Agonist and antagonist activities of hydroxyflutamide and casodex relate to androgen receptor stabilization

BASIC SCIENCE

ELSEVIER

AGONIST AND ANTAGONIST ACTIVITIES OF HYDROXYFLUTAMIDE AND CASODEX RELATE TO ANDROGEN RECEPTOR STABILIZATION* JON A. KEMPPAINEN AND ELIZABETH M. WILSON

he androgen receptor (AR) is a ligand-acti-

T vated transcriptional activator required for male sex development. Recent studies have correlated AR protein stabilization with androgen occupancy, in that slowly dissociating androgens such as dihydrotestosterone (DHT) more effectively stabilize AR against degradation relative to a less potent, more rapidly dissociating androgen such as testosterone.1 In the absence of androgen, AR degrades at a faster rate, suggesting that receptor stabilization is a regulatory mechanism in androgen action. The role of androgen in prostate cancer progression remains unclear. Prostate cancer usually responds to androgen withdrawal and antiandrogen therapy but recurs during the course of treatment. 2 Antiandrogens used in prostate cancer treatment include flutamide and Casodex (bicalutamide) (Zeneca Pharmaceuticals, Wilmington, Del); however, in 40% of patients undergoing prolonged treatment with high-dose antiandrogen, remission occurs after antiandrogen withdrawal, a phenomenon known as flutamide or antiandrogen withdrawal syndrome. 3'4 Mutations in AR can alter the antagonist/agonist activities of antiandrogens. For example, the AR mutation in the human prostate cancer cell line LNCaP, Thr 877 to Ala, enhances the agonist activity of hydroxyflutamide 5,6 and has been identified in prostate cancer specimens. 7"8 Other mutations in the AR ligand-binding domain from prostate cancer samples include Leu 701 to

His, 9 Val 730 to L e u , 12 His

to Met, 10 Val 715 to Met, 11 Arg 726 874 to Tyr, 13 and others. 2'8 Some of these increase transcriptional activation by hydroxyflutamide, 14 as observed for LNCaP mutant AR, 15 and some alter the ligand-binding specificity of AR activation. Although AR mutations may contribute to the development a n d / o r progression of hormone-independent prostate cancers 16.17and to the antiandrogen withdrawal syndrome, the frequency of these mutations in prostate cancer varies from relatively l o w 10-12'18 to high. 19 Moreover, it is not known how many of these mutant ARs increase the relative binding affinity for hydroxyflutamide or other antiandrogens because only a few have been functionally characterized. Thus, the possibility remains that mechanisms other than AR mutations contribute to the antiandrogen withdrawal syndrome. In the present report we investigated the mechanism whereby certain antiandrogens and steroids that are typically androgen antagonists acquire agonist activity. The results indicate that the ability of a ligand to stabilize AR directly relates to its agonist activity. When an AR mutation increases binding affinity for an antagonist, the antagonist gains both AR stabilizing and agonist activity. Furthermore, the antiandrogen hydroxyflutamide at sufficiently high concentrations becomes an agonist with wild-type AR, accounting perhaps for most cases of flutamide withdrawal syndrome. MATERIAL AND METHODS

*This work was supported by Grants HD16910 and P30HD18968 from the National Institute of Child Health and Human Development Center for Population Research, NIH, Bethesda, Maryland. From the Laboratories for Reproductive Biology and Departments of Pediatrics and Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina Reprint requests: Elizabeth M. Wilson, Ph.D., Laboratories for Reproductive Biology, CB 7500 MacNider Building, Room 374, Medical Sciences Research Building, University of North Carolina, Chapel Hill, NC 27599 Submitted: February 22, 1996, accepted (with revisions): April 10, 1996 COPYRIGHT 1996 BY ELSEVIERSCIENCE INC. ALL RIGHTSRESERVED

