Advances in Brief Mutations in the Androgen Receptor Gene Are Associated with Progression of Human Prostate Cancer to Androgen Independence I

Progression to androgen-independent growth of human prostate cancers may be mediated by alterations in the structure and/or expression of the androgen receptor (AR) gene. To date, mutations in the AR gene have largely been identified in hormone refractory tumors. In this study, single-strand conformational polymorphism analysis and DNA sequencing of the entire AR gene coding region was performed on 25 primary prostate tumors sampled prior to initiation of hormonal (i.e., androgen ablation) therapy. Base changes leading to amino acid substitutions in the AR were identified in 11 (44%) tumors. The presence of AR amino acid substitutions was associated with decreased immunohistochemical staining for AR in tumor cells and the rapid failure of subsequent hormonal therapies. Singlestrand conformational polymorphism analysis of exons 2, 3, and 8 of the X-linked hypoxanthine guanine phosphoribosyl transferase (HPRT) gene in the same samples revealed no bandshifts, suggesting that the high frequency of AR gene mutations detected was not a consequence of generalized genetic instability. These data indicate that AR gene mutations occur commonly in advanced prostate cancers prior to endocrine treatment of disease and may contribute to altered androgen responsiveness of the tumors. Introduct ion Prostate cancer is the most frequently diagnosed invasive cancer and the second leading cause of cancer deaths in men in Western countries (1). The predominant form of systemic treatment for patients with metastatic disease is hormonal or androgen ablative therapy (i.e., orchidectomy, luteinizing hormonereleasing hormone analogues, and AR 3 antagonists; Ref. 2). Received 9/22/95; revised 12/18/95; accepted 12/21/95. 1This work was supported by grants from the National Health and Medical Research Council of Australia, Anti-Cancer Foundation of the Universities of South Australia, and Clive and Vera Ramaciotti Foundations. 2 To whom requests for reprints should be addressed. Phone: 61-8-2045511; Fax: 61-8-374-0832; E-mail: suwdt@cc.flinders. edu.au. 3 The abbreviations used are: AR, androgen receptor; SSCP, singlestrand conformational polymorphism; BPH, benign prostatic hyperplasia; PSA, prostate-specific antigen; HPRT, hypoxanthine guanine phosphoribosyl transferase; MIOD, mean integrated optical density; AIS, androgen insensitivity syndrome. Although an initial response to hormonal therapy is observed in 70-80% of patients with advanced disease, most tumors progress rapidly to androgen-independent growth and only 1020% of patients are alive 5 years following diagnosis (3). At the present time, the molecular changes in tumor cells that lead to resistance to endocrine treatments and independence of androgens for growth are poorly understood. The effects of androgens on the development of the normal prostate gland and the growth of prostate tumors are mediated by the AR, which is a member of a superfamily of ligandactivated nuclear transcription factors (4, 5). Proposed mechanisms of progression to androgen-independent growth include loss of AR expression (6, 7), amplification of the AR gene (8), and structural alterations in the receptor protein (9). Other mechanisms that do not involve the AR pathway [e.g., activation of androgen-independent growth factor pathways (10)] have also been proposed. Immunohistochemical studies have demonstrated expression of AR in primary, advanced, and hormone refractory prostate cancers, suggesting that development of androgen-independence is unlikely to be a consequence of loss of AR expression (11). Indeed, AR gene amplification, which possibly facilitates disease progression by enhancing the growth-promoting effects of androgens at low cellular concentrations, has been demonstrated in a subset of hormone refractory prostate cancers (8). The often rapid progression of advanced prostate cancers from androgen-dependent to androgen-independent growth has led to investigation of structural and/or functional alterations in the AR that could account for the development of resistance to hormonal therapies and disease progression. Mutations in the AR gene have been identified in a stage B (12) and a small number of advanced (i.e., stage D2) prostate cancers (13-15). In addition, a recent report has demonstrated that 5 of 10 hormone