Androgen Action in Prostate Cancer
Donald Tindall
l
James Mohler
Editors
Androgen Action in Prostate Cancer
Editors Donald Tindall 200 1st Street SW., Rochester MN 55905 USA
[email protected]
James Mohler Elm and Carlton Streets Buffalo NY 14263 USA
[email protected]
ISBN 978-0-387-69177-0 e-ISBN 978-0-387-69179-4 DOI: 10.1007/978-0-387-69179-4 Springer Dordrecht Heidelberg London New York Library of Congress Control Number: 2008942188 # Springer Science+Business Media, LLC 2009 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)
Contents
Introduction: Proper Nomenclature Facilitates Clinical and Translational Research in Prostate Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 James L. Mohler and Donald J. Tindall Part I: The Role of Androgens in Developement of the Normal Prostate and Progression of Prostate Cancer Androgen Action and Modulation of Prostate and Prostate Cancer Growth: An Historical Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Shutsung Liao, John M. Kokontis, Chih-Pin Chuu, and Richard A. Hiipakka Clinical Progression to Castration-Recurrent Prostate Cancer . . . . . . . . . . . . . 57 Mark Pomerantz and Philip Kantoff Differential Roles of Androgen Receptor in Prostate Development and Cancer Progression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 Shuyuan Yeh, Yuanjie Niu, Hiroshi Miyamoto, Tamin Chang, and Chawnshang Chang Imaging Androgen Receptor Function In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 Michael Carey and Lily Wu Part II: Androgen Metabolism Increased Expression of Genes Converting Adrenal Androgens to Testosterone in Castration-Recurrent Prostate Cancer . . . . . . . . . . . . . . . . 123 Steven P. Balk
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Androgen-Metabolic Genes in Prostate Cancer Predisposition and Progression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141 Juergen K.V. Reichardt and Ann W. Hsing Effect of Steroid 5a-Reductase Inhibitors on Markers of Tumor Regression and Proliferation in Prostate Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . 155 Lynn N. Thomas and Roger S. Rittmaster 5a-Reductase Isozymes in Castration-Recurrent Prostate Cancer . . . . . . . 175 Mark A. Titus and James L. Mohler Improving Intermittent Androgen-Deprivation Therapy: OFF Cycle and the Role of Steroid 5a-Reductase Inhibitors . . . . . . . . . . . . . 187 Shubham Gupta, Daniel Shevrin, and Zhou Wang Part III: Androgen Receptor Structure/Function Relationships Insights from AR Gene Mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207 Grant Buchanan, Eleanor F. Need, Tina Bianco-Miotto, Norman M. Greenberg, Howard I. Scher, Margaret M. Centenera, Lisa M. Butler, Diane M. Robins, and Wayne D. Tilley Functional Motifs of the Androgen Receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241 Elizabeth M. Wilson The Role of the Androgen Receptor Polyglutamine Tract in Prostate Cancer: In Mice and Men . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269 Diane M. Robins The Androgen Receptor Coactivator-Binding Interface . . . . . . . . . . . . . . . . . . . 297 Eva Este´banez-Perpin˜a´ and Robert J. Fletterick Part IV: Co-Regulators of the Androgen Receptor Co-regulators and the Regulation of Androgen Receptor Action in Prostate Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315 Irina U. Agoulnik and Nancy L. Weigel Androgen Receptor Coregulators and Their Role in Prostate Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341 Latif A. Wafa, Robert Snoek, and Paul S. Rennie Interaction of the Androgen Receptor Ligand-Binding Domain with the N-Terminal Domain and with Coactivators . . . . . . . . . . . . . 375 Jan Trapman
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Multitasking and Interplay Between the Androgen Receptor Domains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385 F. Claessens, T. Tanner, and A. Haelens Chromatin Remodeling and Androgen Receptor-Mediated Transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 405 Li Jia, Omar Khalid, Baruch Frenkel, and Gerhard A. Coetzee Part V: Ligand-Independent Activation of the Androgen Receptor Ligand-Independent Androgen Receptor Activity . . . . . . . . . . . . . . . . . . . . . . . . . 427 Scott M. Dehm and Donald J. Tindall Role of IL-6 in Regulating the Androgen Receptor . . . . . . . . . . . . . . . . . . . . . . . . 451 Zoran Culig and Alfred Hobisch The Role of Cyclic AMP in Regulating the Androgen Receptor . . . . . . . . . . 465 Marianne D. Sadar Part VI: Role of the Androgen Receptor in Prostate Cancer During Castration Cellular and Molecular Signatures of Androgen Ablation of Prostate Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 507 Clifford G. Tepper and Hsing-Jien Kung Tissue Levels of Androgens in Castration-Recurrent Prostate Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 553 James L. Mohler and Mark A. Titus Unique Effects of Wnt Signaling on Prostate Cancer Cells: Modulation of the Androgen Signaling Pathway by Interactions of the Androgen Receptor Gene and Protein with Key Components of the Canonical Wnt Signaling Pathway . . . . . . . . . . . . . . . . . . . . 569 Matthew J. Tanner, Elina Levina, Michael Shtutman, Mengqian Chen, Patrice Ohouo, and Ralph Buttyan The Role of Foxa Proteins in the Regulation of Androgen Receptor Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 587 David J. DeGraff, Xiuping Yu, Qian Sun, Janni Mirosevich, Ren Jie Jin, Yongqing Wang, Aparna Gupta, Srinivas Nandana, Thomas Case, Manik Paul, Hong-Ying Huang, Ellen Shapiro, Susan Logan, Kichiya Suzuki, Marie-Claire Orgebin-Crist, and Robert J. Matusik
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Part VII: Androgen-Regulated Genes during Prostate Cancer Progression Androgen Receptor as a Licensing Factor for DNA Replication . . . . . . . . . 619 Donald J. Vander Griend and John T. Isaacs Androgen-Regulated Genes in the Prostate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 631 Nigel Clegg and Peter S. Nelson Mapping the Androgen Receptor Cistrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 663 Qianben Wang and Myles Brown Differential Regulation of Clusterin Isoforms by the Androgen Receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 681 Tanya K. Day, Colleen C. Nelson, and Martin E. Gleave Androgen Regulation of Prostate Cancer Gene Fusions . . . . . . . . . . . . . . . . . . . 701 Rou Wang, Scott A. Tomlins, and Arul M. Chinnaiyan Androgens and the Lipogenic Switch in Prostate Cancer . . . . . . . . . . . . . . . . . 723 Johannes V. Swinnen, Koen Brusselmans, Hannelore V. Heemers, and Guido Verhoeven Part VIII: The Androgen Receptor as a Drugable Target Molecular Biology of Novel Targets Identified Through Study of Castration-Recurrent Prostate Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . 743 Philip A. Watson and Charles L. Sawyers Selenium and Androgen Receptor in Prostate Cancer . . . . . . . . . . . . . . . . . . . . . 755 Nagalakshmi Nadiminty and Allen C. Gao Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 781
Introduction: Proper Nomenclature Facilitates Clinical and Translational Research in Prostate Cancer James L. Mohler and Donald J. Tindall
This book serves as a tribute to the work of the many investigators who have attempted to understand the role of the androgen receptor (AR) in the development and progression of prostate cancer. The relationship between prostate cancer and androgen status was initially appreciated in the late 1800s, and then awareness reawakened in the 1940s (Huggins and Hodges 2002) by the studies of Charles Huggins and colleagues, for which the Nobel prize was awarded in 1966. Androgen deprivation therapy by surgical or medical means using methods that remove the source of testicular androgens or inhibit the production of testicular androgens, respectively, produced remission in most, but not all, men. Moreover, clinical studies have cast doubt on whether ‘‘combined androgen blockade’’ (Labrie et al. 1982) is any more effective than androgen deprivation monotherapy (Prostate Cancer Trialists’ Collaborative Group, 1995), except when an antiandrogen is used to block testosterone flair in men with bulky metastatic disease. Nonetheless, responses to androgen deprivation therapy vary widely. In order to explain this clinical variation, it was proposed initially that prostate cancer consists of ‘‘androgen-dependent’’ and ‘‘androgen-independent’’ cells (Carter and Isaacs 1988; Isaacs and Coffey 1981). The argument went as follows: The relative distribution of prostate cancer cells between these two phenotypes determine whether there is a clinical response to androgen deprivation therapy and the duration of any response. If the tumor consists of androgen-dependent cells entirely, androgen deprivation therapy would prove curative since all androgen-dependent cells would undergo apoptosis upon androgen deprivation. If a prostate cancer is composed exclusively of androgenindependent cells, then no clinical response to androgen deprivation therapy would occur and that patient would ‘‘resist’’ androgen deprivation therapy and succumb to his prostate cancer as if androgen deprivation therapy had never been administered. Almost, if not, all patients fall between these two extremes and exhibit clinical remissions, which may be complete and sustained if the androgen-dependent cells far outnumber the androgenindependent cells. Because apoptosis does not occur in as high a proportion of cells as predicted, a third phenotype of prostate cancer cell was proposed, the androgen-sensitive cell (Pollack et al. 1983; Grossman 1986). The ‘‘androgen-sensitive’’ cell is responsive to androgen deprivation therapy by becoming quiescent until such time that androgens become available. Thus, the extent of regression of prostate cancer after androgen
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J.L. Mohler, D.J. Tindall deprivation therapy is influenced by the relative proportion of androgen-dependent, androgen-sensitive and androgen-independent cells. The eventual emergence of the ‘‘androgen-independent phenotype’’ is predicted by the proportion of androgen-independent cells.
The development of prostate cancer cell lines, prostate cancer cell line xenografts, and human prostate cancer xenografts has enabled translational researchers to model manifestations of clinical disease progression. The availability of these models has resulted in increased complexity in terminology. Many commercially available tissue culture media contain levels of androgens sufficient to activate even a normal androgen receptor. However, a truly androgen-independent state can be achieved in a cell culture environment by careful charcoal stripping of media and the avoidance of inclusion of phenol red. Hence, investigators should prove that their androgen-free media is truly androgen free lest results be compromised. In the in vitro situation, proper terminology allows for androgen-dependent, androgensensitive, or androgen-independent growth. An androgen-dependent cell line is one that is comprised exclusively of androgen-dependent cells, which undergo apoptosis upon androgen withdrawal. Restoration of androgen after a certain period would have no effect since all cells would have died. Even the LNCaP cell line does not meet this definition. LNCaP cells vary their growth rate many fold depending on androgen levels and exhibit a well-known bimodal growth response to androgens; androgen levels either too low or too high inhibit growth (Gregory et al. 2001). Thus, LNCaP should be considered an androgen-sensitive cell line in which the vast majority undergoes apoptosis but some cells become quiescent, i.e., growth ceases until such time that androgens are restored in the media. LNCaP cells grown in progressively androgen-depleted media selects for ‘‘androgen-independent’’ variants. But androgen response may be even more complicated; although xenografted cells grow equally well in castrate or intact hosts, they decrease growth rate approximately twofold upon androgen stimulation (Lee et al. 2007). Other ‘‘androgen-independent’’ cells, such as CWR-R1 and LNCaP-C4-2, increase growth twofold when androgens are added to the media (Gregory et al. 2001). Even greater difficulties ensue when the terms androgen-dependent, androgensensitive, and androgen-independent are applied to xenografts and patients. The mouse has widely fluctuating levels of testosterone, and most investigators control testosterone levels by surgical castration and implantation of sustained-release pellets to provide physiologically relevant (human) androgen stimulation of xenografted cell lines or clinical specimens. However, the laboratory mouse differs markedly from the human. Testicular androgen levels in the castrate mouse are below the level of detection using radioimmunoassay and routine mass spectrometric measurement (Van Steenbrugge 1991; Jin et al. 2004). However, clinically significant circulating DHEA levels may be similar in intact and castrate BALB/c nude mice (Jin et al. 2004). Removal of sustained release androgen pellets creates an androgen deprivation therapy situation different than in the human due to the availability in the human of very high levels of circulating low-affinity andrenal androgens. In addition, many androgen-sensitive cell lines (LNCaP, LAPC4), androgen-dependent xenografts (CWR22, LUCaP), or clinical specimens (fresh
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tissue samples) require orthotopic seeding, which is challenging technically, or coadministration with Matrigel1 for establishment and growth, even in the face of normal circulating levels of testicular androgens. In the human patient, advanced prostate cancer is treated with androgen deprivation therapy. In rare cases, no clinically measureable response will occur, and the tumor should be labeled appropriately ‘‘castration-resistant prostate cancer’’ (Scher and Sawyers 2005). However, most men experience a clinical and/or biochemical regression and, hence, their tumor should be considered either androgen-dependent or -sensitive. Unfortunately, an androgen-dependent tumor cannot be distinguished from an androgen-sensitive tumor in a human because of the inability to assess accurately the degree of apoptosis vs. cell quiescence, since biopsy specimens are not obtained routinely to measure apoptosis directly, and neither spatial nor functional imaging is reliable for accurate response assessment. Thus, the treatmentnaive prostate cancer is best called ‘‘androgen-stimulated,’’ thus avoiding an assumption of androgen dependence, sensitivity, or resistance. Almost no men with advanced prostate cancer are cured by androgen deprivation therapy. Thus, the relative distribution of malignant epithelial cells among the androgen-dependent, androgen-sensitive, and androgen-independent compartments is really only of academic interest. Of more practical interest is the proper terminology for prostate cancer that returns clinically during androgen deprivation therapy. What should the tumor be called when it recurs after some degree of response to androgen deprivation therapy? ‘‘Androgen-independent’’ is an inappropriate term since it is now well established that prostate cancer, which recurs during surgical or medical androgen deprivation therapy, has tissue levels of testicular androgens that are sufficient for activation of even a normal androgen receptor (Geller et al. 1979; Mohler et al. 2004; Titus et al. 2005). In fact, a recent publication has suggested that benign prostate tissue also contains levels of testicular androgens sufficient to activate the androgen receptor within 4 weeks after initiation of androgen deprivation therapy (Page et al. 2006). ‘‘Hormone refractory’’ is an equally imprecise terminology since the prostate cancer dependence upon androgens far outweighs the magnitude of its dependence of any of the other steroid hormones and, furthermore, some recurrent prostate cancers will respond to secondary treatment with nonandrogenic steroid hormones. When prostate cancer recurs after radiation therapy, it is often called ‘‘radiationrecurrent.’’ When prostate cancer recurs after radical prostatectomy, it is often deemed recurrent, which should more appropriately be termed ‘‘radical prostatectomy-recurrent.’’ Hence, a nomenclature is proposed when a patient with unsuccessfully treated, early-detected prostate cancer may eventually be described as having Gleason grade 4 + 3 = 7, radical prostatectomy-recurrent, salvage radiationrecurrent, castration-recurrent, anti-androgen-recurrent, taxotere-recurrent, and prednisone-recurrent prostate cancer. A similar nomenclature could be applied to xenografted cell lines or human tissue specimens. In contrast, when modeling prostate cancer in vitro, use of the term ‘‘androgen-independent’’ is appropriate when media has been properly charcoal-stripped by the investigator or androgen levels have been confirmed below the limit of detection using appropriate mass
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Table 1 The terms used and preferred (bold) for clinical prostate cancer and its treatment Treatment Naı¨ve Treatment Clinically recurrent cancer Androgen-dependent Androgen ablation therapy Hormone-relapsed Androgen-sensitive Hormone therapy Androgen-independent Hormone-dependent Androgen depletion therapy Castration-resistant Hormone-stimulated Castration Recurrent Androgen-stimulated Combined androgen Hormone-resistant blockade Total androgen blockade Androgen-deprivation therapy current Hormone withdrawal Lethal phenotype Androgen withdrawal Androgen depletion-independent Androgen deprivation Castration-recurrent therapy
spectrometry techniques. More effective communication between translational scientists, clinical scientists, clinicians, and patients will be facilitated by proper description of androgen status, and response to treatment, of prostate cancer. Thus, the editors recommend a uniform nomenclature (see Table 1) that hopefully allows the reader to more easily grasp the excellent reviews provided by the contributors.
References Carter, H. B., Isaacs, J. T.: Experimental and theoretical basis for hormonal treatment of prostatic cancer. Semin Urol, 6: 262, 1988 Geller, J., Albert, J., Loza, D.: Steroid levels in cancer of the prostate-markers of tumour differentiation and adequacy of anti-androgen therapy. J Steroid Biochem, 11: 631, 1979 Gregory, C. W., Johnson, R. T., Jr., Mohler, J. L. et al.: Androgen receptor stabilization in recurrent prostate cancer is associated with hypersensitivity to low androgen. Cancer Res, 61: 2892, 2001 Grossman, H. B.: Hormonal therapy of prostatic carcinoma: is there a rationale for delayed treatment? Urology, 27: 199, 1986 Huggins, C., Hodges, C. V.: Studies on prostatic cancer: I. The effect of castration, of estrogen and of androgen injection on serum phosphatases in metastatic carcinoma of the prostate. 1941. J Urol, 168: 9, 2002 Isaacs, J. T., Coffey, D. S.: Adaptation versus selection as the mechanism responsible for the relapse of prostatic cancer to androgen ablation therapy as studied in the Dunning R-3327-H adenocarcinoma. Cancer Res, 41: 5070, 1981 Jin, R. J., Wang, Y., Masumori, N. et al.: NE-10 neuroendocrine cancer promotes the LNCaP xenograft growth in castrated mice. Cancer Res, 64: 5489, 2004 Labrie, F., Dupont, A., Belanger, A. et al.: New hormonal therapy in prostatic carcinoma: combined treatment with an LHRH agonist and an antiandrogen. Clin Invest Med, 5: 267, 1982 Lee, S. O., Dutt, S. S., Nadiminty, N. et al.: Development of an androgen-deprivation induced and androgen suppressed human prostate cancer cell line. Prostate, 67: 1293, 2007 Mohler, J. L., Gregory, C. W., Ford, O. H., III et al.: The androgen axis in recurrent prostate cancer. Clin Cancer Res, 10: 440, 2004
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Page, S. T., Lin, D. W., Mostaghel, E. A. et al.: Persistent Intraprostatic Androgen Concentrations after Medical Castration in Healthy Men. J Clin Endocrinol Metab, 2006 Pollack, A., Block, N. L., Stover, B. J. et al.: In vivo selection of androgen-insensitive cells in R3327-G rat prostate tumors: diethylstilbestrol diphosphate treatment versus orchiectomy. J Natl Cancer Inst, 70: 907, 1983 Prostate Cancer Trialists’ Collaborative Group: Maximum androgen blockade in advanced prostate cancer: an overview of 22 randomised trials with 3283 deaths in 5710 patients. Lancet, 346: 265, 1995 Scher, H. I., Sawyers, C. L.: Biology of progressive, castration-resistant prostate cancer: directed therapies targeting the androgen-receptor signaling axis. J Clin Oncol, 23: 8253, 2005 Titus, M. A., Schell, M. J., Lih, F. B., et al.: Testosterone and dihydrotestosterone tissue levels in recurrent prostate cancer. Clin Cancer Res, 11: 4653, 2005 Van Steenbrugge, G. J.: The Nude Mouse in Oncology Research. In: Hormones. Edited by E. Boven and B. Winograd. London: CRC, p. 224, 1991
Androgen Action and Modulation of Prostate and Prostate Cancer Growth: An Historical Perspective Shutsung Liao, John M. Kokontis, Chih-Pin Chuu, and Richard A. Hiipakka
Abstract Early models of steroid hormone action emphasized potential effects of these hormones on metabolic pathways to explain their effects on cells. However these models were abandoned soon after the discovery of the pathway for information transfer from DNA to protein through RNA. Testosterone (T), the major circulating androgen in blood, increases mRNA levels in target tissues and is metabolized to 5a-dihydrotestosterone (DHT) by 5a-reducatse in many target organs. DHT is selectively retained as a protein complex, the androgen receptor (AR), in nuclei, the site of RNA synthesis. Metabolism of T to DHT is critical for androgen action in certain tissues based on the phenotype of individuals with mutations in the gene for 5a-reductase. The cloning of the cDNA for AR has revealed its primary structure and its similarity to other steroid receptors, all members of a superfamily of transcription factors controlled by small lipophilic molecules. Various mutations in the gene for AR are responsible for androgeninsensitivity in men and a potential cause of prostate cancer progression. Certain natural products, like the polyunsaturated fatty acid, g-linoleic acid and the green tea catechin, epigallocatechin gallate (EGCG) are inhibitors of 5a-reductase and may be useful for treatment of disorders dependent on DHT. EGCG also affects appetite and may have a role in the treatment of obesity. Clinical prostate cancer progression can be mimicked in vitro using the LNCaP human prostate cancer cell line. These cells become hypersensitive to androgens, elevate expression of AR and are repressed by physiological doses of androgens after long-term androgen deprivation or treatment with the antiandrogen, Casodex. Although androgen receptor signaling is important for prostate cancer growth and progression, and a target of current therapies, other nuclear receptor signaling pathways may have utility in the treatment of prostate cancer. Activation of liver X
S. Liao(*) The Ben May Department for Cancer Research, The University of Chicago, The Gordon Center for Integrative Science, 929 East 57th Street, Chicago, IL, 60637, USA, E-mail: sliao@huggins. bsd.uchicago.edu
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receptor signaling modulates the growth and progression in human prostate tumor xenografts.
1 Introduction This chapter provides an historical account of many studies regarding the mechanism of action of androgens in the prostate and efforts toward uncovering the molecular and cellular mechanisms controlling prostate cancer growth and function. Some of these studies have been described in more detail in earlier reviews (Liao and Fang 1969; Liao 1975, 1994; Liao et al. 1989, 2001a; Hiipakka and Liao 1998; Kokontis and Liao 1999; Chuu et al. 2007). A time line for many of the discoveries cited in this account is presented in Fig. 1. The studies described in this chapter were performed over a period of about 50 years and were carried out at the Ben May Laboratory for Cancer Research. Dr. Charles B. Huggins established this laboratory at the University of Chicago in 1950 with generous support from Ben May, a businessman and philanthropist from Mobile, Alabama. The Ben May Laboratory became the Ben May Institute for Cancer Research in 1985 and the Ben May Department for Cancer Research in 2007. In September 1957, I (S.L.) was a graduate student in the Biology Department at the Illinois Institute of Technology and had accepted a fellowship from Cornell University at Ithaca, NY, and planned to transfer to the laboratory of Robert Holley (Nobel Laureate 1968, shared with M.W. Nirenberg and H.G. Khorana) to work on a research project dealing with nucleic acids. About a week before my scheduled departure for Ithaca, I visited the University of Chicago campus in the Hyde Park neighborhood on the south side of Chicago. After admiring the very impressive gothic university buildings, I visited, without a prior appointment, Earl Evans, then chairman of the Department of Biochemistry. Dr. Evans kindly showed me around his laboratory and discussed a few research projects in his laboratory. Later that afternoon, Dr. Evans called and suggested that I visit the next morning with Paul Talalay, an associate professor in the Department of Biochemistry, who was working in the Ben May Laboratory. Drs. Evans and Talalay had had a short meeting prior to my meeting with Dr. Talalay, and they made all the necessary arrangements, in one afternoon, for me to become a graduate student at the University of Chicago, even though I never requested or applied for admission. I met Dr. Huggins on my first day in the Ben May Laboratory. His first question was: ‘‘What did you discover today?’’ He then remarked: ‘‘You must make a discovery before you go home.’’ Huggins was a founding member of the University of Chicago Medical School, a surgeon specializing in urology, and a medical researcher who pioneered hormonal therapy for prostate and breast cancer. He received the Nobel Prize in Medicine in 1966 for his work on prostate cancer. He was my mentor until passing away in 1997 (Liao 1997, 2002; Talalay 1997). A photograph of Dr. Huggins and myself (S.L.) is presented in Fig. 2.