MATERIALS Cell lines were from the American Type Culture Collection; fetal calf serum for cell propagation from Irvine Scientific, Santa Ana, Calif; bovine calf serum for transfections from HyClone Laboratories, Inc., Logan, Utah; Trans35S-label (L-methionine, [3sS], L-cysteine, [3sS]), 1000 Ci/mmol from ICN Biomedicals, Inc., Costa Mesa, Calif); Pansorbin cells from CalbiochemNovabiochem International, La Jolla, Calif; [ 3H] methyltrienolone ([17-alpha-methyl-3H]R 1881, 80 Ci/mmol) from DupontoNew England Nuclear, Boston, Mass; hydroxyflutamide from Schering Corp., Kenilworth, NJ; Casodex ICI176334 from Zeneca Pharmaceuticals, Wilmington, Del; and steroids and chemicals from Sigma Corp., St. Louis, Mo. 0090-4295/96/$15.00 PII S0090-4295(96)00117-3

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FIGURE 1. Competitive binding of unlabeled Iigands for [SH]R 18B I with wild-type ancl LNCaP androgen receptor (AR). COS cells plated at 2 × 105 cells/well in 12-well tissue culture plates were transiently transfected with I #g/ well of pCMVhAR or pCMVLNCaPhAR expression vector DNA using diethylaminoethyl dextran, as described in Methods. Unlabeled ligand concentrations ranged from 5 to 500 nM, as indicated, and incubations were for 2 hours at 37°C in the presence of 5 nM [3H]R 1881. Nonspecific binding was accounted for by using parallel incubations with I O0-fold excess unlabeled R 188 I. Data are expressed as percent inhibition of binding observed in the absence of competing ligand. Shown are competitive binding curves with wild-type (WT) and LNCaP AR for dihydrotestosterone (DHT), hydroxyflutamide (OH-FL), Casodex, progesterone (Prog), and 17-beta-estradiol (E2).

TRANSIENT EXPRESSION AND A R ASSAYS Cell transfections were performed using monkey kidney COS-1 and CV1 cells maintained in 10% fetal calf serum in Delbecco's modified essential medium and transfected using calcium phosphate for CV1 cells or diethylaminoethyl dextran for COS ceils, as previously described. 2° Androgen receptor ligand binding was performed on transfected COS cells in 12well tissue culture plates containing 2 × 105 cells/well, as previously described. 21 Forty-eight hours after transfection, cells were incubated in serum-free, phenol red-free medium containing 5 nM [3H] R 1881 for 2 hours in the presence and absence of competing unlabeled ligand. After labeling, cells were washed and harvested in 400 #L of 2% sodium dodecyl sulfate, 10% glycerol, and 10 mM Tris, pH 6.8, and radioactivity was determined by scintillation counting. Transient cotransfection of CV1 cells with a mouse mammary tumor virus (MMTV) promoter-luciferase reporter vector and wild-type human pCMVhAR or mutant LNCaP pCMVLNCaPhAR with coding sequence Thr 877 changed to Ala, as previously reported for AR in the LNCaP human prostate cancer cell line, 5.6.1swas performed as previously described. 22Androgen receptor degradation was determined by plating 1.2 × 106 COS cells/10-cm dish and transfecting using diethylaminoethyl dextran 23 with 10 #g of wild-type and LNCaP AR expression vector deoxyribonucleic acid (DNA). Transfected COS cells were incubated with [35S]methionine/cysteine Trans-label (100 #Ci) for 30 min at 37°C, and cells were washed, incubated in the presence and absence of hormones, and harvested as previously described. 1

RESULTS COMPETITIVE BINDING AND TRANSCRIPTIONAL ACTIVATION Unlabeled ligands were tested for their ability to c o m p e t e for [3H] R 1881 binding to wild-type and

