng of Renilla. Cells were treated with 1 µM mifepristone or ethanol vehicle for 24 h. As shown earlier, mifepristone has a minimal agonist activity in the presence of VP16. Ectopic expression of either VP16/NCoR.ID or VP16/SMRT.ID could induce an approximately 5-fold increase in the reporter expression in the presence of mifepristone, suggesting a strong functional interaction between AR and corepressors. B, Mammalian two-hybrid assay. CV-1 cells were cotransfected with 10 ng of either GAL4/NCoR.ID or GAL4/SMRT.ID, 100 ng of FR-Luc reporter, 100 ng of VP16/AR, and 10 ng of Renilla. Cells were treated with the indicated ligands. Concentrations of ligands used were as follows: 1 nM R1881, 1 µM E2, 1 µM CPA, 1 µM hydroxyflutamide, 10 µM bicalutamide, 1 µM mifepristone. C Inhibition by added full-length corepressors of mammalian two-hybrid interactions of NCoR.ID and SMRT.ID with AR. CV-1 cells were transiently cotransfected with 10 ng of GAL4/NCoR.ID or GAL4/SMRT.ID, 100 ng of VP16/AR, 100 ng of FR-Luc, and 10 ng of Renilla plus the indicated amounts of plasmids for full-length NCoR or SMRT. The cells were then treated with mifepristone (1 µM) or ethanol control, harvested and analyzed as described in Materials and Methods. D, Coimmunoprecipitation assays. COS-7 cells were seeded in 100-mm dishes and cotransfected with Flag-NCoR (5 µg/dish) together with or without pCMVhAR (5 µg/dish). Sixteen hours after transfection, cells were cultured in phenol red-free medium containing 5% CCS and treated for 24 h with 10 nM R1881, 10 µM bicalutamide, 1 µM mifepristone, or ethanol control. Cell lysates were prepared and coimmunoprecipitation was done with anti-Flag antibody and western blotting analysis of the immune complexes was performed with anti-AR antibody (PG21) as described in Materials and Methods. E, Interaction of the NCoR-interacting domain and AR were examined in the presence of R1881 and mifepristone. CV-1 cells were cotransfected with 10 ng pCMVhAR, 100 ng MMTV-Luc, 100 ng VP16, or 100 ng VP16/NCoR.ID, 10 ng Renilla luciferase plasmid and 80 ng pBSK+. Cells were treated with 0.1 nM R1881, 0.1 nM DHT, 1 µM hydroxyflutamide, 1 µM CPA, 10 µM bicalutamide, 1 µM E2, or 1 µM mifepristone for 20 h. F, CV-1 cells were cotransfected with 10 ng VP16/AR, 100 ng MMTV-Luc, 100 ng SMRT, 100 ng VP16/NCoR, or 100 ng pCMX, and 10 ng Renilla luciferase plasmid. Cells were treated with 0.1 nM R1881 or 1 µM mifepristone for 20 h.

 
We further examined differences in the AR/NCoR interaction in the presence of a variety of ligands by using the VP16 transcriptional activator and MMTV-luciferase as a reporter. As shown in Fig. 7EGo in the presence of R1881 and several other ligands, the NCoR-interacting domain bound to AR and interfered with VP-16-assisted activation of MMTV-luciferase. In contrast, in the presence of mifepristone, there was a paradoxical effect whereby the interaction of NCoR.ID and AR facilitated VP16-mediated transcription from the MMTV promoter. However, over expression of both full-length NCoR and SMRT were able to interfere with VP16/AR-mediated transcriptional activation by mifepristone, but to a much lesser degree by R1881 (Fig. 7FGo). These data indicate that mifepristone affects AR in such a way as to cause a different interaction with NCoR than R1881.

