Promotion of Agonist Activity of Antiandrogens by the Androgen Receptor Coactivator, ARA70, in Human Prostate Cancer DU145 Cells

Proc. Natl. Acad. Sci. USA Vol. 95, pp. 7379–7384, June 1998 Biochemistry Promotion of agonist activity of antiandrogens by the androgen receptor coactivator, ARA70, in human prostate cancer DU145 cells HIROSHI MIYAMOTO*, SHUYUAN YEH*, GEORGE WILDING†, AND CHAWNSHANG CHANG*†‡ *George Whipple Laboratory for Cancer Research, Departments of Pathology, Urology, and Biochemistry, University of Rochester Medical Center, Rochester, NY 14642; and †University of Wisconsin Comprehensive Cancer Center, Madison, WI 53792 Communicated by Jack Gorski, University of Wisconsin-Madison, Madison, WI, April 15, 1998 (received for review January 9, 1998) ABSTRACT Although hormone therapy with antiandro- androgens (13–15). However, the mechanisms responsible for gens has been widely used for the treatment of prostate cancer, androgen independence and the antiandrogen withdrawal some antiandrogens may act as androgen receptor (AR) syndrome remain unclear. agonists that may result in antiandrogen withdrawal syn- In the present study, we investigated the influence of six drome. The molecular mechanism of this agonist response, antiandrogens and related compounds on AR transcriptional however, remains unclear. Using mammalian two-hybrid as- activity. Classic steroid binding assays have suggested these say, we report that antiandrogens, hydroxyflutamide, bicalu- compounds can inhibit the binding of androgens to the AR in tamide (casodex), cyproterone acetate, and RU58841, and target cells. Three of these antiandrogens have been used other compounds such as genistein and RU486, can promote clinically in the treatment of prostate cancer: CPA, HF, and the interaction between AR and its coactivator, ARA70, in a bicalutamide (casodex). CPA is a synthetic steroidal antian- dose-dependent manner. The chloramphenicol acetyltrans- drogen and one of the first antiandrogens used clinically in ferase assay further demonstrates that these antiandrogens Europe (16); however, it has many side effects (17). HF is a and related compounds significantly enhance the AR tran- nonsteroidal antiandrogen and seems to be better tolerated scriptional activity by cotransfection of AR and ARA70 in a 1:3 than CPA (5, 16). Casodex is a newer nonsteroidal antiandro- ratio into human prostate cancer DU145 cells. Our results gen originally thought to be a ‘‘pure antiandrogen’’ without suggest that the agonist activity of antiandrogens might occur agonist activity. It has a long half-life (6 days) and a higher with the proper interaction of AR and ARA70 in DU145 cells. binding affinity to the AR than HF (18, 19). In addition to These findings may provide a good model to develop better these three antiandrogens, three other compounds were tested antiandrogens without agonist activity. in this study: genistein, RU486, and RU58841. Genistein is an isoflavonoid phytoestrogen found in some edible plants, in- The androgen receptor (AR) is a member of a large family of cluding soybean (20), that may contribute to the prevention of ligand-dependent transcriptional factors known as the steroid prostate cancer. In vitro studies with genistein have revealed an receptor superfamily (1, 2). Androgens and the AR play an antiproliferative effect in a variety of human cancers, including important role in the growth of prostate cancer and normal prostate cancer (20, 21). However, the molecular mechanisms prostate. Prostate cancer represents the most common male of the anti-tumor action of genistein remain poorly under- malignancy in the United States. In 1998, about 39,200 men stood. RU486 was identified first as antiprogesterone (22) and will lose their lives to this malignancy (3). Since 1941 (4), later determined to have antiandrogen properties as well (9). androgen ablation has been the cornerstone of treatment in It was reported that RU486 can block the interaction of the patients with locally advanced or metastatic prostate cancer. receptor with the transcription initiation complex (23). Recently, antiandrogens such as hydroxyflutamide (HF) in RU58841 is a new nonsteroidal antiandrogen with high affinity combination with surgical or medical castration have been for the ARs from hamster prostate and flank organ. Animal widely used for the treatment of this disease (5). Although the studies suggest that RU58841 is a candidate for the treatment response rate to such androgen ablation therapy is high, the of androgen-dependent skin diseases (24). effect is often short-lived (5), as androgen-dependent prostate Immunohistochemical staining showed AR expression may cancer progresses into androgen independence. On the other contribute to the response to hormone therapy (25, 26). hand, the agonist effect of antiandrogens such as HF and Several