The Prostate Supplement 6:9-12 (I 996) Provocative Aspects of Androgen Genetics Terry R. Brown Department of Population Dynamics, Division of Reproductive Biology, johns Hopkins University School of Hygiene and Public Health, Baltimore, Maryland ABSTRACT: Androgens play a key role in prostate structure and function, leading to the hypothesis that effects of the hormone are an important component in the development of prostatic disease. Differences in serum testosterone levels and 5a-reductase activities between ethnic and racial groups have been implicated in the variable incidence of prostate cancer among certain populations. Androgen receptors transduce the steroid signal within cells, but attempts to correlate differencesin receptor levels with prostatic disease have been unsuccessful. However, molecular studies of androgen receptor gene structure have recently provided new insights toward defining a genetic basis for the pathology associated with three diseases-spinal bulbar muscular atrophy, breast carcinoma, and prostate cancer-affecting middle-aged and older men. In summary, epidemiologic data on androgen biosynthesis, metabolism, and action of androgens and molecular genetic analysis of gene structure have led to a new understanding of the interrelationshipsbetween environmental and genetic factors that may impact on the incidence of certain pathologic conditions in men. 0 19%Wiley-Liss, Inc. KEY WORDS: testosterone, dihydrotestosterone, Sa-reductase, androgen receptor, prostate INTRODUCTION Androgens promote normal male sex differentiation and development, pubertal masculinization, and initiation of spermatogenesis, and maintenance of male sexual function. Testosterone is the primary androgen secreted by the testes, but its conversion by the enzyme, 5a-reductase, to the more biologically active metabolite, 5a-dihydrotestosterone (DHT), is critical for differentiation and development of the male external genitalia and prostate. Both testosterone and DHT bind to the intracellular androgen receptor to regulate androgen-dependent cellular differentiation and function. The role of testosterone and DHT in maintenance of normal prostatic function and in the pathobiology of prostatic carcinoma and benign prostatic hyperplasia has been the focus of many studies [l].At the center of these studies have been measurements of 5a-reductase enzyme activity and intracellular DHT concentrations, and of the intracellular concentration and subcellular localization of the androgen receptor. Pharmacologic approaches to prevention or treatment of prostatic diseases has relied on inhibitors of 5a-reductase and antiandro0 I996 Wiley-Liss, Inc. gens to prevent the conversion of testosterone to DHT and to block the transcriptional activation of gene transcription by the androgen receptor, respectively. A few recent studies of 5a-reductase activity and the androgen receptor have suggested that the production of DHT and the molecular structure of the androgen receptor may provide an unexpected genetic basis for several pathologic conditions in men. ~CX-REDUCTASE ACTIVITY The incidence of prostate cancer varies widely between countries and ethnic groups. Black Americans have the highest incidence rates worldwide, whereas native Japanese have among the lowest. The reasons for this risk differential are unknown; however, cir- Received for publication January 16, 1995; accepted October 5, 1995. Address reprint requests to Dr. Terry R. Brown, Department of Population Dynamics, Division of Reproductive Biology, Johns Hopkins University School of Hygiene and Public Health, 615 North Wolfe Street, Baltimore, MD 21205. 