Effect of Mifepristone on Prostate-Specific Antigen (PSA) Expression in LNCaP Cells
PSA is an AR-responsive gene under control of promoter and enhancer elements that are androgen-regulated (42). To determine whether mifepristone could affect expression of an endogenous androgen-responsive gene LNCaP cells were treated with 0.1 nM R1881 in the absence or presence of 0.1–1000 nM mifepristone and transfected with the PSA/PSE-luc reporter plasmid. R1881 induced more than 10-fold increased PSA expression over baseline. Mifepristone at concentrations of 10–1000 nM inhibited the effects of R1881 on PSA expression (Fig. 8Go). At 1000 nM mifepristone-alone induced as much endogenous PSA expression as mifepristone + R1881. Partial agonist activity of mifepristone for the endogenous PSA promoter was seen at concentrations of 10 to 1000 nM. This is not surprising since other antiandrogens, including hydroxyflutamide and bicalutamide, have been found to be able to induce endogenous PSA expression in LNCaP cells (43). In contrast, mifepristone was less of an agonist for reporter expression under control of the engineered PSA promoter/enhancer construct. When the exogenous plasmid reporter was used, mifepristone alone had agonist activity at 100 and 1000 nM. Moreover, mifepristone exhibited a greater degree of antagonism of R1881 action when the reporter plasmid was used to monitor AR activity. As shown in Fig. 8Go, the PSA promoter construct showed a low level of activation by mifepristone alone and antagonism of R1881 by higher concentrations of mifepristone as seen in Fig. 1CGo. Therefore the effects of mifepristone on an endogenous androgen-responsive PSA promoter were quantitatively different from the effects on the reporter construct.



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  		Fig. 8. The Effects of Mifepristone on PSA Promoter Activity in LNCaP Cells

LNCaP cells were cultured for 48 h with 0.1 nM R1881, mifepristone (1–1000 nM), or 0.1 nM R1881 plus increasing concentrations of mifepristone (0.1–1000 nM) as indicated. Cell supernatants were analyzed for PSA concentration by ELISA assay (open figures, right scale). The graph also shows relative luciferase activity transcribed off the PSA/PSE-luciferase reporter construct transfected into LNCaP cells (solid figures, left scale).

 
Effect of Mifepristone on LNCaP Cell Growth
The LNCaP prostate cancer cell line is dependent on androgen for growth in vitro and will cease to divide in the absence of androgen. We compared the effects of mifepristone and bicalutamide in their abilities to antagonize LNCaP cell growth in the presence of R1881 and charcoal-treated calf serum. Figure 9AGo shows the effects of different concentrations of R1881 on LNCaP growth after 10 d. These data recapitulate published findings of the response of LNCaP cells to different steroid hormones and antihormones (30). Bicalutamide alone had a slightly inhibitory effect on LNCaP cell growth, especially at concentrations of at least 10-6 M (Fig. 9BGo). However, bicalutamide did not antagonize 0.1 nM R1881 on LNCaP cell growth (Fig. 9CGo). Mifepristone alone could stimulate LNCaP cell growth in a dose-dependent manner. However, when 10-5 M mifepristone was used, the cell number decreased, suggesting a toxic effect that this supraphysiologic concentration mifepristone may have toxic effects on the cells (Fig. 9DGo). The effect of 0.1 nM R1881 on LNCaP cell growth was antagonized by mifepristone in a dose-dependent manner (Fig. 9EGo).



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  		Fig. 9. Effect of Ligands on LNCaP Growth

A–E, LNCaP cells were seeded in 96-well plates (5000/well) and cultured for 3 d in phenol red-free medium containing 2.5% CSS. Cells were then exposed to different concentrations of ligands as shown in the figure. Cultures were down in triplicate. Growth after 10 d was assayed as described and is shown as relative OD on the vertical axes.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Based on the nature of the compounds, antiandrogens can be either partial/mixed antagonists (such as the steroidal antiandrogen CPA) or full/pure antagonists (such as the nonsteroidal antiandrogens hydroxyflutamide, nilutamide, and bicalutamide). Antiandrogens can effectively block AR-mediated target gene expression, and have been therefore widely used in the treatment of androgen-dependent prostate cancer. However, antiandrogen monotherapy is generally not as effective as castration or treatment with GnRH agonists (44, 45). Antiandrogens add very little to the efficacy of surgical or chemical castration (46). However, second-line hormonal therapy is now used with increasing frequency because patients with prostate cancer are initiating androgen ablative therapy earlier in the course of the disease. It is therefore important to identify candidates for use in second-line therapy of prostate cancer.

In this study, we demonstrated that mifepristone, an antagonist for progesterone and glucocorticoid receptor, could function as a potent antiandrogen with minimal agonist activity. In contrast to other antiandrogens like hydroxyflutamide and CPA, mifepristone could inhibit the mutant AR(T877A)-mediated transactivation as effectively as it could inhibit the wild-type AR. These results can be observed in both LNCaP cells and CV-1 cells transfected with AR(T877A). This is important since T877A mutation has not only been found in LNCaP cells but has also been detected in prostate cancer patients (47). The AR(T877A) and many other AR mutations found in prostate cancer alter the responsiveness to antiandrogens and to natural low-affinity ligands E2, progesterone, and adrenal androgens. Our results suggest that mifepristone is interacting in a different way with the AR(LBD). Indeed, proteolytic degradation studies have shown that mifepristone protects different AR fragments, compared with agonists and other antagonists (32).