Androgen Action and Modulation of Prostate and Prostate Cancer Growth 1957 1961 1962 1965 1966 1968 1969 1969 1969 1971 1973 1976 1978 1979 1980 1982 1982 1985 1987 1987 1988 1988 1089 1990 1991 1991 1992 1993 1994 1994 1995 1995 1995 1996 1996 1998 2000 2000 2001 2002 2004 2005 2006 2008
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Liao begins graduate studies at the University of Chicago Ph. D. thesis: Discovery, purification and characterization of a new FAD enzyme Testosterone, in vivo, increases mRNA level associated with prostate ribosomes Testosterone, in vivo, increase mRNA content in prostate nuclei Evidence for selective gene transcription by androgen Discovery of nuclear retention and protein (AR) binding of 5α-DHT in prostate Discovery of multiple forms of RNA polymerase in prostate and liver. Antiandrogens, in vivo, reduce nuclear retention of 5α-DHT Androgen specificity of AR Steroid and tissue-specific nuclear retention of 5α-DHT-AR complex Steroid structure and androgenic activity Use of anti-steroid antibodies to assay steroid receptors Discovery of androgen-regulated spermine-binding protein (SBP) Purification and characterization of SBP RNA-dependent release of AR from DNA Discovery of prostate α-protein as a cholesterol binding protein AR recycling in the prostate cells is energy-dependent Discovery of autoimmune antibodies to AR in prostate cancer patients SBP cloned and sequenced Glutathion-S-transferase is androgen-repressed in rat ventral prostate 3-Deoxyadenosine inhibits recycling of AR AR cDNA cloned and sequenced SGP-2 identified as an androgen-repressed in rat ventral prostate 5α-reductase antibodies in prostate cancer patients Mutations in AR are responsible for androgen-insensitivity in men Mutation in AR in LNCaP cells Unsaturated fatty acids are 5α-reductase inhibitors and suppress sebum production Production of antibodies to 5α-reductase Increased AR expression after long term androgen deprivation of LNCaP cells Discovery of UR (the 1st LXR receptor, later called LXRβ), cloned and sequenced Chromosomal localization and immunocytochemical localization of UR Green tea EGCG inhibits 5α-reductase and suppresses sebum production Inhibition of prostate cancer and breast cancer xenografts by EGCG Testosterone suppression of androgen-independent LNCaP xenografts Finasteride stimulation of the growth of androgen-independent LNCaP xenografts Androgen repression of LNCaP cells after long term androgen-deprivation Suppression of endocrine systems, food intake and obesity by EGCG Many hydroxy steroids are ligands for LXR Many topical 5α-reductase inhibitors suppress hamster flank organ growth Many polyphenols inhibit 5α-reductase Anti-proliferative effects of LXR agonists on prostate cancer cells Androgen (A) suppression and reversion of A-independent to A-dependent LNCaP Inhibition of LNCaP xenograft growth and progression by LXR agonist Anti-atherosclerotic effects of a novel synthetic LXR agonist
Fig. 1 Time line of important events and discoveries related to the authors’ laboratory
Although joining the Ben May Laboratory was an accident, it led to a wonderful scientific journey into studies of the mechanism of androgen action and the role of androgens in the growth of benign and malignant prostate. This journey was possible because of the research environment and scientific atmosphere in the
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Fig. 2 The author and Dr. Charles B. Huggins in 1992. Dr. Huggins graduated from Harvard Medical School in 1924 and did his residency in general surgery at University of Michigan. In 1927, he married Margaret Wellman and joined the University of Chicago Medical School where he quickly became the leading urologist in the world. In 1941, Huggins published the classic paper showing that the deprivation of male hormone could lead to regression of metastatic prostate cancer in men. In the 1950s, he showed that removal of female hormones could lead to substantial regression of advanced breast cancer in many women. For these discoveries he was awarded the 1966 Nobel Prize. His discoveries showed that cancer cells were not autonomous and selfperpetuating but are dependent on chemical signals such as hormones to grow, and depriving those signals could restore the health of patients with widespread metastasis. Huggins’s fundamental discoveries led to hormonal therapy and provided an immense stimulus to research in cancer chemotherapy. In 1950, with a generous contribution from Mr. Ben May, Huggins established the Ben May Laboratory for Cancer Research and declared ‘‘Discovery is our business’’ its motto. A proponent of ‘‘small is better,’’ he invited a few young scientists to join him with unambiguous advice: ‘‘Do not go to meetings or the library, do not write books or become a head of anything, they are a waste of your time.’’ ‘‘Discover first’’ he insisted, read afterward to find out whether you can connect your new discovery to established ideas. This creative scientist made many fundamental discoveries (see text). Huggins worked 7 days a week for science and medicine until he was 92. He passed away on January 12, 1997, at his home near the University of Chicago, when he was 95 years old
Ben May Laboratory and at the University of Chicago, which played critical roles in forming the philosophy and scientific approach in my laboratory. During the 1950s, Huggins had already mentored several young scientists, who made many fundamental discoveries, such as identification of mitochondria as the sites of cellular energy production (Albert Lehninger) and elucidation of the biochemical steps involved in phospholipid synthesis (Eugene Kennedy). One of Huggins’ earliest students was William W. Scott, who built the distinguished urology department at the Johns Hopkins University Medical School. In the 1950s, Dr. Talalay, who had been a medical student in the laboratory of Dr. Huggins in the 1940s, was a pioneer in the study of steroid metabolizing
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enzymes. He fed testosterone to Pseudomonas and other bacteria and isolated enzymes that could metabolize testosterone and other steroid hormones. Dr. H. Guy Williams-Ashman, a British biochemist with considerable experience in reproductive biology, joined the Ben May Laboratory during the early 1950s. He was an assistant professor in the Department of Biochemistry in 1957 when I joined Talalay’s laboratory. At that time Talalay and Williams-Ashman were evaluating the possibility that the biological effects of certain steroid hormones, such as estradiol may be related to coupled oxidation and reduction of the hormone. They proposed that a steroid hormone might mediate a pyridine nucleotide-dependent transhydrogenation without consumption of the hormone itself (Talalay and Williams-Ashman 1960). For estrogen action they proposed and showed that the following two reactions could occur in test tubes: (1) Estradiol + NADP+Ô Estrone + NADPH + H+ and (2) Estrone + NADH + H+Ô Estradiol + NAD+. In combination, these two reactions give the following net reaction: NADP+ + NADH Ô NADPH + NAD+. One important implication of this proposed mechanism was that hydrogen produced in the form of NADH by mitochondrial oxidation would be transformed into NADPH that could participate in various synthetic reactions in a cell. However, the steroid hormone-dependent pyridine nucleotide transhydrogenation theory of Talalay and Williams-Ashman was soon found not to be the mechanism involved in steroid hormone action, because (1) diethylstilbestrol, a potent nonsteroidal synthetic estrogen, could not carry out the transhydrogenation, and (2) 17b-estradiol appeared to function without cycling through reduction and oxidation in estrogen-sensitive organs. Gordon Tomkins had proposed another model for steroid hormone action. He showed that a steroid hormone could act as an allosteric effector and modify the function of the active site of an enzyme (Tomkins et al. 1965). Diethylstilbestrol and certain other steroid hormones were shown to alter the quaternary structure of pure glutamic dehydrogenase and caused enzyme heteromers to disaggregate into monomers. This change decreased glutamic dehydrogenase activity and increased alanine dehydrogenase activity of the same enzyme. However, this effect required high concentrations of enzyme and hormone, and so was not likely to occur in vivo. In the Ben May Laboratory, Elwood Jensen, an organic chemist (who was a founding member of the Ben May Laboratory with Huggins, Lehninger, and Talalay) and his associate, Herbert Jacobson, initiated an entirely different approach toward understanding estrogen action in target organs. Their idea was to trace the fate of estrogen in estrogen-sensitive organs. They planned to do this by synthesizing highly radioactive [3H]17b-estradiol and administering this compound to rats. The idea of using tracer technology was not surprising given the University of Chicago’s recent involvement in radioisotope methodologies after the first successful demonstration of controlled nuclear fission at the university. Jensen and colleagues showed clearly that estrogen-sensitive organs, such as uterus and ovary, but not estrogen-insensitive organs, had a specific mechanism to retain estrogen against a concentration gradient between blood and target organ (Jensen and Jacobson 1962; Jensen 1978). This discovery led to the novel idea that estrogen-sensitive cells had receptor(s) that were responsible for retention of
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estrogens and presumably for some aspects of the biological effects of the hormone. The biological significance of this discovery was supported by the demonstration that antiestrogens hindered estrogen retention. Jensen’s pioneering studies led other investigators to discover and study other steroid hormone receptors and ultimately uncover the superfamily of nuclear receptors. Another very important discovery that was made in the Ben May Laboratory was by Samuel Weiss. He discovered that liver cell nuclei contain an enzyme that uses DNA as template to make RNA. This was the first demonstration of a DNAdependent RNA polymerase (transcriptase). Although Weiss’ very important original discovery with a mammalian system was ahead of other reports for bacterial systems, his contribution is rarely acknowledged. Weiss showed that DNase can abolish RNA synthesis and also demonstrated by nearest neighbor frequency analysis that the RNA product synthesized reflected the DNA template sequence (Weiss and Nakamoto 1961a, b). Good scientists making important discoveries provided a fertile research environment in the Ben May Laboratory. I began my studies and research career as a graduate student under Dr. Williams-Ashman’s guidance and after my graduation in 1961, Dr. Huggins persuaded me to stay on in the Ben May Laboratory as an independent researcher.
2 Androgen Regulation of the Synthesis of RNA and Protein 2.1
Hormone–Gene Theory
With the discovery of various enzymes and hormones in the 1930s and 1940s, biochemical studies of the mechanism of action of steroid hormones in the next few decades concentrated on induction of specific enzymes and proteins by different hormones. This approach became very popular after it was understood how genetic information in DNA is transmitted through mRNA to direct specific protein synthesis. Thus, the term, ‘‘Hormone–Gene Theory’’ evolved. One important discovery in this regard was the demonstration that injection of the insect hormone ecdysone into Chironomus tentans (midge) larvae caused a puff to form in specific regions of the chromosomes of the salivary gland (Clever and Karlson 1960). It was suggested that ecdysone induced puff formation by activation of a gene locus to produce RNA. Many subsequent studies of hormonal responses at the cellular and organ level also showed that steroid hormones induce RNA synthesis and subsequently specific protein synthesis.
2.2
Androgen and mRNA Synthesis
To investigate the mechanism by which androgens control RNA and protein synthesis, the rat ventral prostate was used as the target organ for our studies. It was observed that the androgen-dependent increase in protein-synthesizing activity
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of isolated ribosomal particles from the rat ventral prostate was due to an increase in the level of mRNA associated with ribosomes (Liao and Williams-Ashman 1962). Castration led to an increase in the proportion of ribosomes that were not occupied by native mRNA and, therefore, could utilize externally added synthetic polyribonucleotides as mRNA templates to guide radioactive amino acid incorporation into polypeptide linkages. Thus, with prostate ribosomes isolated from castrated animals, poly U stimulated phenylalanine incorporation, whereas poly UG stimulated radioactive valine incorporation into polypeptide linkages. The extent of this synthetic polymer-dependent incorporation was reduced by androgen injection into castrated animals, which suggested that androgen increased the proportion of ribosomes that were occupied by endogenous mRNA. These experiments suggested that androgen increases the amount of mRNA associated with ribosomes and provided an explanation for androgen stimulation of protein synthesis. These experiments were performed shortly after the discovery of the genetic code by Nirenberg and Matthaei (Nirenberg and Matthaei 1961; Nirenberg et al. 1962) and Ochoa and colleagues (Lengyel et al. 1962; Crick 1963), who used synthetic ribopolymers, synthesized by polynucleotide phosphorylase, to determine that bacterial protein synthesis uses a three-base codon to specify amino acid incorporation into proteins. Nirenberg actually visited our laboratory and discussed with us the use of these synthetic polymers. Our findings with a prostate ribosomal system were consistent with his important discoveries and supported the universality of the amino acid codons proposed at that time. In order to show that androgens increase the mRNA level in the nuclei of target cells, nuclear RNA was isolated from the prostate of rats, and a bacterial ribosomal system was used to determine whether this nuclear RNA could act as an mRNA template to stimulate the incorporation of radioactive amino acids into peptide linkages. This method allowed demonstration that androgen in vivo could increase the mRNA template activity of nuclear RNA isolated from the ventral prostate of castrated rats (Liao 1965). The RNA-synthesizing activity of isolated prostate cell nuclei was enhanced within 1 h after androgen injection into castrated rats (Liao et al. 1965).
2.3
Discovery of Multiple Forms of RNA Polymerases
Since very little was known about RNA polymerase at that time, we decided to study the behavior of RNA polymerase in liver and prostate. Cell nuclei from these organs, prepared with buffers containing hypertonic sucrose to prevent leakage of nuclear enzymes, retained a large quantity of RNA polymerase that is not tightly associated with chromatin (Liao et al. 1968). Two different forms of RNA polymerase were identified (Liao et al. 1969). These different forms of RNA polymerase synthesized different types of RNA in vitro. The existence of multiple forms of liver RNA polymerase (RNA polymerases I, II, and III) was later shown by other investigators to be responsible for synthesizing different classes of RNA in mammalian cells.
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Androgen and Modulation of Specific Proteins in Rat Ventral Prostate
In the process of regulating the growth and function of target organs, androgens stimulate the expression of certain genes, but also simultaneously repress the expression of other genes. In the rat ventral prostate two new proteins, a-protein and spermine-binding protein (SBP) were discovered that were positively regulated by androgen. Two other proteins, glutathione S-transferase (GST) and sulfated glycoprotein 2 (SGP-2, also known as clusterin and apo J), were identified that were negatively regulated by androgen. a-Protein was originally discovered in our laboratory in 1971 (Fang and Liao 1971; Chen et al. 1982) but was rediscovered later by other investigators and called prostate-binding protein or prostatein. a-Protein is a cholesterol-binding protein composed of four polypeptide subunits that is a major (>40%) component of the secretory fluid of the rat ventral prostate. One of the smallest subunits that was purified and sequenced inhibited the binding of androgen receptor (AR) to prostate nuclear fractions (Shyr and Liao 1978), but its biological function remains unclear. SBP was discovered in 1977 as a specific polyamine-binding protein (Liang, et al. 1978; Mezzetti et al. 1979; Hiipakka et al. 1984). cDNA for SBP was isolated and the protein sequenced by employing fast atom bombardment mass spectrometry and tandem mass spectrometry because the amino terminus of SBP is blocked (Anderegg et al. 1988). The function of SBP remains unclear, although prostates are rich in polyamines. One of at least four androgen-repressed proteins identified in the ventral prostate is the Yb1 (GST M1-1) subunit of GST that colocalizes with uridylic acid-rich small nuclear RNAs at interchromatinic regions in the cell nucleus (Saltzman et al. 1987; Chang et al. 1987). SGP-2 is another androgen-repressed protein that was identified by cloning and sequencing (Bettuzzi et al. 1989). SGP-2 mRNA increases 17-fold 4 days after castration and decreases rapidly within 6 h after androgen administration.
3 Importance of the Conversion of Testosterone to 5a-Dihydrotestosterone (DHT) 3.1
Selective Retention of DHT
Since androgens rapidly affect nuclear RNA synthesis in the rat ventral prostate, it was reasonable to assume that androgens act in the cell nuclei of the prostate. Based on studies by Jensen, 17b-estradiol is retained by cell nuclei in estrogen target organs, such as uterus without metabolic conversion. However, testosterone produced in testis and circulating in blood is metabolized to many metabolites in liver, prostate, and other organs. Therefore, three simple questions were asked:
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(1) Which metabolite(s) of testosterone are retained by prostate cell nuclei? (2) Is nuclear retention specific for androgen-sensitive cells? and (3) Is nuclear retention of androgens inhibited by antiandrogens? Studies were carried out first in vivo by injecting [3H]testosterone intraperitoneally into castrated rats. Later studies relied on in vitro incubation of isolated rat ventral prostate tissue and other organs with [3H]testosterone. Whether using whole animals or isolated organs, the ventral prostate, but not other organs, such as liver, brain, or thymus that are normally not considered androgen target organs, retained [3H]5a-DHT in nuclei. Retention of 5a-DHT in nuclei lasted for about 12 h. The cytoplasmic soluble (cytosol) fraction of prostate tissue had more than six radioactive metabolites, including a large quantity of steroids more polar than testosterone. When [3H]4-androsten-3,17dione was used instead of [3H]testosterone, the radioactive steroid retained by isolated cell nuclei was exclusively [3H]5a-DHT. A steroid chemist, Josef Fried, verified our identification of the steroids retained by prostate nuclei. Prior to joining the Ben May Laboratory, he had synthesized many fluorinated steroidal drugs in industry. The radioactive steroid retained by the prostate cell nuclei could not be removed easily by incubating the nuclei at 0–4 C with high concentrations of nonradioactive testosterone or 5a-DHT, or washing nuclei with isotonic sucrose containing 1% deoxycholate or 0.4% Triton X-100, which remove cytoplasmic membrane contaminants and nuclear membranes. Proteases, but not RNases, effectively released the [3H]5a-DHT from nuclei. However, [3H]5a-DHT retained in nuclei could be extracted with buffers containing 0.4 M KCl as a protein-bound complex that could be characterized by gel filtration or by sedimentation analysis using sucrose gradient centrifugation. These findings strongly suggested that 5a-DHT was retained in nuclei as a specific 5a-DHT–protein complex that was tightly bound to chromatin. The original findings were submitted to several journals in the United States for publication, but for more than 6 months our manuscript was not accepted for publication. Most of the criticism centered on a perceived lack of evidence to support the biological significance of our observations. For example, one referee’s comment was: ‘‘Authors did not succeed in showing that the phenomena observed were related to growth and function of the ventral prostate.’’ Therefore, the manuscript was submitted to Nature, which accepted the manuscript but with a request to remove the word ‘‘receptor’’ (Anderson and Liao 1968). The use of the term ‘‘androgen receptor’’ was taboo at this time. In the proof, the use of ‘‘androgen receptor’’ was reduced. This research work also was submitted as K.M. Anderson’s Ph.D. thesis in the Department of Biochemistry at the University of Chicago. A meeting abstract for a clinical conference, submitted by N. Bruchovsky and J. Wilson, came to our attention after submission of our manuscript to Nature. The abstract was cited when we received our galley proofs of the manuscript. In this abstract, these investigators reported that although [3H]5a-DHT, androstane3,17-diol and testosterone are found in prostate cytoplasm within 1 min after administration of [3H]testosterone, only [3H]5a-DHT and testosterone are found in prostate nuclei after 2 h (Bruchovsky and Wilson 1968).
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In 1967, Dr. Williams-Ashman, who had moved to Johns Hopkins University Medical School in 1963, was informed of our findings. A set of slides was prepared for him and he presented our findings at a ‘‘Symposium on Hormone Action’’ in Europe in early 1968. Several participants of the meeting communicated with us before our study was published in Nature. Our findings elated two investigators in the Ben May Laboratory. One was Charles Huggins, who had already observed that 5a-DHT is more active than testosterone in stimulating the growth of prostate-like organs in the urinary ducts in female rats. The other was Elwood Jensen. His hypothesis that a specific receptor plays a role in estrogen action was strengthened by another example with a different steroid receptor. A distinct difference between the two sex steroids is that 17b-estradiol interacts with its receptor without metabolism, whereas testosterone needs, at least in some target organs, to be converted to 5a-DHT. Nuclear retention of the 5a-DHT–protein complex was reduced considerably in vivo by coadministration of cyproterone or its acetate, both known antiandrogens that suppress male hormone action, which provided additional evidence that retention of 5a-DHT by prostate nuclei is biologically important (Fang and Liao 1969). Both antiandrogens also suppressed retention of 5a-DHT when they were incubated with minced ventral prostate tissue. A nonsteroidal antiandrogen, flutamide, suppressed nuclear retention of 5a-DHT in vivo, but not when prostate tissue was incubated with androgen and antiandrogen in vitro (Liao et al. 1974). The lack of effect in vitro suggested that the active form of flutamide might be a metabolite. Neri et al. (1979) subsequently described the potent antiandrogenic activity of hydroxyflutamide, a metabolite of flutamide. These studies strongly supported our contention that 5a-DHT was bound to a specific protein, the androgen receptor. In collaboration with the Stumpf laboratory at the University of Chicago, nuclear retention of 5a-DHT in rat seminal vesicles and prostates was demonstrated by dry mount autoradiography (Sar et al. 1970).
3.2
Different Biological Functions of Testosterone and 5a-DHT
Clear biological evidence that the conversion of testosterone to 5a-DHT is important for androgen action was provided several years after our observation that 5aDHT is retained selectively by target cell nuclei. J. Imperato-McGinley and her associates discovered that a 5a-reductase deficiency is the cause of a form of male pseudohermaphroditism, where testosterone levels are adequate for development of the Wolffian ducts but are inadequate for complete virilization of the urogenital sinus from which the prostate and external genitalia develop (Imperato-McGinley et al. 1974; Siiteri and Wilson 1974; Andersson et al. 1991; Imperato-McGinley and Gautier 1992). Like testosterone, certain other natural androgens, such as 4-androstenedione or androstanediols, can be converted to 5a-DHT in androgen target organs and produce androgenic effects. 5a-DHT produced in these organs may be a source of
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circulating 5a-DHT. 5a-DHT in blood, however, is bound to blood proteins and may not be as effective as intracellularly formed 5a-DHT. Some androgen-sensitive tissues, such as muscle, contain little or no 5a-reductase. In these tissues testosterone may function as the active androgen. Specific aspects of embryonic male urogenital tract differentiation and certain testicular and brain functions, which include events related to sexual behavior, may also utilize testosterone or its estrogenic metabolite, 17b-estradiol. Testosterone negatively regulates luteinizing hormone (LH) release from the pituitary in males. Feedback inhibition involves both testosterone and estrogen signaling in the pituitary and hypothalamus (Pitteloud et al. 2008). Negative feedback of LH secretion by testosterone does not appear to require 5a-reduction in humans (Iranmanesh and Veldhuis 2005). In rats there is evidence for (Liang et al. 1984) and against (Nagamoto et al. 1994) a role for 5a-DHT in feedback regulation of LH secretion. In rams there is evidence that 5a-DHT may be more effective than testosterone in feedback control (Schanbacher et al. 1987). Testosterone can also be converted by 5b-reductase to 5b-DHT. 5b-DHT is not androgenic and does not bind to AR. However, liver 5b-reductase is required for the synthesis of bile acids, which are secreted in bile and into the small intestine to facilitate lipid absorption. Some 5b-androstanes are active as hematopoietic agents. They increase the production of heme, hemoglobin synthesis, and erythropoiesis, perhaps by interacting with the nuclear receptor, CAR, which regulates synthesis of the mRNA for delta-aminolevulinic acid synthase, the rate-limiting enzyme for heme synthesis (Yamamoto et al. 2003). Testosterone and 5a-DHT also enhance erythropoiesis by stimulating the synthesis of erythropoietin by the kidney. There are two 5a-reductase isozymes that exhibit strikingly different biochemical behavior and expression patterns (Andersson et al. 1989, 1991; Thigpen et al. 1993; Russell and Wilson 1994). The type 2 reductase has been proposed to play an anabolic role in target organ development, whereas type 1 reductase may function more in catabolism of testosterone (Normington and Russell 1992). Many mutations in the gene for the type 2 reductase have been described, which cause a form of male pseudohermaphroditism. Mutations in the gene for the type 1 reductase are not normally related to androgen insensitivity (reviewed in Hiipakka and Liao 1998; Kokontis and Liao 1999) but do cause defects in murine parturition and increase murine embryonic lethality, which in the latter case appears to be due to high intrauterine estrogen levels (Mahendroo et al. 1997). Testosterone is the precursor for both 17b-estradiol and 5a-dihydrotestosterone, and competition for substrate may limit excessive production of estrogen in certain tissues.
3.3
Medical Uses for 5a-Reductase Inhibitors
If the biological action of testosterone in certain tissues is dependent on its conversion to 5a-DHT and binding of 5a-DHT to a specific receptor, certain medical conditions such as benign prostatic hyperplasia (BPH); baldness; acne;
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female hirsutism; and even androgen-dependent cancers of the prostate, breast, and liver may be controlled by a 5a-reductase inhibitor or by an antiandrogen that prevents androgen receptor binding of 5a-DHT. In 1967, before we submitted our paper on selective retention of 5a-DHT by prostate nuclei for publication, we requested that the University of Chicago file a patent for medical uses of 5areductase inhibitors. However, the university, at that time, was not interested in commercialization of new research findings and rejected our request. However, Merck hired Tehming Liang, who was a Ph.D. student and then a research associate in our laboratory, in 1974. At Merck, Liang was part of a group that identified a synthetic compound, finasteride, which is an effective 5a-reductase inhibitor (Fig. 3). Finasteride became the drug Proscar for the control of BPH. Years later the same compound was also used as a drug (Propecia) to treat male pattern baldness. Finasteride is a 4-azasteroid and is a better inhibitor of the type 2 than type 1 reductase. Dutasteride (Avodart), another drug used for treatment of BPH, is also a 4-azasteroid like finasteride, but it is an effective inhibitor of both isozymes (Bramson et al. 1997). Because diet is often an important component in disease susceptibility and since diet may modulate androgen signaling in unknown ways, we thought it was important to analyze dietary components for effects on 5a-reductase. Our laboratory looked for natural compounds that could function as 5a-reductase inhibitors. Several unsaturated fatty acids were found to be excellent 5a-reductase inhibitors. One of the more potent compounds we identified is g-linolenic acid (Fig. 3). Saturated fatty acids like stearic acid are much less active. To determine if these
Fig. 3 Structures of testosterone, 5a-dihydrotestosterone, and various 5a-reductase inhibitors
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compounds could be used for treatment of a medical condition modulated by androgens, such as acne, their effects were investigated using the hamster flank organ, a modified sebaceous gland dependent on androgen for growth and pigmentation. Male hamsters were castrated to reduce the size of flank organs and testosterone in ethanol with or without g-linolenic acid was applied topically. Flank organ growth was stimulated by topically applied testosterone, but we found that this stimulation was suppressed by 5a-reductase inhibitors, such as g-linolenic acid and other unsaturated fatty acid, but not by saturated analogs, such as stearic acid (Liang and Liao 1997). The catechins, epigallocatechin-3-gallate (EGCG) and epicatechin3-gallate (ECG), isolated from green tea were also potent 5a-reductase inhibitors and suppressed flank organ growth and pigmentation, whereas other green tea catechins were less active or not active (Liao et al. 2001b). Both g-linolenic acid and EGCG suppressed sebum production from human male forehead skin, which suggested that they could be utilized to control sebum production, which in excess contributes to acne. Various acne medicines are available and they work by opening blocked follicles (retinoids) or reduction of skin microflora (antibiotics). Since androgens stimulate sebum production, 5a-reductase inhibitors, such as g-linolenic acid and EGCG, may be useful as a new type of acne medicine (Fig. 3). A variety of polyphenolic compounds are also inhibitors of 5a-reductase (Hiipakka et al. 2002). Some of these compounds are part of a normal diet. It remains to be determined whether any of these compounds are useful for treating androgen-dependent disorders or whether they have any adverse affects on androgen signaling in vivo.