refractory prostate cancer metastases contained mutations in the ligand-binding domain of the AR (16). Where functional analyses of mutated ARs have been performed, broadening of ligand specificity was demonstrated, suggesting that hormone refractory disease may in part be caused by activation of these receptors in the androgen-depleted environment (9, 16). Although previous studies have demonstrated AR gene mutations in hormone refractory disease, their occurrence prior to initiation of androgen ablation treatment has only rarely been reported (12, 17). This finding, while inconsistent with the frequently short-lived responses of prostate tumors to hormonal therapies, may in part be attributed to the examination of small segments of the AR (e.g., the ligandbinding domain) rather than the complete AR coding region. In the present study, the frequency of mutations in the entire AR coding region in advanced primary prostate cancers was examined using the PCR and SSCP analysis. The presence of base substitutions was confirmed by sequencing of cloned Research. on October 31, 2017. © 1996 American Association for Cancer clincancerres.aacrjournals. org Downloaded from 278 AR Gene Mutations in Prostate Cancer PCR-amplified fragments exhibiting SSCP bands of reproducibly altered mobility. Expression of AR protein was evaluated using quantitative immunohistochemistry and video image analysis. Our results indicate that mutations in the AR gene are present in advanced prostate cancers prior to initiation of treatment and are associated with reduced AR imnmnostaining in tumor cells and the rapid failure of subsequent androgen ablative therapy. Materials and Methods Prostate Tissues and Cell Lines. Twenty-five primary prostate tumors and 12 BPH specimens were obtained from patients undergoing transurethral resections of the prostate performed for acute urinary obstruction. The presence of carcinoma or BPH in the tissue blocks was confirmed by pathological assessment on hematoxylin and eosin-stained paraffin sections (Department of Pathology, Flinders Medical Center, Bedford Park, Australia). All tumor specimens were obtained from patients prior to initiation of hormonal therapy. Tumor stage was determined according to the Modified Whitmore Jewett System (18). The cohort consisted of 9 stage C and 16 stage D~ tumors• Following surgery and commencement of hormonal therapy (orchidectomy and/or androgen receptor antagonists), an initial decrease in serum PSA levels was observed in all patients. Serum PSA measurements were determined by the Department of Clinical Biochemistry at Flinders Medical Center using a solid-phase, two-site immunoenzymatic assay (Tandem-E PSA; Hybritech, Inc., San Diego, CA) as described previously (19). The normal range of serum PSA levels in healthy men is 0 4 ng/ml for this assay. In this study, serum PSA levels were categorized into three groups: category 1, levels <4 ng/ml; category 2, levels between 4 and 10 ng/ml; and category 3, levels >10 ng/ml. Patients were followed clinically with serum PSA measurements and bone and computerized tomographic scans as required. Clinical follow-up and survival data of 3-7 years were available for 20 of the patients. Five patients were lost to follow-up. Tumor response was assessed by standard clinical and biochemical features (i.e., sustained reduction in serum PSA measurements, improvement in bone and computerized tomographic scans, and survival). Outcome groups were defined at 2 years following initiation of hormonal therapy as follows: (a) response--consisted of those patients with stable disease (i.e., no clinical evidence of disease progression, with PSA levels remaining in the original posthormonal therapy category), or improving disease status (i.e., clinical evidence of decreasing tumor mass and a fall in PSA category) or (b) failure--consisted of those patients with progressive disease (i.e., increasing tumor mass with or without a change in PSA category) and/or those who died from prostatic carcinoma within the first 2 years following initiation of hormonal therapy. The human prostate cancer cell lines PC-3 and LNCaP were obtained from the American Type Culture Collection (Rockville, MD) and maintained in culture as previously described (6). Amplification of Genomic DNA. Genomic DNA was isolated from frozen or paraffin-embedded prostate specimens, peripheral blood lymphocytes from normal volunteers, and the _J