As reported previously, mifepristone was found to have a low level of AR agonist activity (31, 32, 34). However, when cells are exposed to R1881 in the presence of 100- to 1000-fold excess mifepristone, substantial inhibition of AR activity was seen. This effect is independent of cell type as we have demonstrated the effect in CV-1 cells and qualitatively similar results were obtained previously in HeLa cells (32). The effect is also seen with several different androgen-responsive promoters. The relative affinities of mifepristone and R1881 for AR are calculated from our data to differ by a factor of 12 and from the data of Kemppainen et al. (31) by a factor of about 32. These affinity calculations are consistent with the molar excesses of mifepristone required to inhibit R1881 agonist activity.

To clarify the molecular basis of the mifepristone AR antagonism, we first demonstrated that mifepristone mediated DNA binding of the AR. Consistent with its limited ability to activate AR transcriptional activity, mifepristone cannot induce the AR N/C-terminal interaction. Moreover, mifepristone blocked the N/C-terminal interaction in a dose-dependent manner. In addition, we have shown with transactivation and mammalian two-hybrid assays that mifepristone could inhibit the R1881-induced recruitment of coactivators (TIF2 and ß-catenin) by wild type or AR(T877A). Because both the N/C interaction and the recruitment of coactivators play critical roles in AR-mediated transactivation the effects of mifepristone on these interactions may underlie its AR antagonism.

Antiandrogens had differing effects on the interaction between AR and coactivator molecules. Hydroxyflutamide induced an interaction between of AR(T877A) and either TIF2 or ß-catenin in both mammalian two-hybrid (Fig. 6BGo) and transactivaton assays (Fig. 6CGo). In contrast, CPA induced an interaction between AR(T877A) and TIF2, but not ß-catenin. The different effects of hydroxyflutamide and CPA may be due to differences in conformation changes in the AR(LBD) induced by different antiandrogens. These results further support our recent report that the binding surfaces in AR(LBD) for ß-catenin binding are overlapping but not identical to that for TIF2 binding (39).

AR also binds to corepressors, indicating that corepressors might further contribute to the antagonist activity of antiandrogens (13, 38). We also found that the physical interaction between NCoR and AR could be stimulated by agonists as well as antagonists, consistent with other observations that NCoR and AR can interact in the present of agonists (48, 49). SMRT has also been shown to interact with AR in the absence of ligand (14). Because mifepristone is well known for its antagonist effect on progesterone and glucocorticoid receptor mediated action, and it has been reported that corepressor NCoR interacts with the mifepristone-bound progesterone and glucocorticoid receptors, it is of particular interests to determine whether mifepristone bound AR could also interact with corepressors. CPA, bicalutamide, and hydroxyflutamide all induced mammalian two-hybrid interactions between AR and SMRT (50). The discrepancy between the effects of CPA and bicalutamide on the interaction between AR and corepressors among different reports are unknown at the present time, but might be due to different constructs, assays, and/or cell lines used. We have not detected any interaction between AR and corepressors even in the presence of mifepristone when the mammalian two-hybrid assay was done with reciprocal constructs GAL4/AR(LBD) and VP16/corepressors (data not shown). Importantly, the profile of interaction between AR and the NCoR-interacting domain in the presence of mifepristone was different from the interaction mediated by other ligands. This suggests that mifepristone induces a conformation of AR that differs from that induced by a variety of other ligands.

Mifepristone has been shown to have a variety of effects on growth of androgen-responsive cell lines. Mifepristone stimulated growth of a clone of Shionogi mammary carcinoma cells in a manner that was additive with glucocorticoids, suggesting that more than one steroid hormone receptor was responsible for the observed effects (51). Moreover, mifepristone antagonized androgen-mediated effects on gene activation in the mouse kidney (52). Mifepristone inhibited growth of both androgen-sensitive and -insensitive human and rat prostate carcinoma cells, further suggesting that the compound can act via interaction with more than one steroid hormone receptor (29, 30, 53). Mifepristone displaced androgen from its receptor in androgen-responsive breast cancer cells, inhibited proliferation, and antagonized the effects of R1881 in those cells but did not activate an androgen-responsive reporter gene, thus complicating the interpretation of the mifepristone effects (54). Mifepristone acted as an agonist for wild type, but not for mutant AR(T877A) in reporter gene assays performed in PC-3 prostate cancer cells, suggesting that cell milieu may influence steroid biological effects (55). In combination with tamoxifen, mifepristone induced apoptosis of LNCaP prostate cancer cells. However, the interpretation of these effects are unclear because LNCaP cells do not undergo apoptosis in response to androgen withdrawal or to treatment with the antiandrogen bicalutamide (28). Mifepristone has a complex effect in male rats where the drug induces a decrease in FSH and an increase in LH (56). The latter effect confirms that in vivo androgenic effects of mifepristone may be dose dependent. It is also possible that mifepristone has differential agonistic and antagonistic effects in different organs. It was also proposed that mifepristone had a direct effect on the pituitary by inhibiting LH secretion (56).