4 Modulation of Prostate Tumor Growth, Endocrine Systems, and Food Intake by EGCG Green tea catechins have cancer chemopreventative activity in many laboratory models of cancer initiation and progression (Liao et al. 2001a). We have shown that EGCG, but not other green tea catechins, repress prostate and breast tumor xenograft growth in nude mice (Liao et al. 1995). Green tea catechins also inhibit the development of prostate and breast cancer in TRAMP and C3(1) SV40 transgenic mice that express oncogenic SV-40 large T antigen under the control of androgenregulated promoters (Gupta et al. 2001; Leong et al. 2008). Green tea catechins may have similar properties in humans, since tea catechins have been shown to prevent progression of prostate intraepithelial neoplasia to more advance cancer when given orally to men (Bettuzzi et al. 2006). In contrast to these laboratory studies, some epidemiological studies have been equivocal in their support of cancer chemopreventative effects of green tea (Kuriyama et al. 2006). Since green tea catechins have been advocated for health promotion, we studied the acute physiological effects of green tea catechins in rats. EGCG but not other green tea catechins reduced food intake, body weight, blood levels of testosterone,
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estradiol, leptin, insulin, IGF-I, glucose, cholesterol, and triglycerides. The growth of the prostate, uterus, and ovary was also reduced. Similar effects were observed in normal and leptin receptor-defective Zucker rats. Therefore, EGCG does not cause these affects by modulating leptin signaling. Decreased food intake is known to modulate many endocrine systems (Schreihofer et al. 1993). How EGCG regulates appetite is not clear, but EGCG is an inhibitor of fatty acid synthase (FAS; Tian 2006). Inhibition of FAS elevates the level of malonyl CoA in the hypothalamus and this metabolic intermediate can regulate signaling mechanism controlling appetite perhaps by regulating the activity of carnitine palmitoyl-transferase-1 (Wolf 2006).
5 Identification and Biochemical Characterization of Androgen–AR Complexes 5.1
Radioactive Androgens for Identification and Characterization of AR
Highly radioactive 5a-DHT was not commercially available in the late 1960s as a tool to identify and characterize AR in various target tissues. Therefore, [3H] testosterone was incubated with a prostate cytoplasmic membrane fraction or prostate cell nuclear fraction to obtain [3H]5a-DHT for our early studies. Fortunately, [3H]5a-DHT was soon commercially available. Another androgen identified as a potent ligand for AR (Liao et al. 1973a) that has been widely used in characterization of the AR is 17a-methyl-17b-hydroxy-estra-4,9,11-trien-3-one, now commonly known as R1881 or methyltrienolone. R1881 is stable to metabolic conversion and binds to AR with high affinity. Another synthetic androgen, 7a, 17a-dimethyl-19-nortestosterone, DMNT or mibolerone, also proved useful for study of AR (Liao et al. 1973a; Schilling and Liao 1984). Besides its metabolic stability and low affinity for TeBG, DMNT is more receptor selective than R1881.
5.2
Sedimentation Analysis of AR by Gradient Centrifugation
Introduction of gradient centrifugation for the study of estrogen receptor (Toft and Gorski 1966) stimulated the use of this technology in the characterization of various other steroid hormone receptors. When the cytosolic fraction of rat ventral prostate was incubated with [3H]5a-DHT, the radioactive complexes formed migrated with sedimentation coefficients (Svedberg value) of 3–9 S. (Fang et al. 1969; Liao and Fang 1969; Fang and Liao 1971). The larger complexes dissociated into smaller 3–4 S units in the presence of high salt, which suggested that AR was present as a dimer or associated with other cytosolic components. Adventitious interactions of
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AR with other cellular components in tissue extracts can occur. In addition protease action may alter the native receptor structure and alter biochemical characteristics of the receptor. [3H]5a-DHT complexes extracted by buffers containing 0.4 M KCl from cell nuclei of the ventral prostate of rats injected with [3H]testosterone or from prostate tissue incubated with [3H]testosterone or [3H]5a-DHT migrated as 3–4 S entities. Similar complexes were formed when other radioactive androgens (R1881 or DMNT) were used instead of [3H]5a-DHT.
5.3
Assay of AR Using Insolubilized Antisteroid Antibodies
The gradient centrifugation technique is not easily used for quantitative determination of steroid receptors because it is a laborious procedure and because receptors specifically and nonspecifically interact with other cellular components, which makes accurate quantification of steroid-bound receptors difficult because they are dispersed in many fractions of the gradient. Gel filtration of the steroid-receptor complexes and methods using charcoal for removal of free steroids also have limitations when used in quantification of steroid receptors. Anti-5a-DHT antibodies coupled to dextran-agarose beads were used to separate AR complexes from free steroid. The mixture is simply centrifuged and receptor-bound radioactivity in the supernatant measured. This method (Castaneda and Liao 1974) works well because the radioactive DHT is bound tightly in the androgen-binding cavity of AR, and the complex has a very low rate of dissociation. The complex is also not recognized by 5a-DHT antibodies. The method is applicable to the quantitative assay of other steroid receptors because commercially available steroid antibodies can bind to their respective steroids, in free form or weakly bound to nonreceptor proteins, but not steroids in the ligand-binding pocket of the receptor (Castaneda and Liao 1975a, b). The method is very simple and can handle many samples.
5.4
Using a Hydroxylapatite Filtration Assay to Measure AR
A very simple method was developed to assay the androgen-receptor complex using hydroxylapatite adsorption and washing the adsorbed receptor free of unbound steroid on filters (Liao et al. 1984). Radioactive androgen–AR complexes were mixed with slurry of hydroxylapatite and the mixture was transferred onto glass filters that were mounted on a multiple sample filter manifold and washed with a cold buffer solution. The washed filters were transferred into vials for measurement of radioactivity. Protease inhibitors were added to samples to minimize proteolysis. The procedure is economical, simple, fast and requires only small amounts of sample. More than 50 samples could be processed in 30 min. Other steroid receptors, such as glucocorticoid or estrogen receptors in different tissue or cell
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extracts, also could be measured using this method. This method has been employed in many of our studies of AR.
5.5
Structural Requirements for Interaction of Androgens with AR
Various steroid-binding studies have shown that 5a-DHT interacts differently than testosterone with AR. This must be due to differences in steroid structure at the A and B ring junction where these two steroids differ. Dreiding stick and CPK space-filled molecular models were used to compare the structures of these two steroids (Liao et al. 1972, 1973a). These molecular models show that the thickness of the A/B ring area including the C19 methyl angular group at C10 is approxi˚ for testosterone and 3.2 A ˚ for 5a-DHT. At that time, several very active mately 4 A synthetic androgens called ‘‘super androgens’’ were known, and they did not have a C19-methyl group attached to C10. Without an angular C19-methyl group, the structure of the A/B ring area is less bulky. Although these synthetic super androgens have a double bond at C4, as in testosterone, they can interact with the prostate AR even more tightly than 5a-DHT. This observation strongly supported our contention that spatial geometry is an important characteristic of androgens that bind strongly to AR. These studies suggested that the need for 5a-reductase to convert testosterone to 5a-DHT is simply to make the A/B ring area of the molecule less bulky so that it could be accommodated in the androgen-binding cavity of AR. The orientation of methyl groups at other position is clearly very important, since 7b-methyl derivatives are poor competitors, while 7a-methyl derivatives compete well for binding to AR. In addition, cis-testosterone, which has a 17a-OH, does not compete for binding to AR and is not androgenic, an indication of the importance of the configuration of the 17-hydroxyl group for binding activity.
5.6
Androgen and Antiandrogen Binding to the Hydrophobic Cavity of AR
Structural studies on AR binding to androgens and the results from antiandrogen antibody experiments strongly suggest that androgens interact with AR as if the androgen is being totally enveloped in a hydrophobic cavity of AR. This should reduce the rate of dissociation, increase the binding affinity, and increase the biological activity of the androgen. Certain chemicals may resemble in part active androgens and may be capable of interacting with the androgen-binding cavity with sufficient strength to exhibit androgenic or antiandrogenic activity. A transparent plastic box having nine faces was made to illustrate the required geometric
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structure, sufficient to accommodate from all sides a CPK molecular model of 5aDHT, but not testosterone (Liao et al. 1972). Two holes were made to accommodate the 7a- and 17a- methyl groups of methylated androgens. A light and battery was placed at a corner so that the light would go on when the box cover was closed completely indicating a good fit for the ligand. For example, the CPK model of cimetidine (Tagamet), a histamine H2-receptor antagonist prescribed for gastric and duodenal ulcers, fits very well inside the box. In line with this analysis, cimetidine inhibits [3H]R1881 binding to AR, which may explain why cimetidine exhibits antiandrogenic activity in some male patients. The ability of various small molecules to compete with [3H]R1881 for binding to AR in the cytosolic fraction of rat ventral prostate was investigated to explore the possibility that the steroid-binding site of a steroid receptor may have a significant affinity toward small molecules representing portions of the natural ligand. More than 80 mono- and polycyclic aromatic hydrocarbons were tested and certain reduced phenanthrenes are strikingly active at 20 mM, while many anthracenes are inactive at 2 mM. Interestingly, 9,10-dihydrophenanthrene is about 20 times more effective than phenanthrene or phenanthrenes reduced in the two end aromatic rings. The 9- and 10-hydrogenation may alter the conjugated phenanthrene in such a way that the nonconjugated molecule is more bulky, and this decreases the rate of dissociation from the AR ligand-binding pocket (Chang and Liao 1987). These comparisons are biologically meaningful because 9,10-dihydrophenanthrene, but not phenanthrene, injected into rats inhibits the androgen-dependent growth of accessory reproductive organs. 9,10-Dihydrophenanthrene did not compete with [3H]estradiol for binding to estrogen receptor of rat uterus or from human breast tumor MCF-7 cells or glucocorticoid receptor of rat liver (Liao et al. 1983; Chang and Liao 1987). These studies indicate that while the hormonal action of a steroid may be dependent on the interaction of a functional group of the hormone with the receptor, the presence of such a group might not be required for antagonistic activity of a compound that can physically block hormone binding to the receptor. These studies also led to a similar conclusion for estrogen and glucocorticoid receptors. These observations suggest that certain environmental compounds might affect the actions of various steroid hormones through interactions with their receptors (Chang and Liao 1987).
6 Intracellular Transformation and Recycling of AR It is widely accepted that steroid-receptor complexes act by recognizing and interacting with specific promoter regions in target genes through the DNA-binding region of the steroid receptors. Precisely how this occurs is not yet very clear especially because of the multitude of proteins present at chromatin loci where RNA synthesis and processing are carried out. One subject often discussed is how a steroid-receptor complex is recycled or processed after its involvement in transcription initiation. In 1969, we proposed that the androgen–AR complex may bind to
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RNA and is involved in the processing and transport of newly synthesized RNA (Liao and Fang 1969). Ribonucleoprotein particles in rat ventral prostate or calf uterus interacted with androgen–AR complex or estrogen–estrogen receptor complexes, respectively (Liao et al. 1973b; Liang and Liao 1974). Certain polyribonucleotides, such as poly UG, are much better that other polyribonucleotides, such as poly C, poly A, or poly U, in removing androgen–AR complex from DNA-cellulose or from prostate chromatin aggregates, suggesting that androgen–AR complex can interact specifically, depending on nucleotide sequences, with ribopolymers (Liao et al. 1980). Therefore, steroid receptors could play an important role in removing specific newly synthesized RNA from the DNA template for post-transcriptional processing and allow access to DNA for further RNA synthesis. Androgen–AR complex appears to be trapped and accumulates in the nuclei and is not recycled back to the cytoplasm, if RNA synthesis is inhibited by actinomycin D. In the presence of 30 -dA, a potent inhibitor of RNA polyadenylation and nucleocytoplasmic RNA transport, androgen–AR complex is maintained in an RNA-bound form and recycling of androgen–AR complex is prevented. The level of chromatin-bound androgen–AR complex is therefore reduced, and the androgen–AR complex accumulates in the cytoplasm compartment (Rossini and Liao 1982; Hiipakka and Liao 1988; Liao et al. 1989). Some of our other studies also suggest that AR is controlled in target cells by an energy-dependent activation process. A significant portion of AR in prostate cells is in a form that cannot interact with 5a-DHT unless it is reactivated by an energy-dependent process. In the presence of respiratory poisons such as 2,4dinitrophenol, AR is rapidly deactivated (half-life, 2 min) to an inactive form that does not bind 5a-DHT unless it is reactivated by an energy-dependent process to the androgen-binding form. A majority of 5a-DHT–AR complexes formed do not bind to DNA/chromatin unless they are ‘‘transformed’’ by a temperature-dependent process. Isotope-chasing experiments with labeled androgen indicate that the steps involved in androgen–AR complex recycling between chromatin-bound and cytosolic forms are slow compared to other steps in the recycling process and that the 5a-DHT–AR complex has a half-life of more than 50 min in this second part of the cycle (Rossini and Liao 1982). This slow process may reflect the participation of 5a-DHT–AR complex in a time-consuming mechanism that is essential for the hormonal response. Work from many other laboratories has shown subsequently that heat-shock protein/chaperone interactions with nuclear receptors during receptor folding and maturation are dependent upon ATP hydrolysis (see review by Pratt and Toft 1997), and may underlie some of our observations of energy-dependent AR activation.
7 Cloning of AR cDNA Our laboratory has always been small and initially did not attempt the cloning of the cDNA for AR. In April 1987, I was invited to the ‘‘International Symposium on Hormonal Therapy of Prostatic Diseases: Basic and Clinical Aspects’’ in
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Milan, Italy. During the meeting, a group presented an interesting lecture and detailed experimental results in a poster claiming success in the cloning of a cDNA for AR. The AR cDNA was transcribed and then the RNA translated in an in vitro system to generate AR protein. The synthesized AR protein was used to show specific steroid binding and other interesting properties, some of which led me to question whether AR cDNA had in fact been cloned. After returning from the meeting, I decided that it was time to attempt the cloning of AR cDNA in our own laboratory because the current claim would discourage other investigators to take on the task. The cDNAs for estrogen and glucocorticoid receptors had been cloned and it was apparent that the cDNAs for these steroid receptors contained regions with similar DNA sequences. In particular, the DNA encoding the DNA-binding domain of these receptors was very similar. Degenerate oligonucleotide probes that were complementary to the DNA-binding domain of these receptors were generated and used to probe cDNA libraries from the testis and prostate. Several unique cDNAs were isolated. After sequencing various clones, one composite cDNA contained an open reading frame encoding a protein that was homologous to other steroid receptors (Chang et al. 1988a, b). When RNA was transcribed from this cDNA and translated it in a rabbit reticulocyte lysate, the product bound [3H]R1881 and the sedimentation rate of the labeled complex was shifted by an IgG fraction from the serum of a prostate cancer patient known to have antibodies against AR. Our sequence for AR cDNA was confirmed by Dr. D.P. Ma at Texas A&M University. Several other laboratories also cloned cDNAs for AR at this time or shortly thereafter (Lubahn et al. 1988; Brinkmann et al. 1989; Tilley et al. 1989).
8 AR Structure and Mutations The cDNA for both rat and human AR were cloned and sequenced in our laboratory (Chang et al. 1988b; Liao et al. 1989). The cDNA for human AR encodes a protein containing 918 amino acids with a molecular weight of about 99,000. Human AR clones from different laboratories encode proteins that vary in length due to variation in the length of polyglutamine and polyglycine repeats in AR (Fig. 4). Like other steroid receptors, AR is composed of three functional domains, which include an amino-terminal domain, a central DNA-binding domain, and a carboxylterminal ligand-binding domain. The amino-terminal domain of human AR consists of 555 amino acids and contains a ligand-independent transactivation function. This domain contains polyglutamine and polyglycine repeats that are 17- and 27-residue long in the clones we sequenced. A number of other smaller repeats are present including repeats of alanine, glutamine, leucine, proline, and serine (Fig. 4). Some of these repeats are also present in mouse (He et al. 1990) and rat AR. Expansion of the polyglutamine repeat to greater than 40 residues is associated with X-linked spinal and bulbar
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Fig. 4 Amino acid repeats present in human (h), rat (r), and mouse (m) AR (top) and two mutations in AR causing androgen insensitivity and one mutation altering the ligand specificity of AR in LNCaP prostate cancer cells (bottom)
muscular atrophy (Kennedy’s disease; La Spada et al. 1991). Expansion of polyglutamine repeats in other proteins has been linked to several other neurological diseases (Shao and Diamond 2007). Repeat expansion appears to endow the protein with a gain of function that leads to neuron-specific toxicity. Affected proteins are
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found often as insoluble aggregates within nuclei and cytoplasm. Toxicity may be the result of a disruption of the function of cell chaperones (Brignull et al. 2007). Although direct effects of the abnormal protein on neurons are likely, tissuespecific overexpression of wild-type AR in muscle leads to neuronal degeneration similar to that found in mice expressing an AR transgene containing a long polyglutamine repeat (Yu et al. 2006; Monks et al. 2007). Polyglutamine repeats are present in a variety of proteins, which include several transcriptional regulators, but a function for this structure has not been determined clearly (Butland et al. 2007). The DNA-binding domain of AR is composed of about 70 amino acids. This domain shares 56–79% identity with other steroid receptors. This domain contains two zinc atoms that are coordinated to the sulfurs of eight cysteines. The amino acids of this domain are present in a helix–loop–helix structure that interacts with specific DNA sequences called ‘‘androgen response elements.’’ Three amino acid residues within the amino-terminal helix that make critical contacts with the DNA response element are conserved among androgen, progestin, glucocorticoid, and mineralocorticoid receptors, which bind to a similar sequence of DNA. The carboxyl-terminal ligand-binding domain contains about 290 amino acids. The ligand-binding domains of ARs from humans, rats, and mice are identical, and homology to other steroid receptor ligand-binding domains is 15–45%. Like other steroid receptors, this domain is composed of 12 alpha helices that form three layers that create a hydrophobic ligand-binding pocket. The ligand-binding domain of AR bound to several agonists and antagonists has been studied using x-ray crystallography (Matias et al. 2000; Sack et al. 2001; Bohl et al. 2005a, b; Askew et al. 2007). Helices 3, 4, 5, 7, 11, and 12 form the ligand-binding pocket. Polar residues within this pocket form hydrogen bonds to the 3-keto and 17-hydroxy groups. Side chains of nonpolar amino acids form van der Waals contacts with various carbons of the steroid nucleus. Upon ligand binding, helix 12 moves to cover the ligand pocket and creates a new surface for coactivator recruitment or for interactions with the amino-terminal domain of AR (Langley et al. 1995). This process activates the second activation domain (AF-2) of AR. Differences in the ability of testosterone and dihydrotestosterone to activate AR may be related to differences in the ability of these ligands to modulate AF-2 activity (Askew et al. 2007). Cloning of the AR cDNA allowed us and many other investigators to define mutations in AR that are responsible for androgen-insensitivity in humans (Marcelli et al. 1990; Ris-Stalpers et al. 1990; Sai et al. 1990; Trifiro et al. 1991; McPhaul and Marcelli 1992; Bruggenwirth et al. 1996; Gottlieb et al. 1997). More than 300 different naturally occurring germ line and somatic mutations in the AR gene have been described that alter AR’s response to potential ligands (Gottlieb B. The Androgen Receptor Gene Mutations Database World Wide Web Server http:// androgendb.mcgill.ca). Most of these mutations reside in the ligand-binding domain of AR. Mutations in the AR ligand-binding domain cause premature termination or transitions and transversions (Fig. 4) that are responsible for androgen insensitivity (Sai et al. 1990; Ris-Stalpers et al. 1991). Androgen insensitivity in
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the Tfm mouse is due to a mutation (deletion) causing a frame shift mutation leading to premature termination of AR synthesis (He et al. 1991). Our laboratory and others found that human prostate cancer LNCaP cells contain a mutation T877A (Fig. 4) that alters ligand specificity of AR and allows the antiandrogen flutamide to act as an agonist (Veldscholte et al. 1990; Kokontis et al. 1991). This threonine in helix 11 is responsible for hydrogen bonding to the 17b-hydroxy group of active androgens. Mutation to alanine allows other steroids, like progesterone and estradiol, to fit into the binding pocket and activate AR. The prostate cancer cell line CWR22 has the AR mutation H874Y, which also affects ligand specificity (Tan et al. 1997). In contrast to LNCaP cells, this mutation does not directly affect a residue contacting ligand. This amino acid residue lies in a cavity formed between helices 11 and 12. Interactions between these helices are important for coactivator recruitment and the mutation may influence binding of coactivators and modulate their effects on transcription (McDonald et al. 2000).
9 Anti-AR Antibodies 9.1
Autoantibodies to AR in Prostate Cancer Patients
Jensen and colleagues were the first to obtain antibodies against a steroid receptor by immunization of rabbits with estrogen receptor isolated from a very large quantity of calf uteri (Greene et al. 1977). Similar attempts by our group to isolate enough AR for immunization were not successful. One method utilized was to covalently link 5a-DHT through its 17 position to beads by a thioether bridge. This material was used as an affinity adsorbent to bind and isolate AR. Unfortunately, the amount of AR in target tissue extracts is only on the order of 1–100 fmol (0.1–10 ng) per mg protein; therefore, affinity chromatography was not practical for isolating AR for further analysis or for immunization to obtain antibodies. In 1985, we thought that prostate cancer patients, especially those with metastases, might have autoantibodies against AR. About 200 serum samples from prostate cancer patients were obtained from a clinical chemistry laboratory and more than 30% of the samples had antibodies to AR. Three serum samples with very high titers were from prostate cancer patients with metastatic prostate cancers (Liao and Witte 1985). Gradient centrifugation was used to show that purified IgG from one of the patient’s sera interacted with 4 S cytosolic androgen–AR complexes of rat ventral prostate to form 9–12 S complexes. Other serum samples formed 14–19 S units, which suggested that different classes of immunoglobulins might be involved in AR binding or multiple epitopes were being recognized. These antibodies interacted with both cytosolic and nuclear AR complexes, but not with estrogen, progestin, or glucocorticoid receptors from a variety of sources.
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Antibodies also did not interact with many nonreceptor proteins. Later, these autoantibodies played an important role in identification of AR made in a cellfree system through transcription of cloned human AR cDNA and translation of the AR mRNA (see earlier). This provided strong evidence for the identity of the cDNA clone isolated. Tindall and colleagues generated monoclonal antibodies against AR by immortalizing lymphocytes from men with antibodies to AR in their sera (Young et al. 1988).
9.2
Monoclonal Antibodies to AR
Antibodies to AR are useful tools to immunohistochemically localize AR and for techniques like Western blotting. The cloned AR cDNA was used to generate fusion proteins representing different domains of AR linked to the bacterial protein TrpE. Rabbits and rats were immunized with purified fusion proteins to produce rabbit polyclonal and rat monoclonal antibodies to AR (Chang et al. 1989). These antibodies were used to localize AR in various cell types (West et al. 1990).
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10.1
Development of the LNCaP 104 Model of Prostate Tumor Cell Progression Progression of Androgen-Sensitive Prostate Cancer Cells to Androgen-Independent and Androgen-Repressed Cells
After isolation and characterization of the AR cDNA, interest turned to exploring the role of AR in the growth and progression of prostate cancer. Our strategy was to subject androgen-sensitive prostate cancer cells to androgen deprivation in vitro to derive androgen-independent cells, and try to understand what was happening with regard to AR in the progression process. This would mimic the clinical situation in which prostate cancer recurs during androgen deprivation and/or antiandrogen therapy. At the time, around 1990, two choices for androgensensitive cells were the LNCaP human prostate cancer cell line (Horoszewicz et al. 1983) or the Shionogi carcinoma 115 mouse mammary tumor cell line (Minesita and Yamaguchi 1965). The LNCaP cell line was chosen because of its greater applicability to human prostate cancer. A hormone-dependent clonal isolate would minimize the possibility of selection of pre-existent independent clones from a potentially heterogenous population during androgen deprivation. The initial panel of about 15 LNCaP clonal isolates showed that the parental population was indeed quite heterogenous with respect to growth response to
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androgen (Kokontis et al. 1994; Kokontis and Liao 1999). The induction of cell growth, measured by simple cell counts of trypsinized cells after a week of incubation in the presence or absence of an optimal concentration of androgen, 0.1 nM of the synthetic androgen R1881, ranged from 1.5- to 12-fold. The clone that was most androgen sensitive, called 104-S, was used in all subsequent androgen-deprivation experiments. After about 20 passages in medium supplemented with dextran-coated charcoal-stripped fetal bovine serum, LNCaP 104-S cells gave rise to cells, called 104-I cells, that proliferated very slowly in the absence of androgen and showed greatly reduced responsiveness to exogenously supplied androgen (Kokontis et al. 1994). After about 20–25 additional passages, cells that we initially called 104-R cells emerged that grew much more rapidly in the absence of androgen. While 104-R cell growth was stimulated slightly by 0.01 nM R1881, cell growth was unexpectedly repressed by concentrations of androgen (0.1 nM R1881) that were optimal for 104-S cell proliferation. After about 60 additional passages in androgen-depleted medium, LNCaP 104-R cells gave rise to cells, called 104-R2 cells, that proliferated in the absence of androgen at a rate comparable to the proliferation rate of 104-S cells grown in androgen and continued to be repressed by 0.1 nM R1881 (Kokontis et al. 1998). The original 104-R cells were referred to as 104-R1 cells. During the transition of 104-S cells to 104-R1 and 104-R2 cells, AR mRNA and protein level increased dramatically. AR transcriptional activity, measured by androgen induction of prostate-specific antigen (PSA) mRNA or expression of an androgen-dependent reporter gene transfected into cells, also increased up to 20fold during the progression. LNCaP 104-R1 and 104-R2 cells were proliferatively insensitive to the antiandrogen bicalutamide (Casodex). Casodex, unlike flutamide and cyproterone acetate, acts as a true antiandrogen in the LNCaP cell line (Veldscholte et al. 1992a, b). Casodex did not exhibit agonist activity in 104-R1 and 104-R2 cells, as has been reported by other investigators for androgen-independent LNCaP cells (Culig et al. 1999; Chen et al. 2004). Casodex repressed androgen target genes, such as PSA in 104-R1 and 104-R2 cells, and Casodex blocked androgenic repression of growth (Kokontis et al. 1998). This observation suggests that 104-R1 and 104-R2 cells are truly androgen independent and are not just more efficient at scavenging residual androgen in the medium. The basal level of PSA expression did not rise appreciably in 104-R1 and 104-R2 cells, which indicates that AR transactivation of PSA expression continues to be androgen sensitive, even when proliferation is androgen independent. When 104-R1 cells were incubated for several weeks in a high concentration of R1881 (20 nM), cells adapted after a period of growth arrest to grow at a rate equivalent to the parental 104-R1 cells (Kokontis et al. 1998). These cells, called 104-R1Ad cells, grew optimally in 10 nM R1881 and Casodex inhibited R1881-induced growth. 104-R1Ad cells exhibit drastically reduced levels of AR, which suggests that elevated AR expression is responsible for the repressive effect of androgen in 104-R1 and 104-R2 cells.