1 58

LNCaP AR in competitive binding studies using AR transiently expressed in m o n k e y k i d n e y COS cells. R 1881 ( m e t h y l t r i e n o l o n e ) is a high-affinity synthetic a n d r o g e n agonist. 24 In a g r e e m e n t with previous reports, 6'15 LNCaP AR had increased binding affinity for h y d r o x y f l u t a m i d e and progesterone relative to wild-type AR, with less significant differences in the binding of DHT, Casodex, or estradiol (Fig. 1). Agonist and antagonist activities of the ligands were investigated in transient cotransfection assays using an MMTV-luciferase r e p o r t e r vector that reflects a n d r o g e n - r e g u l a t e d transcriptional activity. 22 As e x p e c t e d of an antiandrogen, concentrations of h y d r o x y f l u t a m i d e b e t w e e n 0.2 and 1 # M inhibited D H T - i n d u c e d transcriptional activity (Fig. 2A). However, as the c o n c e n t r a t i o n of h y d r o x y f l u t a m i d e increased from ] to 10 #M, agonist activity was observed with wild-type AR. These results are consistent with o u r recent r e p o r t that high c o n c e n t r a t i o n s of h y d r o x y f l u t a m i d e ( 10 # M ) are agonists with wild-type AR resulting from h y d r o x y f l u t a m i d e - i n d u c e d AR DNA binding. 22 Agonist activity of 10 # M h y d r o x y f l u t a m i d e was also evident with wild-type AR in the absence of DHT (Fig. 2A). W i t h LNCaP AR, greater agonist activity was observed with h y d r o x y f l u t a m i d e and progesterone, w h i c h correlated with the increase in relative binding affinity by the LNCaP m u t a n t AR. H y d r o x y UROLOGY 48 (1), •996

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Agonist and antagonist activities of androgen receptor (AR) ligands in a luciferase reporter transcriptional assay. CVI cells plated at 3 × 105 cellsl6-cm dish were transfected with I00 ng of pCMVhAR or pCMVLNCaPhAR using calcium phosphate DNA precipitation. Ligands were added 30 and 6 hours before cell harvest. Optical units reflect luciferase activity and are indicated with standard error and fold induction relative to the activity observed in the absence of dihydrotestosterone (DHT). Increasing concentrations of ligand from 0.2 to I 0 t~M were added in the presence or absence of O. I nM DHT. Shown are optical units for wild-type (WT) and LNCaP AR with or without DHT for (A) hydroxyflutamide (OH-FL) and Casodex (Cas) and (B) estradiol (E2) and progesterone. Shown is a representative experiment of at least three independent assays. FIGURE 2.

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flutamide failed to inhibit DHT-induced LNCaP AR agonist activity (data not shown) and was an agonist at concentrations of 10 nM or greater (Fig. 2A). Increased agonist activity with LNCaP AR was also observed with estradiol and progesterone relative to wild-type AR (Fig. 2B). Casodex was a moderate antagonist and displayed relatively little agonist activity with wild-type or LNCaP AR, in agreement with no significant change in relative binding affinity observed in Fig. 1. CORRELATION OF AGONIST ACTIVITY AND A R STABILIZATION

Ligand-induced AR stabilization of wild-type and LNCaP AR was investigated by determin1 60

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ing AR degradation in transiently transfected COS cells after pulse-chase labeling with [35S] methionine in the presence and absence of ligand. As shown in Figure 3B, 100 nM hydroxyflutamide stabilized LNCaP AR against degradation, with a half-time of approximately 10 hours at 37°C, a degradation rate indistinguishable from that determined in the presence of ]0 nM DHT (Fig. 3A). At 1 #M hydroxyflutamide, LNCaP AR was completely stabilized, whereas only slight stabilization of wild-type AR was evident at concentrations as high as 10 #M (Fig. 3B). Thus, hydroxyflutamide was more effective in stabilizing LNCaP AR relative to wild-type AR in agreement with its agonist activity at low concentrations with UROLOGY

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LNCaP AR. Higher concentrations of ligand were required in the stabilization assay than in the transient transcription assay because of high AR protein expression in COS cells. Unlike hydroxyflutamide, Casodex at concentrations up to 10/.zM weakly stabilized wild-type and LNCaP AR (Fig. 3B), in agreement with its low agonist and significant antagonist activities with both receptors. Progesterone at 10 nM stabilized LNCaP AR but had little effect on wild-type AR stabilization until greater than 100 nM (Fig. 3C). Similarly, estradiol was slightly more effective at stabilizing LNCaP than wild-type AR (Fig. 3D). UROLOGY 48 (1), 1996