Collectively, our data demonstrate that mifepristone is a potent antiandrogen with minimal agonist activity. Compared with other known antiandrogens, mifepristone is a very strong inducer of the interaction between AR and corepressors NCoR and SMRT and therefore, could be used as a selective receptor modulator. The potential of mifepristone as an AR modulator in clinical prostate cancer has yet to be explored. Chronic clinical administration of mifepristone has been studied in phase II trials for treatment of breast and ovarian cancer (24, 26). The drug is well tolerated and can be administered daily. Prostate cancer is highly androgen responsive and requires AR-mediated signaling pathways even when the disease progresses despite androgen ablation (57). In view of the unique molecular interactions of mifepristone with AR compared with other clinical antiandrogens, a phase II trial of mifepristone for treatment of progressive prostate cancer seems justified.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
[3H]-R1881 (methyltrienolone, 83.5 Ci/mmol) was obtained from Perkin-Elmer Life Sciences (Foster City, CA). Nonradioactive R1881, 5{alpha}-dihydrotesterone, mifepristone [17ß-hydroxy-11ß-(4 dimethyl aminophenyl)-17{alpha}-(1-propynyl)-estra-4,9-dien-3-one], cyproterone acetate (6-chloro-1,2-methylene-17{alpha}-hydroxy-4,6-pregnadiene-3,20-dione-acetate), and E2 were from Sigma (St. Louis, MO). Hydroxyflutamide was from Schering (Bloomfield, IL); and bicalutamide [Casodex (2RS)-4'-cyano-3-(4-fluorophenylsulfonyl)-2-2hydroxy-2-methyl-3'-(trifluoromethyl) propionanilide] from Astra-Zeneca Pharmaceuticals (Macclesfield, UK). All cell culture reagents were from GIBCO Life Technologies (Grand Island, NY).

Plasmids
pCMVhAR, pCMVhAR-(507–919) (wild type and T877A mutant), pCMVhAR-(1–503), VP16/AR, VP16/AR(1–660), GAL4/AR(LBD) (wild type and T877A mutant), VP16/ß-catenin, TIF2, MMTV-Luc have been described. PSA/PSE-Luc was a gift from Arie Belldegrun, UCLA Medical Center (42) and probasin-Luc from Robert Matusik, Vanderbilt University (Nashville, TN) (58). The Renilla null Luciferase reporter was from Promega (Madison, WI). FR-Luc is from Stratagene (La Jolla, CA). NCoR was provided by Dr. Michael Rosenfeld, University of California, San Diego and SMRT was received as gift from Dr. Ron Evans (The Salk Institute, La Jolla, CA). GAL4/ and VP16/NCoR-ID (amino acids 1944–2453), GAL4/ and VP16/SMRT-ID (amino acids 982-1495) were from Mitch Lazar (University of Pennsylvania School of Medicine, Philadelphia, PA). VP16/AR(T877A) and pM-FQNLF were kindly provided by Dr. Donald P. McDonnell (Duke University Medical Center, Durham, NC).

Cell Culture and Transfection
Monolayer cultures of CV-1, COS-7, and LNCaP cells were grown as described (39). Transient transfection was performed as described before with Lipofectamine Plus Reagent (Life Technologies, Inc). For each well of a 24-well plate, we use 100 ng of reporter (FR-Luc, MMTV-Luc, PSA-Luc, or Probasin-Luc) and 10 ng Renilla plus various combinations of other expression vectors. Eqimolar amounts of expression vectors were included to keep the molar amount of each vector constant, with the total transfected DNA brought to 300 ng/well with pBSK+ unless otherwise indicated. The cells were then treated for 24 h with 0.1% ethanol ± steroids in media containing 5% dextran-treated and charcoal-stripped fetal calf serum (CCS) and harvested in 1x Passive Lysis Buffer (Promega). Fifty microliters of the cell lysates were used to assay for Luciferase activity using the Dual-Luciferase Assay System from Promega according to the supplier. The data were normalized for the cotransfected Renilla activity.