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Progression of Prostate Cancer Cells to Androgen-Insensitive Cancer Cells
LNCaP 104-S cells were incubated with 5 mM Casodex to more closely mimic the clinical situation of combined androgen deprivation and antiandrogen therapy. After three to four weeks, Casodex-resistant colonies appeared at low frequency as most of the cells appeared to undergo senescent cell death (Kokontis et al. 2005). This frequency increased with increasing 104-S passage number, which suggests that a subpopulation of androgen-independent cells emerges spontaneously over time even in a clonal population of cells grown in the presence of androgen (Fig. 5). The presence of this subpopulation was manifested by selection in antiandrogen. Six independent Casodex-resistant clonal sublines were derived from these colonies, designated CDXR1 through CDXR6. Like 104-R1 and 104-R2 cells, CDXR cells had increased AR expression and activity, and were repressed by androgen (Kokontis et al. 2005). Unlike 104-R1 cells, most CDXR1, CDXR2, and CDXR3 cells grown in 10 nM R1881 underwent apoptotic cell death starting 6–8 days after R1881 exposure. At low frequency, however, R1881-insensitive colonies appeared that were not repressed by R1881 or Casodex. These sublines, designated IS1, IS2, and IS3 (derived from the corresponding CDXR subline), showed greatly reduced
Fig. 5 Frequency of Casodex-resistant cells increases with LNCaP 104-S passage number. Equal numbers of LNCaP 104-S cells at increasing passage numbers were plated in 12-well plates and grown in medium supplemented with 10% charcoal-stripped FBS and 5 mM Casodex. After 2 weeks, cells were trypsinized and replated to allow expansion of rare colonies. After 4 additional weeks of growth in 5 mM Casodex, wells were scored as growth or nongrowth. Values for frequency were calculated from Poisson’s formula: a = (ln p0)/N, where a is frequency of CDXR cells in the population, p0 is fraction of wells with no Casodex-resistant cell growth, and N is initial cell number/well
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Fig. 6 Progression scheme of LNCaP 104-S cells after androgen (A) deprivation or Casodex (CDX) treatment, and androgen replacement
AR expression. But unlike 104-R1Ad cells, the growth of IS cells was not stimulated by R1881. 104-R2 cells, like CDXR cells and unlike 104-R1 cells, gave rise to androgen-insensitive cells when challenged by high concentration R1881. During progression from 104-R1 to 104-R2 stages, therefore, the cells appear to pass a point where cells can no longer recover responsiveness to androgen, but instead progress to androgen insensitivity (Liao et al. 2005). Direct progression of 104-S cells to the CDXR stage by selection in antiandrogen seems to bypass this intermediate 104-R1 stage. This suggests that the more aggressive combined treatment may cause a more rapid and irreversible selection of independent cells that have lost the potential to reacquire androgen responsiveness and become controllable by endocrine strategies. Androgen-deprivation therapy alone, on the other hand, may promote a slow adaptation to androgen independence that is reversible up to an as yet undefined point during the transition from 104-R1 to 104-R2 cells. This progression scheme is summarized in Fig. 6. Knockdown of AR expression in CDXR3 cells by shRNAi methodology, either constitutive or conditional, relieved androgenic repression of growth and did not affect cell growth in the absence of androgen (Kokontis et al. 2005). These data suggested that elevated AR expression is not required for androgen-independent growth, contrary to reports by others (Zegarra-Moro et al. 2002; Haag et al. 2005; Yuan et al. 2006). Retroviral overexpression of AR in IS2 and IS3 cells, on the other hand, restored the androgen-repressed phenotype in these cells, which confirms that elevated AR expression alone was sufficient for conferring this response in LNCaP cells. Conditional overexpression of AR in 104-S cells also caused androgeninduced growth repression and did not confer androgen-independent growth. Constitutive overexpression of AR is not tolerated in 104-S cells because the cells cannot proliferate either in the presence or absence of androgen. These observations suggest that enforced overexpression of AR may have therapeutic benefit to repress prostate cancer cell growth. The ability of CDXR cells to down-regulate AR expression during progression to the IS stage and rapidly proliferate in the absence of AR expression may indicate that therapeutic strategies based on reduction of AR expression may be easily bypassed by many prostate cancer cells.
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11.1
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Androgen Suppression of Androgen-Independent Prostate Cancer Androgen Induction of G1 Arrest in Androgen-Repressed Prostate Cancer Cells
In an effort to determine the mechanisms by which androgen modulates LNCaP 104-S, 104-R1, and 104-R2 cell proliferation, the expression of known cdk inhibitors (CKIs; including p15, p16, p18, p19, and p21waf1/cip1, p27Kip1, p57Kip2) was examined in cells treated with or deprived of androgen. Flow cytometric analysis of androgen-treated cells revealed that androgen treatment of 104-S cells relieved a G1 arrest induced by androgen deprivation. Conversely, R1881 induced G1 arrest in 104-R1 and 104-R2 cells beginning after about 24 h of exposure (Kokontis et al. 1998). Casodex could block the effect of androgen in all cell lines. Of all tested CKIs, only p21waf1/cip1 and p27Kip1 levels were induced by androgen (Kokontis et al. 1998). p21waf1/cip1 was induced transiently in 104-R1 cells only, while p27Kip1 was induced persistently about threefold in both 104-R1 and 104-R2 cells. In contrast, expression of p21waf1/cip1 and p27Kip1 was repressed by androgen in 104-S cells, which suggests that these inhibitors function in androgen-deprivation-induced G1 arrest. Cdk2 was the most androgen-sensitive cdk. Similar results were obtained with the CDXR sublines (Kokontis et al. 2005). Androgens regulate expression of the F-box protein Skp2 that binds phosphorylated p27Kip1 (Carrano et al. 1999; Tsvetkov et al. 1999; Lu et al. 2002) leading to its ubiquitination and proteolysis. Androgen also down-regulates Skp2 in 104-R1, 104-R2, and CDXR cells, which leads to accumulation of p27Kip1. Androgen also rapidly down-regulates c-Myc at the mRNA level in 104-R1 cells, and enforced retroviral overexpression of c-Myc blocks androgenic repression of 104-R1 growth (Kokontis et al. 1994). c-Myc may have an indirect effect on p27 Kip1 expression through the induction of Cks1, a component of the SCFSkp2 complex responsible for p27 Kip1 degradation (Keller et al. 2007).
11.2
Androgen Suppression of Prostate Cancer Xenografts
The tumorigenicity of LNCaP 104-S and 104-R2 cells was examined in normal and castrated athymic mice (Umekita et al. 1996). LNCaP 104-S cells grow well in normal male mice but do not form tumors in castrated male mice, and this reflects their androgen dependence. On the other hand, 104-R2 cells grow well in castrated hosts but not in normal male mice, consistent with their androgen independence and sensitivity to the repressive effects of androgen. In fact, finasteride reversed the repressive effects of testosterone implants on 104-R2 tumor growth, similar to the blockage of androgen repression by Casodex in vitro. When mice bearing 104-R1
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Fig. 7 Growth of 104-R1 and 104-R1Ad tumors in castrated athymic mice. Open symbols represent tumors grown without testosterone replacement and closed symbols represent tumors grown in mice subcutaneously implanted with testosterone propionate pellets (20 mg). Circles represent 104-R1 tumors grown without initial testosterone replacement and squares represent 104-R1Ad tumors grown with initial testosterone replacement
tumors are treated with testosterone, reversion to an androgen-stimulated phenotype occurs following a period of growth repression (Chuu et al. 2005), similar to the effect of androgen on 104-R1 cells in vitro. If testosterone pellets are removed from mice bearing 104-R1Ad tumors, tumor growth ceases until pellets are reimplanted, at which time growth resumes (Fig. 7). On the other hand, CDXR3 tumors are repressed by androgen treatment, and tumors either regress or relapse after about 90 days of treatment (Kokontis et al. 2005). The relapsed tumors now show diminished expression of AR, but no longer require androgen for growth, essentially identical to the behavior of IS3 cells that emerged after androgen exposure in vitro.
11.3
AR as Tumor Suppressor
Other groups have derived androgen-repressed sublines from the LNCaP cell line after androgen deprivation (Joly-Pharaboz et al. 1995, 2000; Soto et al. 1995; Geck et al. 1997; Culig et al. 1999; Shi et al. 2004). Abreu-Martin et al. (1999) reported that a constitutively activated form of MEKK1 could induce apoptosis in AR-positive cells but not in AR-negative cells. Lin et al. (2006) showed that AR was necessary for Bax-induced apoptosis in LNCaP cells and that androgen enhanced Bax-induced cell death. Recently AR was reported to repress expression of c-Met, the receptor for hepatocyte growth factor, in LNCaP cells by interfering with Sp1 binding to the c-Met promoter region (Verras et al. 2007). Increased c-Met signaling has been associated with prostate cancer progression (Knudsen et al. 2002; Knudsen and Edlund 2004). Evidence is increasing for the androgenrepressed phenotype in addition to that from the LNCaP cell line. The ARCaP cell line (Zhau et al. 1996) is another androgen-repressed prostate cancer cell line. Hara et al. (2003) reported suppression of MDA PCa 2b-hr prostate cancer cell
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growth by high-dose androgen after long term androgen deprivation. PC-3 cells ectopically expressing AR are repressed by androgen (Yuan et al. 1993; Heisler et al. 1997; Litvinov et al. 2004). Bruchovsky et al. (2000) observed possible androgenic repression of serum PSA in an interim analysis of the Canadian Prospective Trial of intermittent androgen-deprivation therapy. Decreased androgen levels during aging may contribute to the onset of prostate cancer in some men, and androgen replacement may benefit many patients with castration-recurrent prostate cancer as well as improve quality of life (Prehn 1999; Algarte-Genin et al. 2004). In an early study of androgen replacement therapy in prostate cancer patients, 7 of 52 patients experienced symptomatic benefit, while the remainder had unfavorable responses (Fowler and Whitmore 1981). Since this study predated convenient quantification of AR expression levels and analysis of AR expression was not performed, the seven patients with favorable responses may represent a patient subgroup with tumors expressing higher levels of AR than the patients who showed unfavorable responses. A more recent phase I study demonstrated no adverse effects of testosterone replacement administered by skin patch or topical gel in patients with castration-recurrent and metastatic disease (Morris et al. 2004). Phase II studies are planned. A recently published case study has findings similar to our animal studies and supports the use of testosterone to treat certain prostate cancer patients (Mathew 2008). In this case study, a prostate cancer patient had undergone radical prostatectomy and received LH-RH therapy. Testosterone levels remained at castrated levels and PSA was undetectable for 15 years. PSA levels then began to rise and the patient was given parenteral testosterone replacement therapy to attain a normal range of serum testosterone. After an initial flare, PSA levels gradually declined over 18 months. After 27 months, PSA level started to increase. When testosterone replacement therapy was discontinued, PSA levels dropped. Testosterone therapy also improved the patient’s quality of life. In the Prostate Cancer Prevention Trial, treatment with finasteride was reported to decrease the incidence of low-grade prostate cancer, but increase the incidence of high-grade prostate cancer (Thompson et al. 2003). The latter finding, however, may have resulted from detection bias due to finasteride-induced reduction in prostate volume (Cohen et al. 2007; Lucia et al. 2007). In experimental systems, androgen has been reported to repress cell growth during androgen-induced differentiation of immortalized human and rodent prostate epithelial cells in vitro (Ling et al. 2001; Whitacre et al. 2002). Bruchovsky et al. (1975) also described the repression by androgen on rat ventral prostate epithelial cell proliferation in the hormonal regulation of prostate homeostasis. In testosterone-induced rat ventral prostate regeneration, secretory epithelial cells enter a state of quiescence after an initial burst of cell proliferation (Waltregny et al. 2001). The proliferative status of cells correlated inversely with p27Kip1 expression. Normal differentiated prostate luminal secretory cells express high levels of p27Kip1 (Cote et al. 1998), and a strong correlation exists between low p27Kip1 expression in tumor cells and poor prognosis clinically (Guo et al. 1997; Cote et al. 1998; Yang et al. 1998; Vis et al. 2000). Recently, a low testosterone level in Nkx3.1:Pten mutant mice was reported to accelerate the progression of prostate cancer compared with a normal testosterone level
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(Banach-Petrosky et al. 2007). AR has been suggested to play a direct inhibitory role in the licensing of DNA for replication (Litvinov et al. 2006; Vander Griend et al. 2007). In this role, AR degradation in prostate tumor cells is hypothesized to be required during mitosis to allow origins of replication to acquire the capacity to fire in the next round of DNA replication. Overexpression of AR may prevent complete degradation of all AR and thereby repress cell cycle progression. This mechanism may operate parallel to the action of AR in regulating c-Myc and p27 levels in prostate cancer cells. c-Myc itself has recently been reported to function nontranscriptionally in the activation of origins of replication (Dominguez-Sola et al. 2007). Much evidence therefore exists for AR functioning as a liganddependent tumor suppressor in prostate cancer cells when it is expressed at high levels and is fully activated.
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12.1
Modulation of Liver X Receptor Signaling as Novel Therapy for Prostate Cancer Discovery and Characterization of Ubiquitous Receptor (UR or LXR)
The cloning of cDNAs for various steroid receptors led to the realization that conserved structure and sequence existed within this protein family and that new members of this family could be identified by using techniques relying on cDNA sequence homology especially within the DNA-binding domain. In 1993 as part of a project to identify a receptor for the weak androgen D5-androstenediol that produces estrogen-like effects on the rat vaginal epithelium (Shao et al. 1975), a novel member of the nuclear receptor superfamily was discovered (Song et al. 1994). This new receptor was named ubiquitous receptor or UR because of its expression in many tissues. cDNAs for the same receptor or paralogs of this receptor were isolated around this time or shortly after including LXR (Willy et al. 1995), NER (Shinar et al. 1994), RIP15 (Seol et al. 1995), RLD-1 (Apfel et al. 1994), and OR-1 (Teboul et al. 1995). Since one isoform of this receptor was present in liver at high levels, it was called liver X receptor (LXR). Eventually, the isoform most abundant in liver became known as LXRa (NR1H3), and the more ubiquitously expressed isoform became known as LXRb(NR1H2). LXRa is also abundant in other tissues besides liver, which include adipose tissue, adrenal gland, brain, intestine, kidney, testis, spleen, and macrophages (Bookout et al. 2006). LXRb is expressed in many mouse tissues and is expressed at higher levels than LXRa in most tissues (Bookout et al. 2006). LXRb is expressed in many cultured cell lines and most of the developing tissues of the mouse embryo based on immunocytochemistry (Song et al. 1995). The human gene for LXRb was localized to chromosome 19q13.3 using fluorescence in situ hybridization, (Le Beau et al. 1995). The gene for LXRa is located at 11p11.2.
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UR/LXR is an Oxysterol Receptor
LXRa and LXRb form heterodimers with the obligate partner retinoid X receptor that binds the ligand 9-cis retinoic acid (Song et al. 1994; Willy et al. 1995). The LXR/RXR heterodimers can be activated with either LXR or RXR agonists. Naturally occurring oxysterols, such as 22(R)-hydroxy-, 24-hydroxy-, 25-hydroxy-, 27-hydroxy-, and 24(S), 25-epoxycholesterol, bind to and activate transcription by LXR (Janowski et al. 1996, 1999; Lehmann et al. 1997; Fu et al. 2001; Spencer et al. 2001). Cholestenoic acid is also a natural ligand for LXR (Song and Liao 2000). A few synthetic LXR agonists have been developed, which include nonsteroidal compounds, such as T0901317 (Schultz et al. 2000) and GW3965 (Collins et al. 2002). Analogs of 6a-hydroxy bile acids activate LXR (Song et al. 2000) and oral administration of a 6a-hydroxy bile acid analog, hypocholamide (3a, 6a-dihydroxy-5b-cholanic acid-N-methyl-N-methoxy-24-amide), produces hypolipidemic effects in hypercholestermic rats, mice, and hamsters (Song and Liao 2001). Hypocholamide also enhances cholesterol and phospholipid transport to apoE in cultured mouse astrocytes (Peng et al. 2003). The compound 3a, 6a, 24trihydroxy-24, 24-di-(trifluoromethyl)-5b-cholane is a potent agonist for LXR, reduces atherosclerosis in LDLR-/- mice fed a Western diet, and does not induce triglyceridemia, a side effect of synthetic LXR agonists, like T00901317 that has complicated development of LXR agonists as drugs to treat cardiovascular disease (Peng et al. 2008). The auto-oxidized cholesterol sulfates, 5a, 6a-epoxycholesterol3-sulfate and 7-ketocholesterol-3-sulfate, often present in atherosclerotic plaques are antagonists of LXR (Song et al. 2001). LXRs are important regulators of cholesterol, fatty acid, and glucose homeostasis and modulate inflammation (Zelcer and Tontonoz 2006). LXR has an important role in reverse cholesterol transport, since it regulates the expression of the cholesterol transporters ABCA1, ABCG1, ABCG5, and ABCG8 and the lipid acceptor lipoprotein apoE (Zelcer and Tontonoz 2006). Composition of lipoproteins may also be controlled by LXR, since it regulates the expression of cholesterol ester transfer protein and phospholipid transfer protein, which are involved in lipid transfer between different lipoproteins (Zelcer and Tontonoz 2006). Treatment with an LXR agonist lowers the cholesterol level in serum and liver and inhibits the development of atherosclerosis in murine models of this disease (Peet et al. 1998; Alberti et al. 2001; Joseph et al. 2002). LXR also regulates fatty acid synthesis by modulating the expression of sterol regulatory element-binding protein-1c (SREBP-1c) and downstream lipogenic genes, which include acetyl CoA carboxylase and fatty acid synthase (Repa et al. 2000; Yoshikawa et al. 2001; Liang et al. 2002). LXR agonists suppress gluconeogenesis, induce expression of glucokinase in liver, stimulate insulin secretion, reduce plasma glucose, and improve glucose tolerance and insulin resistance in murine and rat obesity models (Cao et al. 2003; Laffitte et al. 2003; Efanov et al. 2004). Glucose is a low-affinity ligand for LXR and can modulate its transcriptional activity at physiological concentrations (Mitro et al. 2006). LXR agonists can have an anti-inflammatory effect (Joseph et al. 2003), and LXR signaling is important for brain function (Wang et al. 2002; Andersson et al. 2005).
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Antiproliferative Effect of LXR Agonists
Production and secretion of cholesterol increases in prostate cancer compared to healthy prostate tissue (Sporer et al. 1982). Expression of fatty acid synthase, the enzyme responsible for converting acetate to fatty acids, is up-regulated in early stage prostate cancer and further increases in advanced prostate cancer (Epstein et al. 1995; Shurbaji et al. 1996; Swinnen et al. 2002). Since LXR signaling regulates both cholesterol and fatty acid homeostasis, it is possible that LXR agonists may be good candidates for treatment or management of prostate cancer. Since 2000, our laboratory has had projects focusing on identification of new LXR agonists for cardiovascular and Alzheimer’s disease therapy and novel therapies against prostate cancer. In 2004, several natural compounds including oxysterols used in the LXR project were tested in an assay of prostate cancer progression. Unexpectedly, we found that 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and T0901317 suppressed the proliferation of both androgen-dependent 104-S cells and androgen-independent 104-R1 cells (Fukuchi et al. 2004). These LXR agonists also suppressed the proliferation of AR-negative prostate cancer cell lines PC-3 and DU145. The suppression of cell proliferation using LXR agonists was via induction of G1 cell cycle arrest. T0901317 decreased the percentage of cells in S-phase, increased the percentage of cells in G1-phase, suppressed the expression of Skp2, and promoted the accumulation of cell cycle inhibitor p27Kip1. In vivo, administration of T0901317 suppressed prostate tumor growth in athymic mice. T0901317 (10 mg/kg/day) inhibited the growth of androgen-sensitive LNCaP 104-S tumors, which resulted in a twofold difference in mean tumor volume between the control and the LXR agonist-treated mice (Fig. 8). T0901317 was found later to suppress the proliferation of many commonly used cancer cell lines (Chuu et al. 2007).
12.4
Inhibition of Prostate Cancer Progression Using LXR Agonists
Since expression of LXRa and its target gene ABCA1 was lower in androgenindependent 104-R1 and 104-R2 cells compared to androgen-dependent 104-S cells (Fukuchi et al. 2004), down-regulation of LXR signaling-related genes may assist the progression of prostate cancer cell toward androgen independency. A prostate cancer progression model was developed in athymic mice using LNCaP cells that mimic the cell culture model. LNCaP 104-S cells were inoculated into athymic mice to form tumors. Androgen-sensitive 104-S tumor xenografts progressed to androgen-independent tumors (104-Rrel) in athymic mice after castration (Chuu et al. 2006). The growth of 104-Rrel tumors was suppressed using testosterone. AR expression in tumors and serum PSA increased during progression from 104-S to 104-Rrel (Chuu et al. 2006). Expression of LXRa, ABCA1, and another LXRa target gene, sterol 27-hydroxylase (CYP27), decreased during progression from
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Fig. 8 Inhibition of proliferation and progression of prostate cancer by the LXR agonist T0901317. (a) Inhibition of androgen-sensitive LNCaP 104-S tumor growth in intact mice by treatment with the LXR agonist T0901317. Mice were administered 10 mg/kg T0901317 ( filled square, 10 mice with 13 tumors) or vehicle alone ( filled circle, 10 mice with 15 tumors) using gavage once a day during the experimental period. Relative tumor volumes were expressed as mean SE. (b) Inhibition of progression of androgen-sensitive LNCaP 104-S tumors toward androgen-independent 104-Rrel tumors in castrated mice by treatment with the LXR agonist T0901317. After castration of control mice that were used in Fig. 5a, mice were administered 10 mg/kg T0901317 ( filled square, 9 mice with 15 tumors) or vehicle alone ( filled circles, 9 mice with 13 tumors) by gavage five times a week during the experimental period. Relative tumor volumes were expressed as mean SE
104-S to 104-Rrel (Chuu et al. 2006). The development of androgen-independent relapsed tumors in athymic mice bearing 104-S tumors after castration was delayed by 4 weeks in mice treated with T0901317 (10 mg/kg; Fig. 8) compared to the control group (Chuu et al. 2006). Therefore, LXR agonists can retard progression of prostate cancer in vivo.
12.5
Phytosterols and LXR Signaling
Phytosterols, plant equivalents of mammalian cholesterol, are essential components of all plants. Phytosterols are abundant in plant oil, seeds of legumes and nuts, as well as vegetables and fruits. The most common phytosterols include b-sitosterol, campesterol, and stigmasterol. Dietary phytosterols are believed to be beneficial to health and are reported to have anticancer activity. Treatment with b-sitosterol also retards the growth and metastasis of ARnegative PC-3 tumors in mice (Awad et al. 2001) and the proliferation of ARpositive LNCaP cells in culture (Awad et al. 2000). The mechanism involved in the anticancer activity of phytosterols is not clear. However, plant sterols and stanols
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from the 4-desmethylsterol family activate LXRa and LXRb (Plat et al. 2005) with EC50 of 30–150 nM. Phytosterols, therefore, may suppress growth and metastasis of prostatic cancer through activation of LXR signaling. LXR agonists inhibit proliferation and progression of prostate cancer cells both in vitro and in vivo. LXR agonists also suppress the proliferation of breast cancer cells in vitro. Phytosterols, a recently discovered group of LXR agonists, inhibit proliferation of several prostate and breast cancer cell lines. Synthetic nonsteroidal LXR agonists T0901317 and GW3965 exhibit more potent agonistic effect than natural oxysterols. Although T0901317 and GW3965 were reported to be beneficial for treatment of atherosclerosis, diabetes, and Alzheimer’s disease in murine disease models, both T0901317 and GW3965 have been reported to increase plasma and liver triglycerides in some mice models. The synthetic steroidal LXR agonist hypocholamide, a 6a-hydroxylated analog of bile acids, shows an overall hypolipidemic effect but does not increase the serum triglyceride level (Song and Liao 2001). YT-32, a synthetic LXR agonist developed by modifying the phytosterol structure, selectively activated intestinal ABC transporters in mice without increasing plasma triglyceride levels (Kaneko et al. 2003). These findings with hypocholamide and YT-32 suggest the possibility of developing potent and useful LXR agonist drugs through modification of bile acid and phytosterol structure. Although the exact mechanism responsible for inhibition of prostate cancer progression by LXR agonists requires further study, modulation of LXR signaling may be a novel and potentially important therapy for prostate and other cancers.