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COMMENT The present results support the hypothesis that agonist activity of an AR ligand relates directly to its ability to stabilize AR against degradation. In the case of LNCaP AR, a mutation in the ligandbinding domain changes Thr to Ala at residue 877, increasing AR binding affinity for hydroxyflutamide, progesterone, and, to a lesser extent, estradiol, concomitantly increasing the stabilizing and agonist activities of these ligands relative to wildtype AR. Casodex, in contrast, remained an antagonist with LNCaP AR and did not have increased 161

binding affinity or stabilizing activity with this m u t a n t receptor. Nevertheless, antagonists like h y d r o x y f l u t a m i d e b e c o m e agonists with wild-type AR at sufficiently high concentrations, m o s t likely t h r o u g h AR stabilization as well as activation. The c o n c e n t r a t i o n of ligand required for AR stabilization exceeded that for AR i n d u c t i o n of luciferase activity, possibly because only a small percentage of AR needs to be stabilized to detect a transcriptional response in the luciferase assay. F u r t h e r more, there is an a p p r o x i m a t e l y 100-fold higher level of AR expression in COS cells used for the stability assay than in CV1 ceils used for the transcription assays. W h e r e a s 10 # M h y d r o x y f l u t a m ide increased wild-type AR stabilization o n l y slightly, its agonist activity at this c o n c e n t r a t i o n in CV1 cells suggests that AR stabilization o c c u r r e d b u t b e c o m e s detectable only at h i g h e r c o n c e n t r a tions. This is predicted o n the basis of the observation that LNCaP AR stabilization is measurable at c o n c e n t r a t i o n s higher than those r e q u i r e d for agonist activity in the transcription assay. L o n g - t e r m flutamide t r e a t m e n t for prostate cancer results in s e r u m levels of h y d r o x y f l u t a m i d e in the m i c r o m o l a r range 2s that could be sufficient to i n d u c e agonist activity with wild-type AR. Furt h e r m o r e , h y d r o x y f l u t a m i d e m a y a c c u m u l a t e in tissues after p r o l o n g e d treatment, m a k i n g higher a m o u n t s available to b i n d AR. Indeed, patients w i t h d r a w n from flutamide after p r o l o n g e d treatm e n t are m o r e likely to u n d e r g o remission. 26 In patients w h o u n d e r g o a n d r o g e n withdrawal therapy b y castration or leuteinizing h o r m o n e - r e l e a s ing h o r m o n e agonist suppression of testosterone p r o d u c t i o n , the absence of c o m p e t i n g a n d r o g e n m a y c o n t r i b u t e to the agonist activities of h y d r o x y f l u t a m i d e , as previously suggested. 22 A hypothesis for a n t a g o n i s t / a g o n i s t activities of steroid r e c e p t o r ligands proposes that mixed-ligand dimers mediate antagonism, whereas same-ligand dimers mediate agonist activity. It was shown, for example, that the p r o g e s t e r o n e r e c e p t o r forms mixed-ligand dimers with a progestin and antiprogestin and that these mixed-ligand dimers b i n d p o o r l y to r e s p o n s e - e l e m e n t DNA, resulting in ant a g o n i s m Y H y d r o x y f l u t a m i d e m a y be m o r e effective as an AR antagonist in the presence of DHT than in its absence because it inhibits AR binding to a n d r o g e n r e s p o n s e - e l e m e n t DNA by forming D H T - h y d r o x y f l u t a m i d e AR dimers. 22 In patients w h o have u n d e r g o n e a n d r o g e n withdrawal therapy for the t r e a t m e n t of prostate cancer, sufficiently high c o n c e n t r a t i o n s of h y d r o x y f l u t a m i d e in the presence of low a n d r o g e n levels c o u l d prom o t e agonist activity t h r o u g h the f o r m a t i o n of same-ligand h y d r o x y f l u t a m i d e AR dimers. T h e present results s u p p o r t the use of low effectivedose flutamide t r e a t m e n t of prostate cancer to 1 62