Radioligand Competition-Binding Assay
Radioligand binding assay of AR was performed as described before. Briefly, COS-7 cells were seeded into 24-well plates at a cell density of 5 x 104cells/well in phenol red-free medium containing 5% CCS and were transfected with pCMVhAR expression vectors (500 ng/well). After 16 h, cell monolayers were cultured in fresh phenol red-free medium containing 5% CCS and maintained for 24 h. After washing twice with PBS, cells were cultured in phenol red-free, serum-free medium containing 5 nM [3H]-R1881 in the absence or presence of 0.1- to 1000-fold molar excess of unlabeled competitor ligands for 90 min at 37 C.

Coimmunoprecipitation and Western Blotting
Protein-protein interactions were assessed by coimmunoprecipitation of factors from whole cell extracts. COS-7 cells were plated into 100-mm2 dishes with a cell density of 1.5 x 105cells/dish. Twenty-four hours later, cells were transiently transfected with 5 µg Flag-NCoR, together with or without 5µg pCMVhAR. Sixteen hours after transfection, cells were treated with of with 10 nM R1881, 1 µM mifepristone, 10 µM bicalutamide, or ethanol control. Twenty-four hours after treatment, cells were chilled on ice, washed with ice-cold PBS, and lysed in lysis buffer [20 mM Tris-HCl (pH 7.4), 120 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 1 mM Na3VO4, supplemented with protease inhibitor mixture (Roche Molecular Biochemicals, Mannheim, Germany)]. Cell lysates were cleared by centrifugation and the total protein concentration was determined by the DC protein assay (Bio-Rad, Hercules, CA). Coimmunoprecipitation assays were done with the Catch and Release Immunoprecipitation System (Upstate Biotechnology, Lake Placid, NY) according to the supplier. Briefly, cell lysates (500 µg) diluted in 1x Catch and Release Lysis/Wash Buffer, 4 µg of anti-Flag antibody (M2, Sigma) and 1 µg of Antibody Capture Affinity Ligand were mixed and placed on a rocking platform for 15 min at room temperature. The lysate, antibody, and antibody capture affinity ligand mixture were transferred to the Catch and Release Spin Column and centrifuged 10 min at 1500 x g. Spin columns were washed three times with 500 µl of 1x Catch and Release Lysis/Wash Buffer for three minutes at 2000 x g. The immune complexes were eluted by adding 30 ml of 1x elution buffer and centrifugation at 500 x g for 2 min. The immune complexes were used for western blotting analysis with the AR antibody (PG21, Upstate Biochemicals). Western blotting analysis of PSA expression was performed in LNCaP cells after treatment of cells with 0.1 nM R1881, increasing concentrations of mifepristone or R1881 plus mifepristone for 48 h, using monoclonal PSA antibody (BioGenex, San Ramon, CA). An antibody for ß-actin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used as the internal control.

ELISA Assay for PSA
LNCaP cells (40,000/well) were plated were plated in 24-well plates in 0.4 ml of medium with 5% CCS onto 24-well plates in 0.4 ml of medium containing 5% CCS and cultured for 24 h. The cells were then treated with 0.1 nM R1881, 1–1000 nM mifepristone, or both for additional 2 d, and medium was collected. A PSA ELISA kit (Alpha Diagnostic International, San Antonio, TX) was used to measure PSA in the medium according to the manufacturer’s directions. Ten-fold dilutions were made to ensure that the sample readings fell within the standard curves.

Cell Growth Assays
LNCaP cells were plated in 96-well tissue culture plates at a cell density of 1000 cells/well and maintained in phenol red-free medium containing 2.5% CCS for 3 d. The cells were then left untreated or treated for 10 d with increasing concentrations of either R1881 mifepristone, bicalutamide, R1881 + mifepristone, or R1881 + bicalutamide. Cell proliferation was determined by Cell Proliferation Kit I (Roche Diagnostics GmbH, Mannheim, Germany) according to the protocol provided.


   ACKNOWLEDGMENTS
 
Christina Fang provided technical assistance.


   FOOTNOTES
 
This work was supported by NIH Grant CA96854 (to E.P.G.).

Abbreviations: AF, Activation function; AR, androgen receptor; ARE, androgen response element; CCS, charcoal-stripped 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.

Received for publication May 23, 2003. Accepted for publication October 20, 2003.


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