10 Brown Androgen Receptor Prostate Cell R608K blood etfect blood Fig. I. Androgen metabolism and effects on prostate cells. Testosterone (T), the primary androgen secreted by the testes into blood, enters prostate cells, where it can bind directly to the androgen receptor (AR) or be converted by the Sa-reductase enzyme to the more potent androgen, 5a-dihydrotestosterone (DHT). DHT binds t o AR with higher affinity than T. AR regulates androgen effects in cells. Measurement of DHT or of the Sareduced products, 3a-androstanediol glucuronide and androsterone glucuronide. provides an index of androgenic potency. culating testosterone concentrations are higher in young black men than in young white men and may help explain the underlying differences in subsequent prostate cancer incidence between these two populations . By comparison (Fig. l), serum testosterone concentrations of young Japanese men are not significantly different from those in young white or black men. In one study, both black and white men had significantly higher serum levels of 5a-reduced androgen products, androstanediol glucuronide, and androsterone glucuonide, than were found in Japanese men . In a second study, both precursor androgens (dehydroepiandrosterone sulfate and androstenedione) and 5a-reduced androgen products (androstanediol glucuronide and androsterone glucuronide) were higher in the serum of Caucasian men than of Chinese men . These studies provide circumstantial evidence for genetic differences in androgenic activity among men of different racial backgrounds that may have a role in prostatic carcinoma. ANDROGEN RECEPTOR The androgen receptor is a member of the superfamily of ligand-activated nuclear transcription factors that mediates androgen action at the transcriptional level. Cloning of the cDNA and deduction of the structure of the androgen receptor have contributed significantly to our understanding of the molecular basis of androgen action. The androgen receptor protein is composed of 919 amino acids and, like all members of this family, it is structurally organized as three domains; an N-terminal transactivation domain, two zinc-finger motifs within a central DNA-binding domain, and a C-terminal ligand binding domain (Fig. 2). The gene encoding the androgen receptor consists of eight exons, the first encoding the large N-terminal Fig. 2. Mutations in the androgen receptor alter androgen action. In spinal bulbar muscular atrophy, the polymeric glutamine region in the transcriptional activation domain is expanded from the normal number of glutarnine residues ( I 1-30) to more than 40; in male breast cancer, two different amino acid substitutions have been identified in the second zinc finger of the receptor; and in prostate cancer, several different amino acid substitutions in the steroid-binding domain have been associated with altered cell responsiveness t o antiandrogens, progesterone and adrenal androgens. domain, the second and third encoding each of the two zinc fingers required for binding to DNA, and the fourth through eighth exons encoding the hormone binding domain. The gene is localized to the X chromosome at q11-12. Whereas in vitro deletion mutagenesis of the AR cDNA has confirmed the importance and role of the functional domains of the androgen receptor, more detailed understanding of its structure and function has derived from the naturally occurring mutations present in subjects across the phenotypic and clinical spectrum of the androgen insensitivity syndromes . The mutations of the androgen receptor gene associated with the various forms of androgen insensitivity inevitably result in loss of function, either partial or complete, and are manifested by abnormalities in sex differentiation. The present discussion of genetic alterations in the androgen receptor focuses on potential “gain of function” mutations associated with extension of the polymorphic glutamine repeat region of the N-terminal transactivation domain in spinal bulbar muscular atrophy (Kennedy syndrome), mutation of the DNA-binding domain in male breast carcinoma, and somatic cell mutations in the ligand-binding domain from prostate tumors (Fig. 2). X-Linked Spinal Bulbar Muscular Atrophy X-linked spinal bulbar muscular atrophy is a form of adult-onset progressive motor neuron degenerative disease uniquely associated with male hypogonadism (gynecomastia, oligospermia, testicular atrophy, impotence) . Affected men have a characteristic expansion of the trinucleotide repeat, (CAG),, in the 5’ translated portion of the androgen receptor gene from Genetics of Androgen Action a normal, polymorphic length of n = 11-31 to n 240. The resulting androgen receptor protein has an expanded polyglutamine tract in its N-terminal transactivation domain and is postulated