minimize potential agonist activity of the active flutamide metabolite with wild-type AR. ACKNOWLEDGMENT.To Dr. Frank S. French for helpful suggestions and review of the manuscript; Michelle Cobb for culturing the cell lines; and Jonathan Hopkins for preparing the illustrations. REFERENCES 1. Zhou ZX, Lane MV, Kemppainen JA, French FS, and Wilson EM: Specificity of ligand-dependent androgen receptor stabilization: receptor domain interactions influence ligand dissociation and receptor stability. Mol Endocrinol 9: 208-218, 1995. 2. Gelmann EP: Androgen receptor mutations in prostate cancer, in Hait W (Ed): Cancer Treatment & Research: Drug Resistance. Norwell, Mass, Kluwer, 1996 (in press). 3. Scher HI, and Kelly WK: Flutamide withdrawal syndrome: its impact on clinical trials in hormone-refractory prostate cancer. J Clin Oncol 11: 1566-1572, 1993. 4. Kelly WK, and Scher HI: Prostate specific antigen decline after antiandrogen withdrawal: The flutamide withdrawal syndrome. J Urol 149: 607-609, 1993. 5. Harris SE, Harris MA, Rong Z, Hall J, Judge S, French FS, Joseph DR, Lubahn DB, Simental JA, and Wilson EM: Androgen regulation of HBGF-1 (~FGF) mRNA and characterization of the androgen receptor mRNA in the human prostate carcinoma cell line LNCaP/A-Dep, in Karr JP, Coffey DS, Smith RG, and Tindall DJ (Eds): Molecular and Cellular Biology of Prostate Cancer, Plenum Press, New York, 1991, pp 315-330. 6. Veldscholte J, Ris-Stalpers C, Kuiper GGJM,Jenster G, Berrevoets C, Claassen E, van Rooij HCJ, Trapman J, Brinkmann AO, and Mulder E: A mutation in the ligand binding domain of the androgen receptor of human LNCaP cells affects steroid binding characteristics and response to anti-androgens. Biochem Biophys Res Commun 173: 534-540, 1990. 7. Gaddipati JP, McLeod DG, Heidenberg HB, Sesterhenn IA, Finger MJ, Moul JW, and Srivastava S: Frequent detection of codon 877 mutation in the androgen receptor gene in advanced prostate cancer. Cancer Res 54: 2861-2864, 1994. 8. Taplin ME, Bubley GJ, Shuster TD, Frantz ME, Sponner AE, Ogata GK, Keer HN, and Balk SP: Mutation of the androgen receptor gene in metastatic androgen independent prostate cancer. New Engl J Med 332: 1393-1398, 1995. 9. Suzuki H, Sato N, Watabe Y, Masai M, Seino S, and Shimazaki J: Androgen receptor gene mutations in human prostate cancer. J Biochem Mol Biol 46: 759-765, 1993. 10. Newmark JR, Hardy DO, Tonb DC, Carter BS, Epstein JI, Issacs WB, Brown TR, and Barrack ER: Androgen receptor gene mutations in human prostate cancer. Proc Natl Acad Sci U S A 89: 6319-6323, 1992. 11. Culig Z, Hobisch A, Cronauer MV, Cato ACB, Hittmair A, Radmayr C, Eberle J, Bartsch G, and Klocker H: Mutant androgen receptor detected in an advanced stage prostatic carcinoma is activated by adrenal androgens and progesterone. Mol Endocrinol 7: 1541-1550, 1993. 12. Elo JP, Kvist L, Leinonen K, Isomaa V, Henttu P, Lukkarinen O, and Vihko P: Mutated human androgen receptor gene detected in a prostatic cancer patient is also activated by estradiol. J Clin Endocrinol Metab 80: 3494-3500, 1995. 13. Tan JA, Sharief Y, Hamil KG, Gregory CW, Zang DY, Sar M, Mohler JL, Pretlow TG, and French FS: Altered ligand specificity of a mutant androgen receptor (AR) in the androgen dependent human prostate cancer xenograft CWR-22: comparison with the LNCaP mutant AR (abstract). Proceedings of the American Urological Association, Annual Meeting, Orlando, Fla, 1996. UROLOGY 48 (1), 1996