13
Conclusion
The journey into the realm of androgen biology has taken us to many different areas, and much has been learned about the mechanism of action of this steroid hormone over the past 50 years. Many individuals and many different laboratories have contributed in this effort. Even though our knowledge in this field has increased enormously, much more remains to be learned and perhaps some surprises await us.
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Clinical Progression to Castration-Recurrent Prostate Cancer Mark Pomerantz and Philip Kantoff
1 Introduction Since Huggins and Hodges proved unequivocally that prostate cancer (CaP) regresses in the castrate state, androgen deprivation therapy (ADT) has been the cornerstone of treatment for advanced CaP (Huggins and Hodges 1941). This treatment strategy has taken many forms. Bilateral orchiectomy reliably diminishes androgen production and was the mainstay of treatment in the 1940s. In 1941, Huggins and Hodges published their seminal work showing that therapy with estrogen, in the form of diethylstilbestrol (DES), was as effective as orchiectomy (Huggins and Hodges 1941). Chemical castration via DES was widely used until the mid-1960s, when the estrogen formulation was directly compared to other strategies, including bilateral orchiectomy. DES was associated with an improved cancer-related mortality but worse overall survival, due primarily to increased cardiovascular side effects (The Veterans Administration Co-operative Urological Research Group 1967). Despite attempts to find a less toxic but effective dose, DES fell out of favor as an alternative to orchiectomy. In the 1980s, long-acting, synthetic luteinizing hormone-releasing hormone (LHRH) agonists were developed (Tolis et al. 1982). These agents have emerged as the predominant method for achieving castrate levels of androgen. ADT is highly effective, capable of inducing regression of disease in over 90% of CaP patients (Prostate Cancer Trialists’ Collaborative Group 2000; The Medical Research Council Prostate Cancer Working Party Investigators Group 1997). However, ADT is rarely curative. While the time in remission can vary markedly between patients, CaP invariably becomes refractory to surgical or chemical castration. This chapter will examine clinical progression from androgen dependence to androgen independence, focusing on the clinical factors associated with the
M. Pomerantz(*) Lank Center for Genitourinary Oncology, Dana-Farber Cancer Institute, Dana Bldg, Room 1230, Boston, MA 02115, USA, E-mail:
[email protected]
D. Tindall and J. Mohler (eds.), Androgen Action in Prostate Cancer, DOI: 10.1007/978-0-387-69179-4_2, # Springer Science + Business Media, LLC 2009
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emergence of resistance, and the natural history of and treatment strategies for castration-resistant prostate cancer (CRPC).
2 Definition of Androgen-Independent Prostate Cancer Castration recurrent prostate cancer (CRPC) denotes progression of disease despite chemical or surgical castration. Various terms have been used to describe this disease state, and most have limitations. ‘‘Hormone refractory prostate cancer’’ is a term commonly used but is often inaccurate. Many patients who progress on ADT may still respond to further hormonal manipulation, such as inhibition of ligand– androgen receptor interaction or suppression of adrenal androgens (Scher et al. 1995). The term androgen-independent prostate cancer (AIPC) also can be misleading. AIPC is ‘‘androgen independent’’ in the sense that CaP flourishes at castrate levels of androgen, but the disease remains to some extent dependent on signaling of the androgen receptor. ‘‘CRPC’’ has been adopted as the best descriptor, is becoming more widely used, and will serve as the term describing castrateresistant disease in this chapter. The Prostate Specific Antigen Working Group was organized in 1999 to establish a standard definition for progression of CaP in the castrate state and maintain consistency across clinical trials (Bubley et al. 1999). The group defined progression as an increase in radiographic measurements of soft tissue CaP metastases, an increase in the number of metastatic sites on bone scan, or two consecutive increases in prostate-specific antigen (PSA). Disease progression must occur after bilateral orchiectomy or in the setting of a serum testosterone of 50 ng/dl. These criteria broadly characterize CRPC, yet they do not neatly categorize all men with CRPC. The biology and natural history of CRPC can vary widely from patient to patient. Further categorization is necessary to better assess prognosis and optimal treatment for CRPC patients. Prior to the era of widespread PSA testing, there was considerably more uniformity among CRPC patients. Since biomarkers such as PSA that herald the onset of advanced disease did not exist, surgical or chemical castration was rarely performed prior to the onset of CaP-related symptoms. Thus, virtually all men who developed CRPC reached this state in the setting of metastatic disease. In the PSA era there is considerably more clinical diversity among CRPC patients, and the variation in clinical presentation at the onset of CRPC has important implications. Much of this heterogeneity is related to the substantial stage migration that has occurred since the introduction of PSA testing. The majority of PSA-screened CaP patients are treated with radical prostatectomy (RP) or radiation therapy (RT) for localized disease (Catalona et al. 1993; Farkas et al. 1998), and up to 40% of these patients experience biochemical recurrence, defined as a serial rise in PSA in the absence of clinical metastases (Moul 2000). A rise in PSA is their first manifestation of recurrence, usually occurring well before the onset of metastatic CaP. Despite a lack of convincing evidence of benefit
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of early intervention, at least one-third of patients are treated with ADT within 5 years of PSA-only recurrence without evidence of metastases (Mehta et al. 2004). Analysis of the Cancer of the Prostate Strategic Urologic Research Endeavor (CaPSURE) database, a network of urologists at more than 30 community and academic urology practice sites, showed that the median time from biochemical recurrence to the initiation of ADT is approximately 9 months (Mehta et al. 2004). As a result of starting ADT early in the course of disease, a considerable number of patients develop CRPC before the onset of clinical progression. The natural history of this group of patients is different from that of CRPC patients with metastatic disease, as will be discussed later. Another important factor dividing CRPC patients into separate biologic categories is responsiveness to secondary hormonal manipulations. One group of patients progresses despite castrate levels of testosterone but is still capable of responding to further hormonal manipulation. A second group is deemed insensitive to all forms of hormonal therapy and is truly ‘‘hormone refractory.’’ Finally, it is now well established that ADT in the neoadjuvant and adjuvant setting with radiation therapy (RT) improves outcomes compared with RT alone for those with intermediate- or high-risk CaP. Should these patients recur after treatment, ADT is often reinitiated. This introduces another factor distinguishing one group at risk for CRPC patient from another: those who have been exposed to ADT in the past versus those who have not. Some evidence suggests that such patients progress to CRPC more rapidly. In short, CRPC can be loosely defined as progression of disease despite castrate levels of androgen. However, a more precise definition must take into account other factors. As will be discussed later, responsiveness to other hormonal manipulations, extent of disease, and timing of initial ADT affect progression from androgen-naı¨ve to castration-resistant CaP. These distinctions also affect prognosis and treatment options.
3 Clinical Predictors for the Development of CRPC In the PSA era, clinical progression from treatment for localized disease to prostate cancer-specific mortality (PCSM) typically begins with PSA recurrence, ultimately followed by development of metastases and death (Pound et al. 1999). Several retrospective and prospective studies have examined time to reach these three endpoints (The Medical Research Council Prostate Cancer Working Party Investigators Group 1997; Moul 2000; Messing et al. 1999). Meanwhile, sometime during this series of events, ADT is started and CRPC develops. The length of time between the start of ADT and the development of CRPC, as gauged by PSA levels, may influence the time to clinical progression or PCSM; there is evidence that the time to PSA progression on ADT has prognostic significance (Shulman and Benaim 2004; Svatek et al. 2006). Moreover, evaluating time to PSA progression on ADT may provide important insight into the biology of CaP, as PSA elevation is the first
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indication of CaP growth in the castrate state. However, few studies have comprehensively examined time to ADT resistance as a clinical endpoint, and fewer still have completely captured the heterogeneity of patients developing CRPC. A retrospective series of 553 patients treated with ADT at the Dana-Farber Cancer Institute (DFCI) is among the largest studies to date analyzing time to CRPC (Ross et al. 2008). Most in this cohort were treated in the setting of relapse after local treatment with RP or RT. Approximately half of the patients had developed metastatic disease by the time ADT was initiated, and 16% had previously received ADT as part of neoadjuvant and/or adjuvant treatment. With a median follow-up of over 5 years, the median time to PSA progression from the start of ADT was 23.7 months (Ross et al. 2008). Other, smaller studies have reported time to CRPC among ADT patients (Oefelein et al. 2002; Fowler et al. 1995). One retrospective analysis consisted of 245 patients with localized CaP and 78 patients with metastatic CaP followed at the University of Mississippi. Those with localized CaP were ineligible for definitive treatment with RP or RT due to advanced disease, age, or comorbidity. ADT was the initial CaP treatment for all patients. As in the DFCI cohort, PSA progression was analyzed as a clinical endpoint. Median time to CRPC was only 15 months in the nonmetastatic subgroup and 10 months in the metastatic subgroup. The differences in outcome among these datasets reflect the heterogeneity of patients receiving ADT. Though Gleason scores appear similar across the two studies, shorter time to CRPC is related to the extent of disease at the start of ADT. Virtually all patients in the University of Mississippi cohort, for example, were diagnosed due to physical findings or CaP-related symptoms. The majority of patients classified as having ‘‘localized’’ disease had locally advanced disease, such as lymph node or seminal vesicle involvement (Fowler et al. 1995). The median pre-ADT PSA in the locally advanced group was 49 ng/mL, compared with 8.4 ng/mL in the DFCI subgroup without metastatic CaP. Overall, the differences across studies demonstrate how the development of CRPC is influenced by host factors. This observation was clearly demonstrated within each series, when patients who started ADT in the setting of metastases were directly compared with those who started before objective evidence of metastases. In each study, the extent of disease at the time of initiation of ADT significantly effected the time to development of CRPC (Ross et al. 2008; Fowler et al. 1995). The biological basis for this is not known, but it may be that advanced, metastatic tumors contain a higher number and/or proportion of cells whose growth is androgen independent, accounting for a more rapid detection of CRPC as these cells flourish. Clinical parameters at the time of ADT initiation also predicted time to resistance in these studies. In the DFCI cohort, PSA > 16.3 ng/mL was a predictor of CRPC (p < 0.05) (Ross et al. 2008). A higher PSA level, like metastatic disease itself, perhaps reflects a higher volume of disease, a larger volume of androgenindependent cells, and therefore shorter time to progression. However, this association was limited to only those patients without evidence of metastases at the ADT start date. A similar finding was reported in the University of Mississippi cohort. A direct correlation was noted between pretreatment PSA levels and time to PSA
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elevation in the group with localized disease but not in the group with metastases (Fowler et al. 1995). Why this effect should be limited to those without evidence of metastases is less clear. Perhaps, once CaP tumors reach a volume detectable by CT or bone scan, absolute PSA value is a less meaningful surrogate for the size of the androgen-independent cell population. The presence of detectable metastatic tumors in itself may indicate that an appreciable number of castrate-resistant cells exist, regardless of PSA level. Other pretreatment PSA parameters may provide a more accurate reflection of the biology of the disease and how quickly CRPC will emerge in the metastatic setting. In particular, a shorter PSA doubling time (PSADT) prior to ADT is associated with a shorter time to progression to CRPC. In the DFCI cohort, across both metastatic and nonmetastatic patients, those with a PSADT < 3 months prior to ADT experienced PSA progression sooner than those with a longer PSADT (median 12 months vs. 33 months, p = 0.02) (Ross et al. 2008). As will be discussed later, PSA kinetics are also strongly associated with clinical outcome in patients with CRPC. Another PSA parameter predicting development of CRPC is PSA nadir. A lower PSA nadir (0.2 ng/mL in the DFCI study) is associated with longer time to progression to CRPC when using PSA endpoints (Ross et al. 2008; Oefelein et al. 2002; Fowler et al. 1995). Of note, Gleason score greater than 7 was associated with shorter time to CRPC among patients with metastatic disease in the DFCI cohort (Ross et al. 2008). This suggests that high-grade, androgen-naı¨ve, metastatic CaP becomes resistant sooner than low- or intermediate-grade disease (Morote et al. 2005). Yet, this biologic event may not have significant clinical consequences. Gleason score is not consistently associated with survival in patients with metastatic disease starting ADT (Yossepowitch et al. 2007; Figg et al. 2004). A particularly intriguing finding from the DFCI study suggests that prior ADT for local disease affects the development of CRPC. The median duration of neoadjuvant and adjuvant ADT was 8 months, and the median duration between completion of ADT as a part of local therapy and the beginning ADT for recurrence was about 22 months. Prior therapy was associated with shorter time to CRPC in metastatic and nonmetastatic patients, even when taking other established prognostic factors into account (Ross et al. 2008). This suggests at least two possible models for CRPC. One is that time on ADT is ‘‘fixed’’ no matter when the ADT is administered. If a patient’s disease is ‘‘calibrated’’ to become resistant after a biologically specified period of ADT, the duration of effective ADT can be divided into different segments, for example, a period of adjuvant therapy and a later period of salvage therapy. Time to CRPC is then dependent on the total time treatment was administered. Another possibility is that the time disease remains androgen-sensitive is ‘‘set’’ from the time of first exposure to ADT until the time of CRPC, and includes periods off treatment. In this scenario, adjuvant ADT and ADT after biochemical recurrence are, in effect, part of an intermittent ADT regimen, with a long interval between treatments. Experience with intermittent androgen deprivation (IAD) informs this issue. Several phase II and phase III studies have examined the efficacy of administering
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chemical castration for a proscribed period of time, then withholding treatment until disease progresses to a predetermined PSA threshold. It has been shown that the time from start of hormonal therapy until the development of CRPC is similar for both IAD and continuous ADT (Stewart et al. 2005). This is consistent with the aforementioned latter scenario. On the other hand, these data are not consistent with data from Radiation Therapy Oncology Group (RTOG) trial 86-10. In this trial, men with extensive local involvement of CaP were randomized to 4 months of neoadjuvant ADT followed by RT or RT alone (Pilepich et al. 2001). Secondary analysis was performed to assess overall and disease-specific survival among men in both treatment arms who ultimately required salvage ADT (Shipley et al. 2006). Outcomes were not significantly different between those who received neoadjuvant treatment and those who did not. Unlike the DFCI analysis, the study did not examine time to PSA recurrence and therefore did not capture the earliest marker for CRPC. The findings may not contradict one another. However, they do raise the possibility that there is a threshold duration of prior ADT, which does not compromise outcome. The median duration of neoadjuvant/adjuvant ADT in the DFCI series was twice as long as the length of ADT in the RTOG study. Serum testosterone levels after castration levels may also be a significant predictor for development of CRPC. In a recent study, serial serum testosterone levels were measured over the first 6 months of ADT in nonmetastatic CaP patients. Patients were treated with LHRH agonist alone or in combination with an antiandrogen (Morote et al. 2007). Among those treated with LHRH agonist alone, time to CRPC was significantly shorter for those with a testosterone level greater than 32 ng/dL (88 months vs. 137 months). Interestingly, for patients receiving combined androgen blockade (LHRH agonist and antiandrogen), this difference was not seen, even for patients with a testosterone level greater than 50 ng/dL. This suggests that post-ADT testosterone levels can predict time to CRPC. This study also nicely demonstrates that CRPC patients are not necessarily ‘‘hormone refractory.’’ Further manipulation of the androgen axis, in this case with an antiandrogen, may provide further benefit to the CRPC patient. In summary, patients are started on ADT at widely varying stages of their disease. This significantly influences the development of resistance to castrate levels of testosterone. The extent of disease, PSA parameters, Gleason score, previous exposure to ADT, and adrogen levels are factors that may impact the time to CRPC.
4 CRPC Natural History and Predictors of Clinical Outcome Once CRPC develops, insight into its natural history and predictors of clinical outcome are valuable and may help direct treatment decisions. Extent of disease is, again, a critical consideration and influences the natural history of castrate-resistant disease. Patients with PSA-only CRPC appear to have a better outcome compared than patients with CRPC that develops in the setting of metastases. However, PSA-
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only CRPC is a relatively new entity, a result of changing practice patterns in the PSA era. Few studies have focused directly on CRPC patients with no clinical evidence of disease. The most thorough examination of this patient population was performed as part of an aborted study evaluating the effect of zoledronic acid on time to bone metastases in men with PSA-only CRPC (Smith et al. 2005). The study was closed due to a lower than expected event rate, but valuable insight was gained into the natural history of CRPC in the absence of clinical disease. The study enrolled 201 CRPC patients and at 2 years, only 33% developed bone metastases. Median metastasis-free survival was 907 days, and median overall survival was not reached at 2-years follow-up. The relatively indolent nature of disease in this study stands in contrast to CRPC in the setting of metastasis, as discussed later. It should be noted, however, that interventions with secondary hormonal therapy or chemotherapy were not documented in this cohort. It is therefore unknown if further therapeutic interventions contributed to outcome. PSA levels were well annotated in this study and served as useful predictors of outcome. A longer PSA doubling time, for example, was associated with metastasis-free survival – a parameter that is also a strong predictor of outcome in hormone-sensitive CaP (D’Amico et al. 2003). In multivariate analysis, PSA > 10 ng/mL and increased PSA velocity were associated with shorter time to first metastasis and were predictive of overall survival (Smith et al. 2005). Notably, PSA velocity was also found to be associated with mortality in a similar cohort of CRPC patients analyzed by D’Amico et al. (2005). Patients in this cohort, assembled from two multi-institutional databases, started ADT for PSA recurrence after RP or RT and ultimately developed CRPC. PSA velocity greater than 1.5 ng/mL/year after the start of ADT was associated with increased risk for all-cause and prostate cancer-specific mortality. The natural history of metastatic CRPC has been more widely studied, though it is important to note that even within this population of CRPC patients, there is considerable clinical variability. Much of the data concerning the metastatic CRPC is derived from several studies enrolling a high percentage of patients with significantly advanced disease (Scher et al. 1999; Vollmer et al. 1998; Kantoff et al. 1999; Tannock et al. 2004; Petrylak et al. 2004). Two groups collected data from several such trials to describe the clinical course of metastatic CRPC and to define predictors of outcome (Smaletz et al. 2002; Halabi et al. 2003). The first analysis was derived from clinical series at Memorial Sloan-Kettering Cancer Center (MSKCC). A total of 409 patients were included in a discovery set and 433 in a validation set. Median survival across both groups was less than 16 months (Smaletz et al. 2002). The second analysis was based on a collection of six studies conducted by the Cancer and Leukemia Group B (CALGB). A total of 760 patients were included in the learning sample and 341 in a validation sample. Median survival across these groups was 13 months after a follow-up of 37 months (Halabi et al. 2003). Predictors for survival after multivariable analysis from each study were used to develop nomograms. Both studies found poor performance status, lower
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hemoglobin, higher LDH, and higher alkaline phosphatase to be associated with poorer overall survival. The CALGB study also incorporated Gleason score >7 and presence of visceral metastases into its nomogram, while the MSKCC study did not find that these parameters added information to their model. The MSKCC nomogram included age and levels of albumin, while the CALGB model did not. A higher PSA prior to treatment for CRPC correlated with worse outcome in the MSKCC study, but the effect was very slight. In fact, when comparing the 25% and 75% PSA quartiles, no difference in survival was seen (Smaletz et al. 2002). In an analysis of 143 CRPC cases from DFCI, higher PSA levels predicted improved survival for men with bone metastases and a normal alkaline phosphatase (Xie et al. 2007). PSA in this setting may be a surrogate for two competing biologic processes. On the one hand, a higher PSA represents progressive disease, greater tumor volume and, as a result, worse outcome. As such, PSA also may be associated with worse performance status, higher LDH, and lower hemoglobin – parameters that directly correlate with outcome. On the other hand, a higher PSA may be associated with a more differentiated tumor, and a tumor with greater proportion of androgen-dependent cells. At an advanced stage of disease, this could represent a more favorable biology. Also, PSA itself is reported to have antiangiogenic properties (Balk et al. 2003). Finally, PSA has been implicated in the degradation of PTHrP (Balk et al. 2003), perhaps accounting for the normal alkaline phosphatase observed in the subset of patients in whom PSA appeared protective. The MSKCC and CALGB studies only analyzed single, static PSA levels (Smaletz et al. 2002; Halabi et al. 2003). Since the publication of these studies, numerous groups have demonstrated the predictive value of PSA kinetics. In one series of 160 CRPC patients diagnosed in the PSA era, PSA doubling time was the variable most strongly associated with prostate cancer-specific survival (Shulman and Benaim 2004). Investigators divided the cohort into risk groups and highlighted the heterogeneity in presentation and prognosis among CRPC patients with advanced disease. The group with the most favorable PSA parameters, men with a PSADT >6 months, had a median cancer-specific survival of 89.1 months. Those in the highest risk grouping, which included men with a PSADT <6 months, had a median cancer-specific survival of 14.0 months. Unlike the MSKCC and CALGB studies, no patient in this cohort received cytotoxic therapy, suggesting that castration-resistant disease was discovered relatively early in its course. Likewise, in a similar cohort of 129 men with untreated CRPC, median overall survival was 52 months, and PSADT remained a statistically significant predictor of survival in multivariate models (Svatek et al. 2006). Two recent studies evaluated survival in more advanced CRPC patients, those about to receive chemotherapy (Oudard et al. 2007; Armstrong et al. 2007). These series are particularly informative since most patients received docetaxel-based chemotherapy, the contemporary standard of care. The first study involved a cohort of 250 patients. For patients with a PSADT <45 days, median survival was 16.5 months and with a PSADT of >45 days it was 26.4 months (Oudard et al. 2007). The second study analyzed the cohort from the TAX327 trial, comparing docetaxel-based regimens to mitoxantrone. PSADT was separated into quintiles.
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Median survival for patients with a PSADT <1 month was 13.3 months, and for patients with a PSADT >6 months median survival had not yet been reached (mean survival 22.5 months) (Armstrong et al. 2007).