to lose an intrinsic function that leads to a mild form of androgen insensitivity; however, it also appears to gain a novel extrinsic function that destroys motor neurons. The unexplained mechanism that culminates in this form of neuron-specific death is the prototype for three different adult-onset autosomal dominant forms of neuronopathies caused by (CAG), expansions in the genes associated with Huntington disease, spinocerebellar ataxia, type 1, and dentatorubropallidoluysian atrophy . A number of mechanisms, including reduction in transcriptional activity due to a conformational aberration in the androgen receptor protein, altered translation initiation efficiencies, or differential proteolytic processing of the receptor protein, have been proposed to explain the involvement of the receptor in this neurodegenerative disease. Breast Cancer Breast cancer is very rare in men, and the etiology is unknown. Factors such as hypoandrogenism in Klinefelter syndrome, testicular atrophy, orchitis, undescended testes, testicular trauma, and infertility have been determined as risk factors for male breast cancer. Recently, two germline mutations in the androgen receptor gene of subjects with breast cancer and partial androgen insensitivity have been reported [7,8]. Both mutations occurred within exon 3 encoding the second zinc finger of the DNA-binding domain of the androgen receptor. Each was caused by a point mutation in adjacent codons causing amino acid substitutions of argm7-*glu and arg608+1ys. The mechanism by which these mutant receptors can lead to breast cancer is unknown, as the function of these mutant receptors has not been characterized in a transfection system. One could postulate that these amino acid substitutions within the DNA-binding domain might lead to alternative recognition of hormone response elements such that their respective androgen specificity of transactivation might be altered. Prostate Cancer Prostate cancer is one of the most common cancers in men. The growth of prostatic carcinomas is sensitive to androgen and hormonal manipulation has been used for its treatment. About 75% of prostate cancers initially respond to endocrine therapy; however, more than one-half of responders gradually become resistant to this therapy. Changes in tumors from an androgen-responsive to an androgen-resis- II tant state have been explained by adaptation or clonal selection of cancer cells. Thus, relapsed tumors consist primarily of androgen-independent cells. These cells contain variable levels of androgen receptor by ligand binding assays and often retain immunocytochemically detectable androgen receptor protein. These findings suggest that structural abnormalities may arise in the androgen receptors of tumor cells to alter their function. A mutation in the androgen receptor of LNCaP cells, derived from a metastatic lesion in the lymph node of a subject with prostate cancer, provided the first evidence that such mutations might exist in prostate tumor cells . Subsequently, our laboratory was the first to idenbfy a somatic cell mutation in an organ-confined tumor of a subject with prostatic carcinoma [lo]. The mutation &A of codon 730 was present in approximately 50% of cells from the tumor specimen, whereas only the normal coding sequence was identified in DNA from peripheral lymphocytes of the same individual. The mutation in LNCaP cells occurs in codon 877 (thwala) of exon 8 and the mutation in exon 5 of our patient caused substitution of valnO+met. The mutation of thr877+ala leads to paradoxical stimulatory activity of the androgen receptor in the presence of the antiandrogen, hydroxyflutamide. Recent studies have shown that this same mutation is present in the prostate tumors from other subjects, suggesting that this region is hypermutable during the evolution of tumors to the metastatic form and from androgen-responsive to androgen-independent [ll].One subject with the AR gene mutation, thrsV+ala, in a metastatic tumor also had a second mutation, leu701+his, in the primary tumor . In an androgen-unresponsive tumor from another subject, a G-A transition occurred in codon 715 of the AR gene, causing the substitution of met for Val 1131. In