14. Peterziel H, Culig Z, Stober J, Hobisch A, Radmayr C, Bartsch G, Klocker H, and Cato ACB: Mutant androgen receptors in prostatic tumors distinguish between amino acid sequence requirements for transactivation and ligand binding. IntJ Cancer 63: 544-550, 1995. 15. Veldscholte J, Voorhorst-Ogink MM, Bolt-de Vries J, van Rooij HCJ, Trapman J, and Mulder E: Unusual specificity of the androgen receptor in the human prostate tumor cell line LNCaP: high affinity for progestagenic and estrogenic steroids. Biochim Biophys Acta 1052: 187-194, 1990. 16. Culig Z, Klocker H, Eberle J, Kaspar F, Hobisch A, Cronauer MV, and Bartsch G: DNA sequence of the androgen receptor in prostatic tumor cell lines and tissue specimens assessed by means of the polymerase chain reaction. Prostate 22: 11-22, 1993. 17. Sartor O, Cooper M, Weinberger M, Headlee D, Thibault A, Tompkins A, Steinberg S, Figg WD, Linehan WM, and Myers CE: Surprising activity of flutamide withdrawal when combined with aminoglutethimide in treatment of Uhormone refractory" prostate cancer. J Natl Cancer Inst 86: 222-227, 1994. 18. Sharief Y, Pretlow T, Wilson EM, Hall SH, Hamil KG, Tan JA, Gumerlock PH, de Vere White R, Schalken JA, French FS, and Mohler JL: Androgen receptor gene mutations associated with androgen independent prostatic carcinoma. Proceedings of the Society for Basic Urologic Research: Basic and clinical aspects of prostate cancer (abstract 27), 1994. 19. Tilley WD, Buchanan G, Hickey TE, and Bentel JM: Mutations in the androgen receptor gene are associated with progression of human prostate cancer to androgen independence. Clin Cancer Res 2: 277-285, 1996. 20. Zhou, ZX, Sar M, Simental JA, Lane MV, and Wilson EM: A ligand dependent bipartite nuclear targeting signal in

UROLOGY 48 (1), 1996

the human androgen receptor: requirement for the DNA binding domain and modulation by the NH2-terminal and carboxyl-terminal sequences. J Biol Chem 269: 13115-13123, 1994. 21. Yarbrough WG, Quarmby VE, Simental JA, Joseph DR, Sar M, Lubahn DB, Olsen KL, French FS, and Wilson EM: A single base mutation in the androgen receptor gene causes androgen insensitivity in the Tfm rat. J Biol Chem 265: 88938900, 1990. 22. Wong CI, Kelce WR, Sar M, and Wilson EM: Androgen receptor antagonist versus agonist activities of the fungicide vinclozolin relative to hydroxyflutamide. J Biol Chem 270: 19998-20003, 1995. 23. Kemppainen JA, Lane MV, Sar M, and Wilson EM: Androgen receptor phosphorylation, turnover, nuclear transport and transcriptional activation: specificity for steroids and antihormones. J Biol Chem 267: 968-974, 1992. 24. Bonne C, and Raynaud JP: Methyltrienolone, a specific ligand for cellular androgen receptors. Steroids 26: 227-232, 1975. 25. B~langer A, Giasson M, Couture J, Dupont A, Cusan L, and Labrie F: Plasma levels of hydroxyflutamide in patients with prostatic cancer receiving the combined hormonal therapy: an LHRH agonist and flutamide. Prostate 12: 79-84, 1988. 26. MoulJW, Srivastava S, and McLeod DG: Molecular implication of the antiandrogen withdrawal syndrome. Semin Urol 13: 157-163, 1995. 27. Edwards DP, Altmann M, DeMarzo A, Zhang Y, Weigel NL, and Beck CA: Progesterone receptor and the mechanism of action of progesterone antagonists. J Steroid Biochem Mol Biol 53: 449-458, 1995.

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