5 Treatment of CRPC As described earlier, the clinical course of CRPC may vary considerably. As such, treatment should be individualized based on a variety of factors including the presence or absence of metastases, performance status, and PSA kinetics. However, choosing an appropriate treatment for a particular patient can be challenging since there is an absence of survival data for most CRPC therapies. Moreover, CRPC is a difficult condition to evaluate via clinical trials. Overall survival is the most meaningful clinical parameter, but may not be a realistic study endpoint given the long natural history of many with CRPC, particularly those with PSA-only recurrence. Surrogates for survival in CaP have limitations. Compared with most other common cancers, CaP develops few soft tissue lesions measurable by radiograph. The most common site of CaP metastasis is bone, and bone scans are ill suited for accurately measuring changes in disease and are subject to interobserver variation. PSA would seem a worthy surrogate marker in light of the data emerging from CRPC series, as shown earlier. In addition, there are clinical data suggesting that treatment-induced PSA decreases 50% confer a survival advantage (Scher et al. 1997; Small et al. 2004). Yet there remains a lack of consensus regarding the best use of PSA, and no prospective trial data are available to resolve the issue. There may be a patient population in which a watchful waiting approach is appropriate. CRPC patients with PSA-only recurrence after ADT may experience prolonged metastasis-free survival, particularly in the setting of favorable predictors, such as a long PSA doubling time (Smith et al. 2005). At the opposite extreme, cytotoxic therapy may be indicated for patients with rapidly progressive, symptomatic disease. In particular, docetaxel-based therapy has been clearly shown to improve survival and quality of life in this population (Tannock et al. 2004; Petrylak et al. 2004). For the large number of CRPC patients with a disease profile somewhere in between these two scenarios, a secondary hormonal manipulation may be the treatment of choice. When initiating secondary hormonal treatment, it is helpful to consider the pathophysiology of CRPC. Despite the use of the term ‘‘androgen independence’’ to describe this disease state, the androgen axis remains involved in disease progression. In fact, the androgen axis often appears hyperactive in this disease state, and androgen receptor is consistently overexpressed in CRPC (Chen et al. 2004). Thus, relatively low levels of serum and intraprostatic androgen are required to promote disease progression. Moreover, mutations in the androgen receptor have been described, and certain mutations may confer a growth advantage to a given CaP clone (Taplin et al. 1995). The androgen axis hyperactivity may also result from alterations in cofactor expression, increased ligand promiscuity on the part of
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the androgen receptor, or complete independence from ligand-receptor binding (Feldman and Feldman 2001). Because the androgen axis remains very active in CRPC and is helping drive disease progression, ADT must be maintained no matter which treatment course is chosen, including watchful waiting. Though prospective data are lacking, there appears to be benefit in minimizing the ligand available to the androgen receptor. In a retrospective analysis of 341 patients treated in four randomized controlled trials for CRPC, continued androgen suppression was an independent predictor of survival (Taylor et al. 1993). While secondary hormonal therapies have several mechanisms of action, there are two main methods by which these treatments take advantage of the cancer’s continued dependency on the androgen pathway: antagonism of the androgen receptor (AR) and decrease in adrenal androgen production. For patients progressing after LHRH agonist or bilateral orchiectomy alone, AR antagonism with nonsteroidal antiandrogens is the strategy of choice. In a series of over 200 patients receiving the antiandrogen flutamide for metastatic CRPC, response rate was 34.5% and mean duration of response was 24 months (Labrie et al. 1988). Among patients progressing while receiving an antiandrogen (either as part of initial treatment or as salvage treatment) up to 20% experience a greater than 50% decrease in PSA in response to withdrawal of the antiandrogen (Small and Carroll 1994; Small and Vogelzang 1997). The withdrawal effect occurs because the antiandrogen, which once served as an antagonist of the androgen receptor, begins to act as an agonist. It has been shown that over time antiandrogens select for androgen receptor mutations, fundamentally changing their effects. However, this may not be the mechanism of action in all cases (Taplin et al. 1999). Response, when it occurs, generally lasts 3–5 months before further progression. Despite the low rates of withdrawal response, almost all CRPC patients should be given a trial period off antiandrogen before new therapy is started. This transformation from inhibition to activation appears to be a molecular event specific to each individual antiandrogen agent. This is evidenced by the fact that many patients respond to a second antiandrogen after failing a prior one. High doses of the antiandrogen bicalutamide (150–200 mg) are capable of inducing responses in patients who progressed on flutamide (Scher et al. 1997; Joyce et al. 1998). The antiandrogen nilutamide is also effective as a second line antiandrogen. In one study, this agent induced a nearly 50% reduction in PSA in 27% of patients whose disease had progressed on flutamide or bicalutamide (Kassouf et al. 2003). Since the androgen receptor remains active in the setting of CRPC, further suppression of circulating androgen has therapeutic potential. This has been demonstrated with the administration of cytochrome P450 inhibitors, agents that block steroidogenesis in the adrenal gland as well as the testes (Pont et al. 1982). The adrenal gland is the source of approximately 10% of androgen production. The antifungal drug ketoconazole is the most widely used regimen in this class. A phase III trial randomized 260 CRPC patients to antiandrogen withdrawal or antiandrogen withdrawal followed by high-dose ketoconazole 400 mg three times daily with hydrocortisone (Small et al. 2004). Hydrocortisone is routinely included in this
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regimen to prevent adrenal insufficiency. Those progressing on withdrawal alone arm were allowed to receive deferred ketoconazole. PSA response rates were 27% in the ketoconazole arm and 32% when including the patients receiving deferred treatment. Other trials using intermediate to high doses of ketoconazole have shown 50% reduction in PSA in 28–62% of patients (Small et al. 1997; Millikan et al. 2001; Nakabayashi et al. 2006). The agent abiraterone is also a potent inhibitor of adrenal androgen steroidogenesis. It is more focused than ketoconazole, specifically targeting 17-alpha hydroxylase and C17,20-lyase. Early clinical data suggest a profound effect on androgen production, and clinical trials utilizing this agent in patients with CRPC are ongoing (Taplin 2007). Estrogens have known activity in CRPC, a phenomenon reported as early as the 1950s, when Nesbit and Baum reported a 17% response rate in this setting (Nesbit and Baum 1950). Several studies have since demonstrated estrogen’s effects in CRPC, including several in the PSA era (Oh 2002). Estrogens are administered in several forms – diethylstilbestrol (DES), transdermal estradiol, conjugated estrogens (Premarin), and, though no longer available, PC-SPES. Estrogens exert their effects though multiple mechanisms. There is estrogen-mediated inhibition of the pituitary release of gonadotropins, which blocks testosterone production by the testes. It appears unlikely, however, that further decrease in circulating testosterone beyond that achieved by an LHRH agonist is clinically meaningful in CRPC. There is also evidence that estrogen treatment leads to a decrease in adrenal androgen precursors, suggesting a mechanism similar to ketoconazole (Takezawa et al. 2001; Kitahara et al. 1997). The side effects associated with estrogen therapy can be significant. Thomboembolic and cardiovascular events are not uncommon, and even attempts to utilize a lower dose of estrogen (DES 1 mg) result in event rates between 5 and 18% (Oh 2002). Nonetheless, estrogens can induce responses in CRPC, and side effects may be manageable when patients are carefully monitored and treated with antithrombosis prophylaxis. In the PSA era, Smith et al. administered 1-mg DES and reported a 43% PSA response rate in 21 CRPC patients with a 5% rate of deep venous thrombosis (Smith et al. 1998). More recently, 90 CRPC patients were randomized in a phase II crossover study to 3-mg DES or the estrogenic herbal formulation, PCSPES. PSA response to DES was 24% with a thromboembolism rate of 9% after low-dose warfarin prophylaxis (Oh et al. 2004). Less data are available for other estrogen formulations. Transdermal estrogens have an improved safety profile, and no thromboembolic events were noted across two studies following a total of 44 patients (Ockrim et al. 2003; Bland et al. 2005). However, PSA response rates were poor, only 12.5% in one study (Bland et al. 2005). Premarin is composed of conjugated estrogens and has been widely studied as hormone replacement therapy in postmenopausal women. In a phase II trial including 45 CRPC patients, PSA declines of greater than 50% were seen in 25% receiving high-dose Premarin (1.25 mg three times daily) (Pomerantz et al. 2007). Efforts are ongoing to improve the effectiveness of secondary hormonal manipulations. Several agents were developed to better utilize the mechanisms of action described earlier. For example, new androgen receptor antagonists are in early
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phase clinical trials. Other novel agents alter downstream functions of AR. Hsp-90 inhibitors, for example, block a protein–protein interaction necessary for proper AR protein folding (Taplin 2007).
6 Summary The emergence of CRPC marks a critical juncture in the clinical course of the disease. Host factors such as extent of disease significantly impact the development of CRPC. Several clinical parameters, such as PSA kinetics, are associated with time to CRPC and the natural history of CRPC once it develops. For patients whose disease is not rapidly progressive or symptomatic, CRPC may be susceptible to secondary hormonal treatment, a therapy generally less toxic that chemotherapy. Secondary hormonal treatments take advantage of CaP’s continued dependence on the androgen axis. The markers for CRPC development and prognosis and secondary hormonal treatments become useful as increasing number of patients develop CRPC in the absence of any other signs of clinical progression.
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Xie W, Nakabayashi M, Regan MM, Oh WK. Higher prostate-specific antigen levels predict improved survival in patients with hormone-refractory prostate cancer who have skeletal metastases and normal serum alkaline phosphatase. Cancer. 2007;110(12):2709–2715. Yossepowitch O, Bianco FJ, Jr., Eggener SE, Eastham JA, Scher HI, Scardino PT. The natural history of noncastrate metastatic prostate cancer after radical prostatectomy. Eur Urol. 2007;51(4):940–947; discussion 947–948.
Differential Roles of Androgen Receptor in Prostate Development and Cancer Progression Shuyuan Yeh, Yuanjie Niu, Hiroshi Miyamoto, Tamin Chang, and Chawnshang Chang
Abstract The androgen depletion therapy (ADT) has become the major treatment for the cancer patients through the use of chemical castration and/or antiandrogens, yet the therapy eventually fails and cancers progress to more advanced stages. The mutation, amplification, overexpression of AR, and cross-talk between AR, AR coregulators, and other growth factor pathways have provided potential explanations for the failure of androgen ablation therapies in some cases. However, whether the differential AR roles in different types of prostate cells could contribute to the failure of ADT remains unclear and will be the focus of this review. AR expresses in both stromal and epithelial compartments of prostate. It has been shown that there are three basic types of prostatic epithelial cells: (i) cytokeratin 8 (CK8)-positive, CK5-negative luminal cells, (ii) CK5/CK8-double positive intermediate cells, and (iii) CK8-negative, CK5-positive basal cells. In addition to prostatic stromal cells, AR expression could be detected in some basal cells, some intermediate cells, and all luminal cells in prostate. By Cre-LoxP strategy, the prostate epithelium-specific AR knockout (pes-ARKO) and inducible-cre ARKO mice were recently established and have allowed the field to address the differential and distinct AR roles in different types of prostatic cells. These ARKO mice were bred with TRAMP prostate cancer model, and results from these models suggest that (i) prostatic epithelial AR plays dual roles as a suppressor of basal cell proliferation and as a survival factor for luminal cells, and (ii) the stromal AR plays a proliferator role to support the epithelial cell survival and proliferation. Using microarray analysis of primary tumor cells isolated from the prostate tumors of pes-ARKO-TRAMP mice, it was found that a series of metastatic genes were altered and responsible for the higher invasiveness and metastatic rates. These recent ARKO animal studies not only advance our understanding of the differential roles of AR in different type of prostatic cells, but also closely reflect the pathological changes for the patients undergoing the ADT. Together, these findings provide new evidences to support the potential beneficial effects of C. Chang(*) George Whipple Lab for Cancer Research, Departments of Pathology and Urology, University of Rochester Medical Center, Rochester, NY, USA, E-mail:
[email protected]
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intermittent ADT therapy, and they also urge the development of cell type and stage selective anti-AR therapies for the prostate cancer patients.
1 Introduction Prostate cancer remains a major factor of cancer morbidity and mortality in man (Jemal et al. 2008). Nearly 200,000 new prostate cancer cases would be diagnosed in the USA and close to 30,000 men would die of the disease yearly. Androgen and androgen receptor (AR) signals are required for prostate development, differentiation, and normal functioning as well as cancer initiation and progression (Huggins and Hodges 1972; Niu et al. 2008a, b). Androgen exerts its biological effects through binding to the AR and inducing AR transcriptional activity. The androgen-induced AR transactivation is modulated by the interaction of the AR with various coregulators in response to various growth factors (Buchanan et al. 2001b; Heinlein and Chang 2002, 2004; Heemers and Tindall 2007). Initial stages of prostate cancer growth could be suppressed by reducing the availability of androgens to cancer cells via surgical or chemical androgen deprivation therapy (ADT). However, ADT ultimately fails, and prostate cancer progresses to a castration-recurrent (androgen-independent) state that is frequently metastatic and almost always fatal. The AR is expressed throughout prostate cancer progression as well as in the majority of patients with castrationrecurrent disease (Chodak et al. 1992; Hobisch et al. 1996; Mohler et al. 1996; Sadi et al. 1991; van der Kwast et al. 1991). The reasons for failure of ADT are not well understood and require further investigation. This chapter discusses findings that the AR can function to stimulate tumor growth or suppress tumor metastasis depending on its expression in different prostatic cells. These differential functions of the AR in different types of prostatic cells could lead to emerging new concepts to treat prostate cancer.
2 The Classic Concept for an AR Role in Stimulating the Growth and Differentiation of Epithelial Cells During Prostate Development The prostate develops from the endodermal urogenital sinus (UGS) (Cunha et al. 2004). The UGS consists of an outer layer of embryonic connective tissue called the urogenital sinus mesenchyme (UGM), and an inner layer called the urogenital sinus epithelium (UGE). UGM expresses the AR, which could mediate the effect of androgens in the initial step of prostatic development. In response to androgen secreted from fetal testis, epithelial buds emerge and grow into the surrounding UGM. In rodents, solid prostate buds start to elongate and undergo branching morphogenesis in the prenatal period, and the process continues until maturation
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at the end of puberty (Cunha et al. 2004; Kinbara and Cunha 1996; Sugimura et al. 1986a). Beginning at the neonatal period, the epithelial cords undergo ductal canalization during which the epithelial cells differentiate into the luminal and basal cells (Cunha et al. 2004; Prins and Putz 2008). Prostatic epithelial differentiation is accompanied by differentiation of the mesenchyme into fibroblasts and smooth muscle cells (Hayward et al. 1996a, 1997). In 1978, Cunha (Cunha and Lung 1978) reported that when the UGM compartment of UGS from wild-type mouse embryo was combined with epithelium from the neonatal bladder of wild-type or testicular feminized mouse (Tfm, a mouse model lacking functional AR) and transplanted into the kidney capsules of recipient mice, the tissues developed into glandular structures histologically resembling prostatic glands. In contrast, when the UGM of embryonic UGS from Tfm mice was combined with epithelium from bladder of wild-type or Tfm mice, the combined tissues developed into structures histologically resembling vaginal epithelium. These results suggested that (a) the AR in the UGM regulates signals for ductal morphogenesis for the initiation of prostatic development through a paracrine mechanism, and (b) Tfm bladder epithelium is capable of participating in an androgenic response from wild-type UGM cells. In spite of the importance of stromal AR signals, the postnatal development and maturation of the prostate are believed to depend on a reciprocal interaction between the stromal (mesenchymal) and epithelial compartments (Hayward et al. 1998). Indeed, experimental evidence showed that the epithelial AR is required for epithelial cell differentiation, expression of some prostatic secretory proteins (Cunha and Young 1991; Donjacour and Cunha 1993), and differentiation of stromal fibroblasts into the smooth muscle cells (Cunha et al. 1992; Donjacour and Cunha 1988; Hayward et al. 1996b). Other experiments indicated that castration of the adult mice resulted in prostate involution, which was attributed to epithelial cell apoptosis (Isaacs 1984), and androgen supplementation in castrated animals resulted in restoration of prostate size and ductal morphology (Kyprianou and Isaacs 1988; Sugimura et al. 1986b). These experiments indicated that the growth of adult prostate epithelial cells is dependent on AR signaling. Further reported data indicated that apoptosis of prostate tissues recombined from rat UGM + wild-type epithelium or rat UGM + Tfm epithelium was similar in castrated hosts, and androgen supplementation inhibited apoptosis of these recombinants in a similar manner (Kurita et al. 2001). Together, those observations suggested that the regeneration of prostate epithelial cells is dependent on stromal, but not epithelial, AR signaling. Our recent study of transgenic mice, in which the AR was specifically knocked out from stromal smooth muscle cells or fibroblasts, indicated that reducing AR signaling in the prostatic stromal cells resulted in the development of smaller prostate glands due to a moderate decrease in proliferation and substantial increase in apoptosis in the epithelium (Yeh and Chang et al. unpublished observations). These observations strongly support the view that prostatic stromal AR is an important modulator for the epithelial cell survival, proliferation, and differentiation.
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3 Prostatic AR as a Suppressor of Basal Cells and a Survival Factor for Luminal Cells Although results from the embryonic tissue recombination studies suggested that stromal AR plays a dominant role over the epithelial AR, the studies using embryonic tissues would allow only 4–6 weeks of observation. Thus, tissue recombination studies were not adequate to assess the relative contributions of epithelium and stroma in the adult prostate. To study AR functions in adult prostate, a temporal and tissue-specific strategy to control AR gene expression was necessary, and the CreloxP recombination system (Lakso et al. 1992; Nagy 2000; Yeh et al. 2002) is very useful for generating transgenic animal models with conditional AR gene knockout. Prostatic epithelial cells develop from stem cells through proliferation stepwise into basal and intermediate cells that finally differentiate into epithelial luminal cells (Litvinov et al. 2003). Earlier studies have shown that there are three basic types of prostatic epithelial cells: (a) cytokeratin 8 (CK8)-positive, CK5-negative luminal cells, (b) CK5/CK8-double positive intermediate cells (van Leenders and Schalken 2003), and (c) CK8-negative, CK5-positive basal cells (van Leenders et al. 2001). AR expression is detected in some epithelial basal cells, some intermediate cells, and all luminal cells (Mirosevich et al. 1999). By crossing floxed AR mice (Yeh et al. 2002) with probasin-Cre mice (Wu et al. 2001), we have generated prostate epithelium-specific AR knockout (pes-ARKO) mice to study the role of the epithelial AR in prostatic tissue homeostasis (Wu et al. 2007). Results suggest that prostatic epithelial AR plays dual roles as a suppressor of basal cell proliferation and a survival factor for luminal cells. Within the epithelium, the probasin promoter transgene is expressed increasingly from 2 to 7 weeks and expression is sustained throughout life (Wu et al. 2001). Consistent with increasing probasin-Cre expression, our data showed that the epithelial AR expression gradually decreased in the prostate of pes-ARKO mice beginning at 6 weeks of age and was undetectable at 24 weeks of age (Wu et al. 2007). Histological analysis of the ventral prostate in pesARKO mice suggested a progressive decrease in epithelial height, loss of glandular infolding, and increase in luminal epithelial cell apoptosis at 6–32 weeks of age. As the pes-ARKO animals matured, the p63-positive epithelial basal cell population increased during puberty and remained elevated, while the CK8/CK18-positive epithelial luminal cell population declined. The loss of epithelial luminal cells was accompanied by decreased expression of the differentiation markers, probasin, PSP-94, and Nkx3.1. At 24 weeks of age, the glands of pes-ARKO mice exhibited one layer of undifferentiated epithelial cells with increased incorporation of 5-bromodeoxyuridine (BrdU) suggesting an increase in epithelial cell proliferation. Those BrdU-positive cells primarily colocalized with CK5-positive basal cells. To rescue the phenotype, we then generated the AR(T857A)-pes-ARKO double transgenic mice by crossing pes-ARKO mice with AR(T857A) transgenic mice carrying an androgen-responsive AR mutant (Han et al. 2005). These mice exhibited normal prostatic morphology and glandular histology. These results suggested that the increase in epithelial basal proliferation and the loss of epithelial luminal
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cells in pes-ARKO mice are attributable to the loss of epithelial AR signaling. These observations not only support the view that the AR in prostatic epithelial cells is required for epithelial cell differentiation (Donjacour and Cunha 1993), but also suggest that the epithelial AR functions as a suppressor for basal cell proliferation and a survival regulator for differentiated epithelial luminal cells.
4 Differential Roles of AR in Prostate Cancer Progression: The Stromal AR as a Tumor Proliferation Stimulator and Epithelial AR as a Tumor Suppressor Through In Vitro and In Vivo Evidences The success in generating cell-type selective ARKO mice using the cre-loxP strategy (Yeh et al. 2002) permitted further investigation of the role of epithelial AR in prostate cancer progression by creating a pes-ARKO in a mouse TRAMP prostate cancer model. The TRAMP mouse develops spontaneous prostate tumors starting at 10–12 weeks of age (Gingrich et al. 1997). There are three basic types of prostate epithelial cells: luminal, intermediate, and basal cells. We have bred TRAMP mice with the floxed AR transgenic and probasin-Cre mice to generate pes-ARKO-TRAMP mice (Niu et al. 2008a). The knockdown of the AR from the epithelial cells in pes-ARKO-TRAMP mice proceeded gradually with age in a time course very similar to that of pes-ARKO mice (Wu et al. 2007). The prostate of pes-ARKO-TRAMP mice exhibited elevated epithelial cell apoptosis compared to the prostates of wild-type AR (WtAR) TRAMP mice at 16 weeks of age. Double immunostaining indicated that the apoptotic cells were CK8-positive epithelial luminal cells. In contrast, there was an increasing number of proliferating cells in the CK5-positive epithelial basal compartment as indicated by the expression of Ki 67 (a proliferation marker) and BrdU incorporation in pes-ARKO-TRAMP mice compared with that in WtAR TRAMP mice (Niu et al. 2008a). Increased apoptosis in the epithelial luminal cells and increased proliferation in the epithelial basal cells resulted in an increase in a population of cells that were CK5/CK8-double positive intermediate cells. Interestingly and unexpectedly, we observed that pes-ARKO-TRAMP mice developed larger and less differentiated primary tumors in the ventral prostate than TRAMP mice at 16 weeks of age (Niu et al. 2008a). CD44, an early progenitor cell marker, expresses in basal and intermediate prostatic cells (Liu et al. 2004). In addition, it has been found that CD44-positive and AR-negative prostate cancer cells purified from human prostate cancer xenografts are enriched in tumorigenic and metastatic progenitor cells (Patrawala et al. 2006). The primary tumors of pes-ARKO-TRAMP exhibited a higher population of CK5/CK8- and CD44-positive cells than WtAR TRAMP mice. These results suggest that knockout of AR in the prostatic epithelium of TRAMP mice resulted in cell population changes with expansion of intermediate-like tumor cells and
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decrease of secretory epithelial luminal cells in the prostate of pes-ARKO-TRAMP mice (Niu et al. 2008a). This is in agreement with a prior report that CK5-positive intermediate cells increased from 29% to 75% when prostate cancer patients received ADT (van Leenders et al. 2001). Moreover, these results confirm the observations in pes-ARKO mice that epithelial AR functions both as a proliferation suppressor of basal intermediate cells and as a survival factor for epithelial luminal cells. Since prostate cancer arises from epithelial cells in TRAMP mice, the dual opposing functions of the epithelial AR might also influence prostate cancer development by favoring the survival of differentiated luminal cells and suppress the expansion of CK5/CK8-positive basal intermediate epithelial cancer cells. Using animal cancer models, it was also found the AR signaling might influence metastasis of prostate cancer. The size of metastatic tumors in pelvic lymph nodes (PLN) in pes-ARKO-TRAMP mice was increased compared with that in their WtAR TRAMP littermates at 24 weeks of age. We observed that primary cultured PLN tumor cells from pes-ARKO-TRAMP mice were more invasive than those from WtAR TRAMP mice in the matrigel invasion assay. Furthermore, restoring the functional AR by transfection of human AR cDNA reduced the invasiveness of PLN tumor cells from pes-ARKO-TRAMP mice (Niu et al. 2008a). It was further examined whether the AR status affected the invasiveness of several human prostate cancer cell lines, which include PC-3 and CWR22rv1 cells. The AR-negative PC-3 cells were isolated from a bone metastasis and the Ar-positive CWR22rv1 cells were isolated from a prostate tumor growing despite ADT (Nagabhushan et al. 1996). Stable AR-positive transfectants of PC-3 cells (PC-3-AR9 cells) also exhibited less invasiveness than control vector-transfected PC-3 (PC-3-v) cells. AR was knocked down in CWR22rv1 cells using a homologous AR gene recombination strategy (Yeh et al. 2003) to obtain CWR22rv1 cells with knockdown (KD) of AR expression (CWR22rv1-ARKD cells). CWR22rv1ARKD cells express much less AR and are more invasive than parental cells in the Boyden chamber invasion assay. Since PC-3 cells were isolated from a bone metastasis, these cells were injected into the tibia of athymic nude mice according to Corey’s method (Corey et al. 2002). We found that PC-3-v tumors grew more aggressively and more invasively than PC-3-AR9 tumors as determined by X-ray analysis and measurement of tumor weights in the mouse tibia. These data from knockin of the functional human AR show that absence of AR signaling in prostate cancer cells promotes invasion both in vitro and in vivo. PC-3-v or PC-3-AR9 cells were also orthotopically inoculated into the anterior prostate of nude mice. Results showed that mice inoculated with PC-3-v cells developed bigger primary tumors that were less differentiated and larger PLN metastatic tumors than mice inoculated with PC-3-AR9 cells (Niu et al. 2008a). Nelius et al. transfected PC-3 cells with an inducible AR-expressing transgene and observed that induction of AR expression resulted in decreased invasion in vitro and decreased tumorigenecity due to decreased microvescular density and increased tumor cell apoptosis (Nelius et al. 2007). These results showed that loss of prostatic epithelial AR expression leads to the development of more invasive and metastatic prostate cancers and restoration of
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a functional AR reverses these characteristics. Together, these results suggest that the epithelial AR is defined as a suppressor of prostate tumor growth and metastasis. Tumor microenvironment and stromal–epithelial interaction remain important for tumor initiation, progression, and metastasis of prostate cancer (Bhowmick and Moses 2005; Condon 2005). Our recent data also indicated that the AR in prostatic stroma could function as a stimulator of prostate cancer progression and metastasis (Niu et al. 2008b). Furthermore, human prostatic stromal WPMY1 cells (Webber et al. 1999) were stably transfected with AR short hairpin RNA (AR shRNA) that can be used to silence or knock down endogenous AR to generate WPMY1-ARsh cells. The influence of WPMY1-ARsh and vector-transfected WPMY1-v cells on invasion abilities of PC-3-v and PC-3-AR9 cells was studied using coculture: stromal cells were placed in the bottom chamber and cancer cells were placed in the upper chamber of a Boyden chamber. The results indicated that both PC-3-v and PC-3-AR9 cells were less invasive when cocultured with WPMY1-ARsh cells than WPMY1-v cells. These results indicated that AR signaling in stromal cells functions as a stimulator of cancer cell invasion regardless of the expression of the endogenous AR in cancer epithelial cells. Furthermore, PC-3-v or PC-3-AR9 cells and WPMY1-v or WPMY-ARsh cells were orthotopically coinoculated into the anterior prostate of nude mice to investigate the role of the stromal AR in tumor progression and metastasis (Niu et al. 2008b). Both cancer cell lines produced smaller primary tumors and PLN metastases when combined with WPMY1-ARsh compared with WPMY1-v, although tumors derived from PC-3-AR9 cells were smaller than those derived from PC-3-v cells in either combination. These observations suggest that AR signaling in stromal cells can provide growth or paracrine factor(s) for growth of AR-positive and AR-negative prostate cancer cells and influence their metastasis. In contrast, the smaller tumors observed with PC-3AR9 cells compared with PC3-v cells suggest that AR expression in prostate cancer epithelial cells promotes differentiation, controls growth, and inhibits metastasis of epithelial prostate tumors regardless of the presence of stromal stimulation effects. These observations support the concept that stromal AR functions as a stimulator of prostate cancer epithelial cells by promoting proliferation and inhibiting apoptosis. In addition, the stromal AR appears prometastatic as opposed to the epithelial AR, which appears antimetastatic. An inducible ARKO model of TRAMP (ind-ARKO-TRAMP) (Niu et al. 2008b) was produced by mating female TRAMP mice carrying heterozygous floxed AR with male Mx1-Cre mice (Kuhn et al. 1995). Following injection of pI-pC to induce cre expression and knockout of AR gene at 12 weeks of age, we found that the AR mRNA levels in the prostate of ind-ARKO-TRAMP mice at 16- and 20 weeks of age were knocked down 40–50% when AR mRNA expression was assessed in prostate epithelium and stroma isolated with laser capture microdissection. Knockdown of AR expression resulted in smaller and less differentiated primary prostate tumors in ind-ARKO-TRAMP than in control Wt-AR-TRAMP mice 16–24 weeks of age. The tumors of ind-ARKO-TRAMP mice had lower proliferation rates and higher apoptosis rates than tumors of the control TRAMP mice. The tumors from
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ind-ARKO-TRAMP mice had decreased CK8-positive epithelial luminal cells and increased CD44-positive and CK5/CK8-double positive basal intermediate cells compared with control TRAMP mice. Although pes-ARKO-TRAMP and ind-ARKO-TRAMP mice both demonstrated increased epithelial apoptosis and expanded intermediate cells, pes-ARKO-TRAMP mice produced larger and indARKO-TRAMP mice produced smaller metastases compared with control mice. Together, those data support the concept that epithelial luminal ARKO would alter the cell population of prostatic epithelial cells by reducing more differentiated luminal cells and increasing CD44-positive and CK5/CK8-double positive intermediate cells. Epithelial ARKO or AR signal blocking may reduce the survival of luminal epithelial tumor cells, yet would promote the expansion of more malignant intermediate tumor cells. In addition, metastatic tumors were also evaluated when primary tumors reached 1 cm3 in TRAMP (20 weeks of age), pes-ARKO-TRAMP (18 weeks of age), and ind-ARKO-TRAMP (36 weeks of age) mice and found that the well-differentiated primary tumors of TRAMP mice developed small metastases in the PLN. The poorly differentiated tumors of pes-ARKO-TRAMP mice developed much larger PLN metastases and metastasized to multiple organs, whereas those of ind-ARKO-TRAMP mice were smaller and more often invaded into the seminal vesicle and migrated to the liver. Thus, loss of the epithelial AR could promote prostate cancer growth and metastasis. The concurrent knockdown of the stromal and epithelial AR could override partially the affects of the epithelial AR knockout, thereby retarding growth of the primary prostate tumor and suppressing metastasis. Furthermore, ind-ARKO-TRAMP mice with early induction of ARKO had longer survival time than wild-type TRAMP and pes-ARKO-TRAMP mice (Niu et al. 2008b). However, the apparent dominance of stromal AR over epithelial AR function diminished if induction of ARKO happened after the primary tumor had progressed for a period of time in ind-ARKO-TRAMP mice. Together, these observations support the concept that the epithelial AR can function as a differentiation factor and a tumor metastasis suppressor and also suggest that the stromal AR functions as a stimulator of prostate cancer growth and metastasis.