transfection assays, this mutation did not significantly alter transactivation by androgens such as dihydrotestosterone or mibolerone but increased the relative ability of progesterone and the adrenal androgens, androstenedione and dehydroepiandrosterone, to induce androgen-responsive gene transcription. These findings suggest that the mutant receptor leads to a gain of function, rather than a loss. Another AR gene alteration that contracted the number of glutamine repeats from 24 in some cells to 18 in others, occurred in the tumor from a subject with a paradoxical response to flutamide therapy . The functional significance of this example of genetic instability in tumor cells is unknown. SUMMARY Taken together, these results suggest an involvement of AR mutations in tumor progression, rather I2 Brown than in tumor promotion. Functional characterization indicates an increase in receptor activity related to alterations in its ligand-binding properties. Whereas most specimens investigated represent primary lesions of early and intermediate stages, further examination of highly malignant prostatic lesions and tumor metastases is required to determine the full extent of androgen receptor involvement in tumor progression and the design of adequate therapeutic modalities. ACKNOWLEDGMENT The work performed in our laboratory was supported by grant DK43147 from the National Institutes of Health. REFERENCES 1. Montie JE, Pienta KJ: Review of the role of androgenic hormones in the epidemiology of benign prostatic hyperplasia and prostate cancer. Urology 43:892-899, 1994. 2. Ross RK, Bernstein L, Lob0 RA, Shimizu H, Stanczyk FZ, Pike MC, Henderson BE: 5-alpha-Reductase activity and risk of prostate cancer among Japanese and US white and black males. Lancet 339:887-889, 1992. 3. Lookingbill DP, Demers LM, Wang C, Leung A, Rittmaster RS, Santen RJ: Clinical and biochemical parameters of androgen action in normal Caucasian versus Chinese subjects. J Clin Endocrinol Metab 72:12421248, 1991. 4. LaSpada AR, Wilson EM, Lubahn DB: Androgen receptor gene mutations in X-linked spinal and bulbar muscular atrophy. Nature 35297-79, 1991. 5. Brown TR: Androgen insensitivity syndrome. J Androl 16~299-303,1995. 6. Caskey CT,Pizzuti A, Fu Y-H, Fenwick RG, Nelson DL: Triplet repeat mutations in human disease. Science 2561784-789, 1992. 7. Wooster R, Mangion J, Eeles R, Smith S, Dowsett M, Averill D, Barrett-Lee P, Easton DF, Ponder BAJ, Stratton MR A germline mutation in the androgen receptor gene in two brothers with breast cancer and Reifenstein syndrome. Nature Genet 2:132-134, 1992. 8. Lobaccaro J-M, Lumbroso S, Belon C, Galtier-Dereure F, Bringer J, Lesimple T, Namer M, Cutuli BF, Pujol H, Sultan C: Androgen receptor gene mutation in male breast cancer. Hum Mol Genet 2:1799-1802, 1993. 9. Veldscholte J, Ris-Staplers C, Kuiper GGJM, Jenster G, Berrevoets C, Classen E, van Rooij HCJ, Trapman J, Brinkmann AO, Mulder E: A mutation in the ligand binding domain of tahe androgen receptor of human LNCaP cells affects steroid binding characteristics and response to anti-androgens. Biochem Biophys Res Commun 173:534-540, 1990. 10. Newmark JR, Hardy DO, Tonb DC, Carter BS, Epstein JI, Isaacs WB, Brown TR, Barrack E R Androgen receptor gene mutations in human prostate cancer. Proc Natl Acad Sci USA 89:6319-6323, 1992. 11. Gaddipati JP, McLeod DG, Heidenberg HB, Sesterhenn IA, Finger MJ, Moul JW, Srivastava S: Frequent detection of codon 877 mutation in the androgen receptor gene in advanced prostate cancers. Cancer Res 54:28612864, 1994. 12. Suzuki H, Sat0 H, Watabe Y, Masai M, Seino S, Shimazaki J: Androgen receptor gene mutations in human prostate cancer. J Steroid Biochem Mol Biol46:759-765, 1994. 13. Culig Z, Hobisch A, Cronauer MV, Cat0 ACB, Hittmair A, Radmayr C, Eberle J, Bartsch G, Mocker H: Mutant androgen receptor detected in an advanced stage prostatic carcinoma is activated by adrenal androgens and progesterone. Mol Endocrinol 7:1541-1550, 1993. 14. Schoenberg MP, Hakimi JM, Wang S, Bova GS, Epstein JI, Fischbeck KH, Isaacs WB, Walsh PC, Barrack E R Microsatellite mutation (CAG24j-18) in the androgen receptor gene in human prostate cancer. Biochem Biophys Res Commun 198:74-80, 1994.