recent studies have also postulated that the alterations cyproterone acetate (CPA) on proliferation of human prostate of AR functions by mutations of this gene may be associated cancer LNCaP cells was recognized in recent years (6). Sub- with a poor response to antiandrogens (27, 28); the real sequently, it was revealed that antiandrogens could activate the mechanism, however, remains unclear. Furthermore, all the AR with a mutation in its ligand binding domain (7). Reports using transient transfection studies (8–10) suggest that anti- documented results cannot rule out the contribution of cellular androgens may act as agonists on the wild-type AR as well as environment and non-AR factors to the alteration of androgen mutant AR. Indeed, clinical data also indicate that HF can activity. Recently, we successfully isolated the first relatively activate the AR-targeted prostatic specific antigen gene and specific AR coactivator, ARA70, which can enhance AR that serum levels of the prostatic specific antigen can decrease transcriptional activity an additional 7–10-fold in human pros- after discontinuation of HF. This phenomenon is known as tate cancer cells (29). We report here that ARA70 can also flutamide withdrawal syndrome (11, 12). In the past few years, enhance the agonist activity of antiandrogens 3–30-fold in withdrawal responses have also been reported for other anti- human prostate cancer DU145 cells. The publication costs of this article were defrayed in part by page charge Abbreviations: AR, androgen receptor; HF, hydroxyflutamide; CPA, cyproterone acetate; CAT, chloramphenicol acetyltransferase; DHT, payment. This article must therefore be hereby marked ‘‘advertisement’’ in dihydrotestosterone. accordance with 18 U.S.C. §1734 solely to indicate this fact. ‡To whom reprint requests should be addressed at: University of © 1998 by The National Academy of Sciences 0027-8424y98y957379-6$2.00y0 Rochester Medical Center, 601 Elmwood Avenue, Box 626, Roch- PNAS is available online at http:yywww.pnas.org. ester, NY 14642. e-mail: [email protected]. 7379 Downloaded by guest on September 28, 2021 7380 Biochemistry: Miyamoto et al. Proc. Natl. Acad. Sci. USA 95 (1998) MATERIALS AND METHODS ment method (Bio-Rad). Blots were quantitated by COLLAGE IMAGE ANALYSIS software (Fotodyne). Antiandrogens. HF was obtained from Schering, CPA was obtained from Sigma, casodex was obtained from ICI Phar- maceuticals, genistein was obtained from GIBCO BRL, and RESULTS RU486 and RU58841 were generously provided by H. Uno Interactions between AR and ARA70. To demonstrate phys- (University of Wisconsin, Madison). ical interactions between AR and ARA70, we used a mam- Plasmids. pSG5-AR and pSG5-ARA70 were constructed as malian two-hybrid assay. For this study, AR fragments (wild previously described (29). Two mutants of the AR gene type or mutants) were generated and fused to the DNA (mAR877 derived from prostate cancer, codon 877 mutation binding protein GAL4. Similarly, ARA70 fragment was gen- Thr to Ser; and mAR708 derived from partial androgen erated and fused to the transcriptional activation VP16. These insensitivity syndrome, codon 708 mutation Glu to Lys) were fusion plasmids were then coexpressed in DU145 cells with a provided by S. P. Balk (Beth Israel Hospital, Boston) and H. plasmid containing the CAT reporter (Fig. 1A). Transient Shima (Hyogo Medical College, Japan), respectively. pGAL0– transfection of wild-type AR and ARA70 with mock treatment wild-type AR was provided by D. Chen (University of Mas- showed no effect on induction of CAT activity. However, when sachusetts, Worcester). pGAL0–mAR877 and pGAL0– treated with 1 nM DHT, the CAT activity increased about mAR708 were constructed by inserting fragments, mAR877 40-fold over the mock treatment (data not shown). Expression and mAR708, into the pGAL0 vector. Similarly, pGAL4– of either wild-type AR or ARA70 alone resulted in less than VP16 was used to construct the fusion of ARA70. pGAL0 1.5-fold activation even in the presence of androgen. We then contains the GAL4 DNA binding domain, and pGAL4–VP16 tested six different antiandrogens and related compounds. As contains the GAL4 DNA binding domain linked to the acidic shown in Fig. 1B, the addition of 10 mM antiandrogens can activation domain of VP16. induce the CAT activity 3–6-fold over the basal level in the Cell Culture and Transfections. Human prostate cancer presence of ARA70. We then extended these findings to two DU145 cells were maintained in Dulbecco’s minimum essen- different AR mutants, mAR877 from prostate cancer and y tial medium containing penicillin (25 units ml), streptomycin mAR708 from a partial androgen insensitivity syndrome pa- m y (25 g ml), and 5% fetal calf serum. Transfections were tient. Similar results were obtained when mAR877

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