5 Molecular Mechanisms Promoting Metastases Following Depletion of AR Function In addition to characterizing the differential AR roles in the prostate cancer metastasis using cell and mouse models, gene microarrays have been applied to understand the mechanistic changes in metastatic tumors of pes-ARKO-TRAMP and of PC-3-v cancer cell xenografts, and found that several prometastasis genes such as cyclooxgenase-2 (Cox-2) (Attiga et al. 2000), matrix metaloproteinase-9 (MMP-9) (Aalinkeel et al. 2004; Corey et al. 2002; Saleem et al. 2006), interleukin-6 (IL-6)
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(Hammacher et al. 2005; Saleem et al. 2006), and tumor necrosis factor-alpha (TNF-a) (Hammacher et al. 2005; Michalaki et al. 2004) were elevated, while antimetastasis genes such as neutral endopeptidase (NEP) (Nelson and Carducci 2000; Tanimura et al. 2005) and P27(kip1) (Baldassarre et al. 2005; Belletti et al. 2005; Papandreou et al. 1998) were decreased in the tumors of pes-ARKO-TRAMP mice and PC3-v orthotopic grafts compared with the tumors of their AR-expressing counterparts (Niu et al. 2008a). Thus, due to the dual functions of the AR, treatment of prostate cancer patients with ADT should have good responses at an early stage of cancer but the prognosis may worsen when ADT is given at later stages when tumors are less dependent on stromal AR-derived signals for growth, a scenario apparently observed in castrated TRAMP mice (Johnson et al. 2005). These findings may also explain why some patients can benefit from intermittent cycles of ADT and androgen supplementation (Akakura et al. 1993; Feltquate et al. 2006), which may allow the tumor cells to remain sensitive to ADT without pushing the adaptive phenotype changes.
6 Adaptive Phenotypic Changes via AR Somatic Mutations in Prostate Cancer After ADT In addition to managing androgen activity on the prostate cancer patients, the AR is subjected to somatic mutations in prostate cancer, and most of the mutations involve single base change substitution of an amino acid residue and are found more frequently in castration-recurrent, metastatic bone tumors than in primary tumors (Bentel and Tilley 1996; Buchanan et al. 2001a, c; Chen et al. 2005; Gottlieb et al. 2004; Linja and Visakorpi 2004; Marcelli et al. 2000; Taplin et al. 1995; Tilley et al. 1996). Many of these AR mutants display gain-of-function with equal sensitivity toward androgen, increased sensitivity toward dehydroepiandrosterone (DHEA), and a broader range of ligand activation by 17b-estradiol, progesterone, corticosteroids, and/or antiandrogens (Yeh et al. 1997, 1998; Buchanan et al. 2001c; Chen et al. 2005; Fenton et al. 1997; Monge et al. 2006; Shi et al. 2002). Other AR mutants exhibit decreased transactivation, no activity (Shi et al. 2002), or altered interaction with AR coactivatiors (Bentel and Tilley 1996; Duff and McEwan 2005; Li et al. 2005). Some AR mutations produce nonsense codons that lead to expression of truncated AR proteins with significantly altered transcriptional activity (Lapouge et al. 2007). The nonsense AR mutants were detected at higher frequency in metastatic cells, where several of the nonsense mutations coexist with other AR mutants, particularly the promiscuous T877A mutation, and one of the nonsense mutants, Q640X, appeared capable of androgen-independent stimulation of the activity of the AR(T877A) mutant (Alvarado et al. 2005). Somatic mutation of AR also occurs spontaneously in TRAMP mouse prostate tumors at a high rate and the rate of mutation increases further after castration (Han et al. 2001), which suggests an adaptive response. In clinical prostate cancer, AR
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mutants are detected more frequently in castration-recurrent disease and after treatment with antiandrogens (Linja and Visakorpi 2004), which also suggests an adaptive change. Expression of the murine AR mutant, AR (E231G), in mouse prostate resulted in development of prostate intraepithelial neoplasia (PIN) (Pinkas and Teicher 2006), which progressed to invasive and metastatic disease (Han et al. 2005;Yeh et al. 1997, 1998). In view of this observation and those gain-of-function AR mutants, which are considered as providing growth advantages for prostate cancer cells, AR may be regarded as a proto-oncogene. Although the linkages of phenotypic changes via AR somatic mutations in prostate cancer cells remain unclear, these changes may contribute to prostate cancer progression and influence the response to ADT. In addition, AR interacts with a group of proteins, AR coregulators, including coactivators and corepressors. To date, there are numerous AR coregulators identified (Heinlein and Chang 2002; Heemers and Tindall 2007). AR requires the proper interaction with its associated proteins to activate the target gene expression. Several AR coregulators are upregulated in advanced prostate cancer (Culig and Bartsch 2006; Fujimoto et al. 2007; Heemers and Tindall 2007; Hu et al. 2004; Kahl et al. 2006; Nishimura et al. 2003; Yang et al. 2007a, b) and increase androgen sensitivity and ligand promiscuity of wild-type AR and some AR mutants (Yeh et al. 1997, 1998; Heinlein and Chang 2002; Rahman et al. 2004). AR-interacting proteins could alter the AR functions both in the prostate cancer cells and in cancerassociated stromal cells. Regulating the AR protein complex in prostatic stromal or epithelial cells could possibly be applied as an alternative therapeutic strategy in combination with ADT to treat prostate cancer. Together, the studies on AR mutation and AR-associated proteins have added more complexity to the understanding of AR functions in prostate cancer initiation and progression.
7 Clinical Implication and New Concept for Prostate Cancer Treatment There are different stages of prostate cancer development: nonmalignant stage, prostatic intraepithelial neoplasia PIN, invasive prostatic adenocarcinoma, and metastatic tumor (Fig. 1). The classic clinical evidence for AR to function as a proliferation stimulator is the observation by Huggins and Hodges (1972) that treatment of prostate cancer patients with castration or injection of estrogen, to reduce levels of circulating testosterone, resulted in reduction of tumor sizes. The success of ADT for treating prostate cancer patients at earlier stages appears to support the classic view that AR functions as a proliferator that stimulates the growth of prostate cancer. However, ADT treatment eventually fails in most patients, and the cancers recur with high rates of metastasis and almost certain mortality. Current evidence of different AR roles in prostate stromal and epithelial cells using multiple in vitro and in vivo strategies indicates that AR signaling does have differential roles on prostate cancer cell proliferation, survival, and metastasis
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Fig. 1 Histopathology of the human prostate cancer progression. (a) normal/nonmalignant prostate, (b) prostatic intraepithelial neoplasia (PIN), (c) invasive adenocarcinoma, and (d) metastatic tumor in lymph node (See Color Insert)
(Wu et al. 2007; Niu et al. 2008a, b). These findings also provide explanations for the fact that ADT will have some positive effects on earlier prostate cancer but will eventually fail via the dual roles of AR signaling in prostate cancer progression. Treatment regimens targeting solely the AR signaling, such as intermittent ADT (Crook et al. 1999; Egawa et al. 2000; Higano et al. 1996; Hurtado-Coll et al. 2002), or secondary hormonal therapy (Sharifi et al. 2008), may have additional benefits, but may eventually fail. Therefore, new treatment regimens should be developed to use in combination with ADT for the management of prostate cancer. Strategies that selectively target the tumor stromal cells (Bouzin and Feron 2007; Hofmeister et al. 2008), and not epithelial cells, should benefit prostate cancer patients. Furthermore, patients with castration-recurrent prostate cancer usually die of metastatic disease, but their survival can be extended if tumor metastasis is delayed or prevented through additional treatment regimens. Treatments targeting metastasisrelated genes downstream of the epithelial AR signaling should improve results from adjunct ADT alone. For example, treatments targeting TGFb1 and/or its receptor (Pinkas and Teicher 2006), Akt, COX-2, MMP-9 (Miyamoto et al.
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2005), or other relevant targets should produce clinical benefits for patients with prostate cancer, when used in conjunction with ADT. The discovery of the differential roles of the stromal and epithelial AR on prostate development, cancer progression, and metastasis may facilitate the development of new therapeutic approaches to battle this deadly disease.
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Imaging Androgen Receptor Function In Vivo Michael Carey and Lily Wu
Abstract Bioluminescence imaging (BLI) is a facile method for studying androgen receptor (AR) signaling during tumor progression in xenografts and genetic models of prostate cancer in mice. This chapter summarizes work where BLI and positron emission tomography coupled with CT were used to analyze AR-mediated transcriptional activity using gene expression-based imaging approaches.
1 Introduction This chapter will focus on approaches for visualizing androgen receptor (AR) function in preclinical models of prostate cancer (PCa) using bioluminescence imaging (BLI). Two themes that will be discussed include whether AR activates transcription in both the androgen-dependent (AD) as well as the recurrent and hormone refractory forms of PCa in standard tumor models [reviewed in Dehm and Tindall (2007)], and how imaging can be employed to study AR signaling during cancer progression. These are key issues in the PCa field as AR function remains a major target of therapeutic intervention (Taplin 2007). There are numerous methods for visualizing AR location and function. Fluorescent-AR fusion proteins can be employed to directly visualize AR dynamics, subcellular localization, and response to various ligands in cell culture (see Marcelli et al. 2006). AR-responsive reporter genes can also be introduced into animals or xenograft tumors, and transcription can be measured by monitoring the activity of the reporter gene (Wu et al. 2003). The reporter gene can be a fluorescent protein or bioluminescent protein that can be visualized using cryogenically cooled, charge-coupled device (CCD) cameras (Contag et al. 2000; Massoud and Gambhir 2003). Alternately, the reporter protein can be designed to bind a probe molecule that can be visualized in some manner. These methods include probes that can be studied using positron emission tomography (PET) (Gambhir 2002). Finally, AR M. Carey(*) Department of Biological Chemistry, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA, E-mail:
[email protected]
D. Tindall and J. Mohler (eds.), Androgen Action in Prostate Cancer, DOI: 10.1007/978-0-387-69179-4_4, # Springer Science + Business Media, LLC 2009
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itself is a ligand-binding molecule, and a new generation of PET probes have recently been synthesized that bind directly to AR and permit its localization within small animals and man by PET (Dehdashti et al. 2005; Jacobson et al. 2005; Parent et al. 2006, 2007; Schoder and Larson 2004).
2 Gene Expression-Based Imaging 2.1
Overview
Gene expression-based BLI has emerged as a relatively facile approach for studying signaling pathways during tumor progression in live animals (Contag and Bachmann 2002; Contag et al. 2000; Helms et al. 2006; Massoud and Gambhir 2003). In gene expression-based BLI, a promoter is placed upstream of a bioluminescence reporter gene (i.e. firefly luciferase – FLuc or Renilla luciferase – RLuc) (Fig. 1). The reporter cassette is introduced into tumor cells in a live animal either genetically or using DNA/viral vectors. To measure the bioluminescence signal emitted from the animal, the mouse is sedated and injected with D-luciferin (substrate for FLuc) or coelenterazine (for RLuc), placed in a light tight chamber and
Fig. 1 Bioluminescence imaging of animal models of prostate cancer (PCa). The diagram illustrates the general approach for imaging promoter activity in live animals. A promoter is attached to a bioluminescence reporter gene, FLuc, introduced into animals and imaged in a Xenogen IVIS. The output is a photo of the mouse superimposed with a color pseudoimage representing the emitted light in photons/s/cm2/steradian (See Color Insert)
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imaged using a cryogenically cooled, CCD camera. In many of the currently available devices, several mice can be imaged simultaneously.
2.2
Methodology
A popular and widely used device is the In Vivo Imaging System (IVIS1) manufactured by Caliper Life Sciences (Hopkinton MA). IVIS measures bioluminescence generated by the reaction of luciferase with its substrate or measures fluorescence upon irradiation of animals bearing a fluorescent reporter protein. A computer interprets and integrates the light signals over a few seconds to five minutes acquisition period. A program (Living Image1) then superimposes a color pseudoimage, representing the quantity of photons emitted by the tissue, over a grayscale photograph of the animal. The optical signals over a particular region of interest can be specifically queried. As of this writing, there are several models of IVIS, the most recent of which allow 3D reconstruction of fluorescence and bioluminescence signals. The imaging technology is linear with respect to dose of luciferase and light output. Additionally, the substrate clears from the system quickly – although D-luciferin and coelenterazine display different pharmacokinetics – and the animal can be reinjected subsequently to generate a time course of promoter activity over days or weeks. Indeed, the short half-life of FLuc in cells, 2 h (Ignowski and Schaffer 2004), permits multiple measurements in a single day. The ability to repetitively image a single animal has advantages over traditional animal studies requiring sacrifice of animals at each time point followed by pathological and molecular analyses to measure transcription. In effect, each animal can serve as its own internal control over time. Hence, repetitive imaging significantly reduces the number of animals needed for a statistically meaningful study.
2.3
Drawbacks
There are several drawbacks of the technology. To date, in the most widely used versions of IVIS, the scans are two-dimensional and relatively of low resolution. Hence, the scans do not accurately reveal the depth of the cells emitting the light. The newest models of IVIS (Spectrum1 and the 3D series) have partially overcome that drawback via 3D reconstruction techniques, although the reconstruction does have limitations. The reader should consult the Caliper Life Sciences website for additional details of the technology (www.caliperls.com). Nevertheless, when in vivo studies are accompanied by ex vivo imaging of tissues, immunohistochemistry, and pathological analyses, the precise location of the cells emitting light can be ascertained. This latter set of techniques requires sacrificing the animal, but is usually performed at an endpoint rather than during a time course. A second
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challenge in BLI is detecting optical signals in dense tissues. Much of the light is scattered or absorbed by blood, tissue, fur, and skin pigmentation. The successful implementation of the technology requires high luciferase levels to image at depths of 1–2 cm (i.e. as in a tumor). Thus, the method functions most efficiently with small animals. Improvements of the technology using red-shifted reporter proteins should decrease some of the light absorption by blood and tissue. The technology was originally validated using potent viral promoter/enhancers, that is, CMV and SV40, which generate robust signals sufficient to image tissues deep within a mouse. However, cellular promoters are typically weaker than viral ones and more difficult to image. Yet cellular promoters that are AR-responsive are more desirable because they can be used to measure the output of specific signaling pathways.
2.4
Reporter Gene Delivery
The methods used to deliver the reporter gene largely depend upon the question being addressed. The use of cells stably transformed with an imaging vector allows the cells to be placed into the animal and tracked. In such cases, the vector can be constitutively expressed via a strong enhancer/promoter like CMV or SV40, or designed to measure particular signaling pathways in the cell. This method can be performed with cell lines or primary cells. In cancer, such approaches can be used to monitor cancer progression, test therapies on xenografts, and track metastatic lesions. A second approach utilizes viral vectors (e.g. adeno-associated virus, adenovirus, and lentiviruses) or encapsulation devices (e.g. nanoparticles) bearing an imaging cassette. These can be injected systemically to allow the vector to be targeted to a wide range of accessible tissues. In this case, it is preferable that the imaging reporter be under a tissue-specific promoter, otherwise the tissue that is best transduced will display the highest signal, which may or may not be desirable depending upon the tropism of the vector. Viral vectors also have an advantage in that their gene transduction capability is sufficiently robust that direct administration of vectors into the animal is feasible. Thus, they can be injected into the bloodstream or directly into a targeted organ or xenograft. Many cancer xenograft models do not grow as cell lines, and thus, direct infection of the tumor with a virus or other agent provides a means to monitor the xenograft tumor directly. Finally, the imaging reporter system can be placed under tissue-specific promoters and employed to generate transgenic mice to study the biology of a particular transcription pathway. This approach can be used to study signaling in an animal, an issue we discuss below with respect to AR. It is particularly useful when the transgenic reporter animal is crossed with a cancer model, that is, to generate a bigenic animal, or with a knockout model to study how loss of a gene affects reporter activity. In this case, the reporter cassette
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can track cancer progression and metastasis in a natural environment and be used to study drugs that intervene at certain stages of cancer. The transgenic approach is important since many xenografts can grow quite rapidly, and it is frequently necessary to euthanize the animal before fully studying the metastatic potential of a tumor. Additionally, the cancer in transgenic models follows a natural progression, which may be desirable in order to study specific regulatory checkpoints in the process. Several of these issues will be explored in following text.
2.5
Reporter Gene Potency
There are two strategies that have been used to augment transcriptional activity for studying the AR signaling pathway in animals by imaging. First, the promoter and enhancer of an androgen-regulated gene are manipulated to artificially augment their activity – this is termed the chimeric or composite enhancer approach. The second approach is based on a concept termed two-step transcriptional amplification (TSTA). A cellular promoter expresses a potent chimeric activator, GAL4VP16. GAL4-VP16 is a fusion of the high-affinity yeast GAL4 DNA-binding domain to the potent herpes simplex virus VP16 activation domain (Emami and Carey 1992; Sadowski et al. 1988). GAL4-VP16 has a unique potency and specificity. No protein exists in mammalian cells that binds to the GAL4 recognition site, and few cellular activators, if any, match the strength of the VP16 activation domain. GAL4-VP16 then binds a GAL4-responsive reporter gene and generates high levels of gene product. The TSTA approach allows weak cellular promoters to be visualized and, hence, expands the repertoire of signaling pathways that can be measured via imaging. The TSTA system is not toxic in animal models allowing for continuous monitoring and the generation of transgenic models. Furthermore, as discussed below, the TSTA approach recapitulates the signal responsiveness of the promoter, permitting detailed investigation of the pathway.
3 Chimeric/Composite Enhancer Approach 3.1
The PSA Promoter and Enhancer
Prostate-specific antigen (PSA) is a kallikrein family serine protease. Many studies have demonstrated the prostate tissue specificity of PSA, and it is a widely used marker for evaluating treatment and progression of cancer (Bok and Small 2002). The PSA gene represents a model for studying AR-mediated gene expression during PCa because it is active in the AD and early recurrent stages of cancer growth. Biochemical and genetic studies have led to the cloning and genetic
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dissection of the PSA promoter and enhancer by numerous groups with much of the work being completed over a decade ago. Both the enhancer and the promoter display androgen responsiveness consistent with the observation that both contain multiple androgen response elements (AREs), which bind AR. The promoter has been delineated to a 600-bp fragment upstream of the start site. It contains two AREs and other DNA sites important for PSA expression (Cleutjens et al. 1996; Reid et al. 2001; Riegman et al. 1991). The enhancer is centered in a region about 4.2 kb upstream of the transcription start site (Cleutjens et al. 1997; Schuur et al. 1996). The importance of the enhancer is highlighted by the presence of an androgeninducible DNase I hypersensitive site (DHS II) at 4.2 kb (Cleutjens et al. 1997). The DHS II region is within a minimal 440-bp core sequence ( 4,324 to 3,884), which provides strong enhancer activity and contains a high-affinity ARE (AREIII). Analysis of the enhancer by DNase I footprinting and mutagenesis indicated that AREIII is flanked by several lower affinity but functional AREs, all of which contribute to the synergistic activation of PSA gene expression (Huang et al. 1999). The reasons behind the development of an AR-responsive PSA gene imaging system are that (1) PSA is an established, albeit imperfect, marker for early and recurrent PCa; (2) the PSA enhancer and promoter have been incorporated into transgenic mice and shown to maintain tissue specificity; (3) the promoter and enhancer are well characterized as described earlier and hence amenable to manipulations to enhance potency; (4) since the PSA promoter and enhancer are critically dependent on AR function, an imaging system based on the PSA gene would provide a real-time readout of AR activity in vivo.
3.2
Chimeric and Composite Constructs
In a study performed by Wu et al. (2001), the synergistic nature of AR action was employed to improve the activity of the PSA regulatory region (PSE) bearing the larger PSA enhancer region ( 5,322 to 2,855) fused to the proximal promoter ( 541 to +12) (Fig. 2). Chimeric plasmid constructs were generated by three main strategies: (1) insertion of four tandem copies of the PSA promoter-proximal AREI element (ARE4) adjacent to the core enhancer (PBA), (2) duplication of core enhancer region (PBC), or (3) a combination of first and second strategies (PBAC). Some intervening sequences ( 3,744 to 2,855) were removed between the enhancer and promoter (PSE-B series) in all of the constructs. The most potent construct, PBC, was 20-fold more active than the baseline PSE in cell culture and retained activity and specificity when inserted into an adenovirus vector (Ad-PBC-FLuc). Other chimeric approaches have been employed to generate reporter constructs that accurately measure AR function. Latham et al. (2000) employed a larger version of the PSA enhancer in cell culture studies and found that duplication of
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Fig. 2 The chimeric/composite enhancer approach. The diagram shows three chimeric constructs in which the potency of the PSA enhancer promoter was modulated by either addition of four copies of PSA AREI (PBA), duplication of the enhancer (PBC), or addition of four AREs plus duplication of the enhancer (PBAC). These were compared with the starting construct PSE and a version of PSE where additional sequences between the enhancer and promoter were removed (PSE-B). The bar graph shows relative activity of the constructs in LNCaP cells
a 1.2-kb enhancer fragment upstream of the promoter led to a synergistic increase in activity. These investigators inserted the duplicated enhancer construct into adenovirus and demonstrated that reporter gene expression maintained cell selectivity, androgen inducibility, and potency. Additionally, Xie et al. (2001) delimited the regulatory regions of the highly similar prostate-specific human kallikrein 2 gene (hK2) and identified the enhancer and promoter. Like the PSA gene, the hK2 enhancer had a significant effect upon androgen-mediated induction of transcriptional activity and an increase in the number of enhancers led to an increase in the fold induction. The authors generated adenovirus constructs bearing EGFP reporters under either CMV or hK2 regulatory regions. They found that hK2-EGFP virus maintained androgen inducibility and cell type specificity in a panel of cultured cell lines. Moreover, the hK2-EGFP adenovirus maintained tissue specificity when injected into xenograft tumors generated from subcutaneously implanted LNCaP cells versus injection of the virus into control mouse tissues, which included liver and brain (Xie et al. 2001). Finally, Matusik and coworkers performed extensive promoter and enhancer analyses to delimit the native mouse probasin (Pb) enhancer and promoter. They generated constructs with an additional ARE from the proximal Pb promoter (termed ARR in their studies) (Zhang et al. 2000, 2004). The ARR2Pb construct has been used to generate prostate-specific gene expression in several
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transgenic mouse models. In the cases described above, the chimeric enhancers have been applied to develop noninvasive, gene expression-based, molecular imaging systems that measure AR function.
3.3
Imaging Metastasis with a Viral Chimeric Construct
A study by Wu et al. demonstrated the ability of Ad-PBC-FLuc to interrogate the AR signaling pathway in live animals bearing LAPC4 xenografts (Adams et al. 2002). The LAPC (Los Angeles Prostate Cancer) series of xenografts were derived by transplanting various PCa specimens from patients into the flanks of SCID mice (Klein et al. 1997). As described above, for the BLI (bioluminescence imaging) experiments to be described and illustrated herein, a computer-generated pseudoimage of the measured light is displayed over the grayscale photograph of the mouse. The color images will be shown in the online version of this book, while grayscale images will be displayed in the paper version. The animal results established both the specificity and potency of the PBC chimeric enhancer and validated its use in monitoring cancer progression. The study showed that whereas Ad-CMV-FLuc is constitutively active in tumors, liver, and other tissues, Ad-PBC-FLuc displayed prostate tumor specificity in both AD and AI (recurrent and hormone refractory) xenografts. The study also demonstrated that the adenovirus-based imaging cassette could detect distal metastatic lesions in the lung and spine, a result confirmed by immunohistochemical staining with antibodies directed against human cytokeratin (Fig. 3). The virus also detects lung metastases upon direct injection via tail vein. The coxsackie adenovirus receptor is overexpressed in advanced PCa, particularly in metastatic lesions. Although the reason for coxsackie adenovirus receptor overexpression is unknown, McCormick has suggested that this property of advanced PCa may make metastases particularly amenable to adenovector-based imaging and gene therapy (Rauen et al. 2002). Additionally, adenovirus displays lymphotropic properties allowing it to target a major route of metastasis (see below). The imaging results agreed well with other studies, which examined AR expression and function in xenografts and patient samples of advanced cancer (Gregory et al. 2001a, 2001b). In summary, the work established the ‘‘proof of principle’’ that a mechanistic approach of augmenting AR-dependent PSA expression could have direct application in imaging xenograft models of cancer. The capabilities of the system could be further improved because the chimeric enhancers, although more active than native PSA regulatory regions, were still transcriptionally weaker (20-fold) than Ad-CMV-FLuc in tumors. Ad-CMV has been employed as a benchmark in the imaging and gene therapy arenas due to its transcriptional potency, but it lacks tissue specificity (constitutive gene therapy has been used in clinics without clear success). A series of studies were initiated to improve the activity of the PSA enhancers, while maintaining their cell and tissue specificity. This approach led to the development of the TSTA imaging system.
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Fig. 3 Imaging of metastasis. The figure shows a time course where AdPBC-FLuc is injected into the LAPC4 xenograft tumor but over time can detect distal metastases in the spine of mouse 1 and lungs of mouse 2 and mouse 3. Ex vivo imaging of the spine from mouse 1 is shown after sacrifice on day 21. To the right of the images are low- and high-resolution immunohistochemical analyses of the spine with an antibody against human cytokeratin, which was used to confirm the tumor origin of the metastatic lesions. Also shown is a confocal image of immunofluorescent staining of lung of mouse 2. It revealed a cluster of human cytokeratin+ metastatic cells and lectin stained blood vessels (See Color Insert)
4 The TSTA Imaging System 4.1
Cell Culture Studies
In the initial tests of the TSTA imaging system (Fig. 4), the baseline PSA regulatory region, PSE, was designed to express GAL4-VP16 (Emami and Carey 1992). GAL4-VP16 then activated high levels of reporter gene expression. The TSTA system was used to amplify expression of both FLuc and a PET reporter gene – mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk) – in LNCaP cells (Iyer et al. 2001). The TSTA expression studies demonstrated 50-fold (FLuc) and 12-fold (HSV1-sr39tk) enhancement over the conventional one-step vectors, which expressed FLuc and HSV1-sr39tk directly from the PSA enhancer–promoter, PSE. The TSTA system was observed to retain cell selectivity by analysis of a panel of cultured cell lines. Additionally, CCD imaging was employed to visualize the amplified FLuc expression in living mice implanted with transfected LNCaP cells. These imaging experiments revealed a significant gain in signal when comparing the TSTA system versus the one-step system (PSE-FLuc).
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Fig. 4 The TSTA scheme. In the presence of AR and DHT, the PSA promoter/enhancer drives expression of the potent activator GAL4-VP16. GAL4-VP16 then binds to multiple GAL4 sites positioned upstream of a promoter expressing luciferase. This scheme can amplify luciferase up to1000-fold
Fig. 5 Creating a titratable TSTA system. The PSE or PBC constructs were used to express GAL4-VP16 derivatives fused to one, two, or four copies of the N-terminal VP16 activation domains. The activities of the reporters were measured on GAL4 reporters bearing either one, two, or five sites upstream of the adenovirus E4T promoter. By varying the activity of both the promoter and effector proteins, the activity of the TSTA system can be carefully modulated. [Figure adapted from Zhang et al. (2002)]
To further refine the system, the technologies from Emami and Carey (1992), Iyer et al. (2001), and Wu et al. (2001) were merged to develop a titratable, prostatespecific, androgen-responsive system for imaging (Zhang et al. 2002). The ‘‘chimeric TSTA’’ system employed normal and chimeric PSA regulatory regions (Wu et al. 2001) to express GAL4 derivatives fused to 1, 2, or 4 VP16 activation domains (GAL4-VP1, -VP2, and -VP4) (Emami and Carey 1992) (Fig. 5). The resulting activators were cotransfected into a panel of cell lines with another
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Fig. 6 Results from titratable system in LNCaP cells. This bar graph shows the results of an LNCaP transfection experiment, in the presence and absence of R1881, comparing the various one-step and two-step vectors to the benchmark activity of the CMV enhancer/promoter. [Figure adapted from Zhang et al. (2002)] (See color Insert)
plasmid containing reporter templates bearing 1, 2, or 5 GAL4-binding sites (G1, G2, and G5) upstream of the adenovirus E4 promoter (E4T) driving FLuc (Emami and Carey 1992). Activity was monitored in parallel via FLuc assays on transfected cell extracts (via luminometry) and in live nude mice implanted with transfected cells (via the IVIS). Luciferase activities were relatively comparable in the luminometer and IVIS device. FLuc expression in PCa cells could be varied over an 800-fold range using the one- and two-step systems (Fig. 6). The optimal combination of components in LNCaP cells was shown to be the PBC regulatory region driving GAL4-VP2 with a separate plasmid containing the FLuc reporter bearing 5 GAL4-binding sites upstream of the adenovirus E4 core promoter. Remarkably, a single plasmid containing the optimized components expressed FLuc at higher levels than CMV-FLuc. This dramatic increase in activity, while maintaining specificity, again highlights the advantages of applying basic transcription principles toward the cancer imaging problem. On the basis of this result, the optimal TSTA construct was inserted into a replication-defective E1/E3deleted adenovirus and used in cell culture and animal studies (Fig. 7). Note that other variations of the TSTA system could be used. The PBC-GAL4-VP4 variant of TSTA is extremely potent, and the baseline expression in absence of ligand (Fig. 6) likely represents the ability of the system to detect residual androgen in the culture
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Fig. 7 The optimal TSTA system inserted into adenovirus. The optimal TSTA construct was inserted into an E1/E3-deleted adenovirus. The figure shows the basic organization of the functional TSTA modules within adenovirus (See Color Insert)
medium. The ligand-inducible level is lower than with PBC-GAL4-VP2, probably because excess production of GAL4-VP4 is toxic. However, in principle, the system may be more sensitive in detecting trace levels of androgen in dedifferentiating tumors.
5 Imaging AR Function During PCa Progression in Xenografts Using TSTA 5.1
TSTA Animal Studies
An advantage of the AdTSTA approach is the ability to inject the virus into any xenograft animal without having to establish cell lines. Many xenografts are derived directly from patient tumors and do not exist as cell lines. The TSTA adenovirus was used to image human xenografts in SCID mice to address several questions about AR function in recurrent cancer (Zhang et al. 2003). As this study was one of the first to comprehensively use imaging to study the role of AR in PCa progression, it was essential to provide a correlation between the imaging and biology of AR function. This required a combination of in vivo, pathological, and molecular analyses. Thus, the CCD signals were compared to serum PSA levels and tumor AR levels. Immunohistochemistry was used to probe the localization of
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Fig. 8 Androgen deprivation is specific to AdTSTA. A total of 107 pfu of AdCMV-FLuc or AdTSTA-FLuc were injected into LAPC9 tumors. A baseline image was taken on day 5 and the mice were castrated and imaged on day 10. AdCMV signal decreases less than 2-fold while AdTSTA decreases more than 20-fold. [Figure adapted from Zhang et al. (2003)] (See Color Insert)
AR in tumor cells, and chromatin immunoprecipitation (ChIP) was used to probe the binding of AR, pol II, and other factors to the endogenous PSA gene in tumors. Figure 8 shows an experiment where AdTSTA was injected into 0.5 cm tumors (diameter by caliper measurement) of male SCID mice bearing LAPC9 xenografts (Klein et al. 1997). LAPC9 is derived from a bone metastasis. It contains a wildtype AR gene and secretes PSA. LAPC9 xenograft tumors are unusual in that they do not regress substantially upon castration and eventually transition into a recurrent state. Five days after virus injection the tumors were imaged, and the mice were castrated. By the 10-day time point, the imaging signal decreased 20-fold in the castrate animals (Fig. 8) but was unchanged in noncastrated cohorts (not shown). The data strongly support the idea that the system is measuring androgen signaling because injection of an adenovirus bearing CMV-FLuc (AdCMV-FLuc) into the tumor revealed that upon castration there is little decrease in the luciferase signal versus the result observed with AdTSTA-FLuc (Fig. 8). It should be pointed out that unlike LAPC9, many xenografts derived from cell lines or true AD xenografts such as CWR22 tend to regress rapidly upon castration, making the aforementioned experiment very difficult to perform. Another advantage of the LAPC9 model is that it rapidly progresses to the recurrent state in animals. Thus, the TSTA system can be employed to monitor this transition. As shown in Fig. 9, the AdTSTA system detects the decrease in signal upon castration and the increased signal upon transition of the tumor to the recurrent state. In this experiment, a baseline image was taken from a single animal on day 4, after which the animal was castrated. The figure shows the decrease in optical signal several days later and then the rise again as the tumor transitions to a recurrent form within 2 weeks after castration. Note that the optical signal decreases and then rises in parallel with PSA. Additionally, the optical signal is more sensitive than PSA to castration, probably because of the short half-life of FLuc in live cells.
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Fig. 9 Transition to the recurrent state. This figure shows the progression of the tumor signal in a single mouse as it transits from AD to the castrated state (ADc) and finally to recurrent cancer (AI) over a period of 3 weeks. Luciferase activity is in photons/s/cm2/steradian 106. PSAs are the serum PSA levels at the various stages in this same individual (mean of three values). The immunohistograms are stained with AR antibodies and show the localization of AR in animals at similar stages in cancer. The immunoblots of AR were normalized to actin and obtained from tumors at similar stages to the animal shown. [Figure adapted from Zhang et al. (2003)]. Note that the term AI refers to animals where the tumor grows after removal of the major source of androgen (i.e. the testis). The caveat is that androgen may be synthesized intratumorally using adrenal androgens. Thus, as discussed by the editors at the beginning of this book, the proper term is recurrent. This caveat applies to all figures shown here where AI is used in the label (See Color Insert)
The figure also shows representative AR nuclear localization in xenograft tumors obtained at different stages of cancer from different mice. AR was localized to the nucleus in AD animals, diffused between the cytoplasm and nucleus after castration, and reentered the nucleus in the recurrent stage. Additionally, the tumor levels of AR parallel its activity in the imaging assay. Collectively, the study argues that AR activity recovers during the transition to recurrent cancer and shows that the imaging signal correlates with biological benchmarks of AR function. In a separate study, the specificity of the virus was tested in a panel of ARcontaining and -lacking cell lines that have been classified as either AD or hormone refractory (AI). The TSTA system displayed robust activity in AD cell lines LNCaP and LAPC4, but was relatively inactive in PCa cell lines lacking AR such as PC3 and DU145. In contrast, the TSTA virus displayed robust androgen responsiveness in the hormone refractory, AR-positive cell line MDA PCa 2b. Indeed, in vivo, the
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TSTA system seemed to display more activity in LAPC9 AI versus AD tumors, suggesting that it may respond to as yet uncharacterized aspects of AR function in recurrent tumors (Sato et al. 2005). This enhanced activity may be related to both the presence of intratumoral androgens and the hypersensitivity of AR to such androgens in recurrent cancer (Gregory et al. 2001b; Mohler et al. 2004). The data collectively argue that AR signaling remains active in recurrent cancer xenografts.
5.2
Correlating TSTA Activity with AR Binding to the PSA Enhancer in Tumors
A key issue was whether the activity of AR reported by the imaging constructs correlated with the actual binding of AR to its sites within genomic DNA (Zhang et al. 2003). Figure 10 shows an experiment, where AR binding to the PSA enhancer and promoter within the xenograft tumor was measured by ChIP. The multiplex nature of the ChIP PCR (four sets of primers in any given reaction) provided internal controls for any given sample. Thus, the middle regions between the promoter and the enhancer, as well as exon 5, were used as negative controls for AR binding in each sample. The figure shows that AR bound weakly to the
Fig. 10 Chromatin IP of tumor samples. Tumors from AD, AI, and ADc xenografts were minced and treated with formaldehyde. Formaldehyde cross-linked and sheared chromatin was immunoprecipitated with AR antibodies or IgG. The coprecipitated DNA was amplified with primer sets encompassing the PSA enhancer, a middle intervening region, the proximal promoter, and downstream exon 5. The multiplex DNA products were fractionated on polyacrylamide gels and autoradiographed. Input are input samples added to the IP reaction. A graph of a quantitative PCR experiment on the exact same samples using primers against the promoter is shown. [Figure adapted from Zhang et al. (2003)]
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promoter and strongly to the enhancer in AD tumors, decreased upon castration, but bound again in the AI tumor. The result was confirmed by quantitative PCR. The data demonstrate that AR is bound to DNA and activates transcription in a recurrent cancer model, and that the imaging signals accurately report the binding status of AR in vivo.
5.3
TSTA Lentiviruses
A similar study that used direct intratumoral injection of lentiviral versions of TSTA generated very similar results to AdTSTA in monitoring tumor growth (Iyer et al. 2004). Indeed, the ability of lentivirus to stably transduce the tissues allowed visualization of the mouse prostate for up to 3 months postorthotopic injection. Moreover, a subsequent study employing systemic intravenous injection of lentivirus revealed that the virus could stably transduce LAPC9 xenografts within 4 days, while maintaining expression for up to 3 weeks (Iyer et al. 2006). Small amounts of background signal were observed in the spleen and liver, and these signals correlated with increased uptake of the virus as measured by qPCR. The background expression may result from basal expression of TSTA due to the lentivirus regulatory elements or may be due to an inherent background in the TSTA system, which at high MOI (of either adenovirus or lentivirus) can generate aberrant signals in a relatively nontissue-specific manner. The background signal issue is addressed in a later section. Nevertheless, this study showed conclusively that the integrating lentivirus could stably deliver the PSA promoter-based TSTA imaging reporter system into a tumor in a live animal and allow for persistent imaging of AR function.
5.4
Detecting the Effects of PCa Therapy by Imaging in Animal Models
A major advantage of the TSTA imaging system is its ability to directly probe AR function during drug treatment. A good example is the effect of flutamide, a nonsteroidal, antiandrogen prodrug (Ilagan et al. 2005). Figure 11 shows that flutamide (60 mg/kg/day) pellets implanted into the mice greatly decrease the imaging signal in AD but not in AI tumors. The inability of antiandrogen therapy to work in AI cancer contributes significantly to PCa mortality. The data support the ability of an AR-dependent imaging system to measure specific antiandrogen drug effects in a xenograft model. Because the imaging is performed repetitively, it permits measurement of AR signaling cues and their response to inhibitors in live animals over the course of a treatment without sacrificing animals at various points to obtain the information.
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Fig. 11 Effect of flutamide on AD and AI xenografts. AdTSTA was injected directly into AD and AI tumors. On day 3, a baseline image was taken and either flutamide or placebo pellets were implanted onto the backs of the mice. Only the flutamide is shown as placebo displayed no changes. The mice were then imaged every 3 days. The image at the endpoint on day 18 is shown. [Figure adapted from Ilagan et al. (2005)] (See Color Insert)
The TSTA system can also be utilized to deliver PET reporter genes. One such gene, sr39tk, also serves as a potential therapeutic gene. In a recent study, AdTSTA-sr39tk was used in a PET imaging-guided therapy in a preclinical model with mice bearing AR- and PSA-positive LAPC4 tumors (Johnson et al. 2005). The study exploited the dual capability of HSV-sr39tk as a PET reporter gene and as a prodrug (ganciclovir)-activated suicide gene (Fig. 12). 18F-FHBGPET was used to monitor the magnitude and location of vector-mediated HSVsr39tk gene expression prior to and after ganciclovir treatment. The suicide therapy with the prostate-specific AdTSTA-sr39tk was compared to that with the constitutive AdCMV-sr39tk. AdTSTA-sr39tk was superior to AdCMV-sr39tk in that it achieved effective tumor destruction while preventing system toxicity (i.e. liver damage). Tumor-directed injection of AdCMV-sr39tk resulted in leakage of the vector into the systemic circulation and liver transduction. Hepatic cellular damage was observed in the AdCMV-sr39tk-treated animals but not in the AdTSTA-sr39tktreated cohorts. In a subsequent study which exploited the lymphotropic features of adenovirus, the AdTSTA-sr39tk and AdTSTA-FLuc viruses were employed to
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Fig. 12 MicroPET/CT imaging of prostate cancer (PCa) gene therapy using TSTA. A comparison of AdCMV-sr39tk and AdTSTA-sr39tk in LAPC4 xenografts by PET. The scheme indicates treatment time course in days which includes intratumoral virus injection, followed by baseline PET measurement (preRx), followed by treatment with Ganciclovir, and followed by an endpoint PET measurement (postRx). The location of the tumor and liver signals are indicated. The PET signal in the abdomen area of the AdCMV-sr39tk-injected animal is due to accumulation of 18 F-FHBG [9-(4-[18F]-fluoro-3-hydroxymethylbutyl)guanine] after leakage of AdTSTA-sr39tk from the tumor and constitutive expression of the PET reporter in liver. H&E and Tunel staining indicate apoptosis in treated tumors and micrographs show liver damage in the AdCMV-sr39tktreated animal by Tunel. [Figure adapted from Iyer et al. (2005b)] (See Color Insert)
detect sentinel lymph node metastases in xenografts using MicroPET (Fig. 13; Burton et al. 2008). Macroscopic and occult nodal lesions from both subcutaneous and orthotopic xenograft tumor models were visualized. These findings also point out that PSA expression and AR function remain active in nodal metastasis in the LAPC-9 model and correlates with findings in clinical scenarios as determined by rapid autopsy (Roudier et al. 2003; Shah et al. 2004). These data raise the possibility of combining specific imaging of the AR pathway in metastatic lesions with therapy in a clinical environment.
5.5
Modifications of AdTSTA
Both an advantage and a drawback of the TSTA strategy is that the close head-tohead orientation of the PBC-GAL4-VP2 gene and the GAL4-responsive FLuc reporter can generate a feed forward loop, where small amounts of AR signaling
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Fig. 13 The TSTA system detects lymph node metastases. AdTSTA-sr39tk or AdTSTA-FLuc was injected via the paw to detect lymph node metastases resulting from subcutaneously implanted LAPC9 xenografts. The figure shows images of axillary lymph node metastases as detected by bioluminescence (BLI) upon injection of luciferin, and MicroPET after injection of 18F-FHBG. The accompanying panels show gross pathological and microscopic evaluation of the lymph nodes (See Color Insert)
generate GAL4-VP16, which further increases the activity of the potent PBC promoter/enhancer. An advantage is enhanced sensitivity, while a disadvantage is that the system is possibly overly sensitive to very small changes in activity that will be driven forward by an autoregulatory loop. Indeed, high MOI with adenoviral and lentiviral TSTA vectors generates activity in nonprostate cell lines. Studies have shown that such background can be lowered when the PBC-GAL4-VP2 effector and GAL4-responsive FLuc reporter are placed in separate adenoviruses (Sato et al. 2003). However, simultaneous delivery of two vectors into one cell is very inefficient in vivo, making such an approach unfeasible when designing strategies for targeted gene therapy or systemic injection of imaging vectors. To overcome this limitation, the effector and reporter were placed at separate locations within the adenovirus genome. In the original TSTA configuration, the effector and reporter were cloned head to head in the E1 portion of the genome. In the optimized versions, the effector (PBC-GAL4-VP2) is placed in E3, and the reporter or therapeutic gene (G5E4FLuc or G5E4sr39tk) is placed in E1, 25 kb away (Sato et al. 2008). This modified TSTA system displays greater cell selectivity and more
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robust androgen induction. The E1/E3 showed significant selectivity and little background in lung tissues of mice upon systemic injection.
5.6
Imaging AR Activity in Combination with MAPK
The advantages of imaging are clearly important when attempting to visualize a key signaling event that may drive the tumor into the recurrent state. To this point we have focused on AR signaling. However, one advantage of the TSTA system is its binary nature. Both the tissue-specific promoter as well as the GAL4-derived activator can be changed to alter the properties of the system. It is possible to modify the system to simultaneously detect both AR and mitogen-activated kinase (MAPK) signals. Elevated MAPK has been proposed to contribute to the transition to recurrent PCa (Gioeli et al. 1999; Weber and Gioeli 2004). The system was modified to detect MAPK by utilizing the serum response transcription factor Elk1, which contains three functional domains – the N-terminal and central domains mediate DNA-binding and the C-terminal domain activates transcription (Cruzalegui et al. 1999; Marais et al. 1993). The activation domain from Elk1 has been shown to function when fused to the GAL4 DNA-binding domain (Hexdall and Zheng 2001). GAL4-Elk1 responds to serum stimulation and to MEKK, which activates ERK. ERK, in turn, phosphorylates multiple sites in the Elk-1 C-terminal region. Upon phosphorylation, Elk-1 becomes transcriptionally active (Fig. 14). The TSTA-MAPK system replaces GAL4-VP16 with GAL4-Elk1 (Ilagan et al. 2006). This binary system has the unique ability to measure the oncogenic potential of PCa where both AR and MAPK are active. When tested in cell culture, the system responds synergistically to the combination of R1881 to induce GAL4-Elk1 and EGF or dominant active MEKK to activate the MAPK pathway. As predicted, the signals are inhibited by both EGF receptor and AR inhibitors. In the LAPC9 xenograft model, the system detects systemic injection of EGF within 4 h of treatment (Fig. 14). Similarly, the system detects the much higher MAPK activity found in the CWR22 PCa xenografts versus the LAPC9 model (Ilagan et al. 2006). Since activation domains of many transcription factors respond selectively to extracellular signals and kinase cascades, it is possible that varying the promoter and the GAL4-fusion protein could permit cell-specific measurement of many different signaling pathways.
6 Imaging AR Function in Transgenic Mouse Models The use of transgenic mice for imaging of AR function in combination with other transgenic mice that possess the ability to develop prostate malignancies (i.e. transgenic adenocarcinoma mouse prostate – TRAMP) allows for the noninvasive, long-term monitoring of PCa progression and metastasis. The strategies developed may also be useful for transgenic research in general by allowing for amplified tissue-specific gene expression.
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Fig. 14 The GAL4-Elk1 system to measure MAPK. The scheme shows that GAL4-Elk1 is cloned in place of GAL4-VP16. In the presence of AR, GAL4-Elk1 is synthesized in prostate cancer cells. GAL4-Elk1 is transcriptionally inactive until it is phosphorylated by ERK1/2. Phosphorylated GAL4-Elk activates transcription of the GAL4-responsive FLuc reporter gene. In the imaging experiment below, LAPC9 xenografts, which have low levels of active MAPK, were injected with AdTSTA-Elk1 and imaged before and 4 h after systemic administration of EGF. The result demonstrates that the TSTA-Elk1 system responds to MAPK activation in live animals. [Figure adapted from Ilagan et al. (2006)] (See Color Insert)
6.1
The TSTA Transgenic
A transgenic mouse was generated to determine the selectivity of TSTA in an animal (Iyer et al. 2005a). One potential drawback of TSTA was that GAL4-VP16 might be toxic to the animals. However, transgenic mice displayed normal physical characteristics and developmental behavior, indicating that the high level of GAL4-VP16mediated expression is tolerated. Detailed pathological examination of the prostates from transgenic and wild-type animals revealed that the structures of the lobes of the transgenic prostate were normal and there was no microscopic evidence of tissue or gland abnormality. The results further showed that the bioluminescence signal was largely, although not exclusively, localized to the lower abdominal region (Fig. 15). It should be noted that it is difficult to use BLI to decisively identify the exact tissue origin of a signal unless it is a xenograft implanted subcutaneously. This drawback
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Fig. 15 Bioluminescence imaging of TSTA transgenic animals. (a) Male and female littermates bearing the TSTA-FLuc system were imaged after injection with D-luciferin. (b) The effects of castration on TSTA transgenics. (c) Harvesting and ex vivo imaging of select individual organs demonstrate prostate specificity of the signal. Note that in one of the transgenic lines there was a weak signal observed in heart probably due to an integration site effect (See Color Insert)
requires that, at early stages in an imaging study, the animal has to be sacrificed and the various organs imaged ex vivo in a petri dish (Fig. 15). Nevertheless, once a tissue origin is determined, the subsequent studies can utilize that knowledge to evaluate the properties of the imaging system in the live animal. In the case of TSTA, the system was male-specific and AR-dependent, that is blocking androgen availability by castration led to downregulation of FLuc expression in the prostate (Fig. 15). These findings suggest that the GAL4-VP16 transactivator can be used to amplify reporter gene expression from a relatively weak promoter in a transgenic mouse model and raise the possibility that the approach can be applied to study other pathways.
6.2
The ARR2Pb Transgenic
In another example of how chimeric constructs can be used to facilitate imaging, Sawyers and coworkers developed a transgenic mouse in which FLuc was driven directly by Matusik’s ARR2Pb construct (Ellwood-Yen et al. 2006). These mice expressed robust levels of luciferase as measured by imaging. Analysis of individual tissues after dissection revealed prominent expression of FLuc in the
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dorsolateral and ventral prostate consistent with Matusik’s earlier characterization of an ARR2PbCAT transgenic model (Ellwood-Yen et al. 2006). Additionally, imaging of FLuc expression showed that it responded appropriately to castration as well as systemic treatment with the antiandrogen bicalutamide. A similar animal was developed by Spencer and coworkers and will be described below.
6.3
The GAL4-AR Transgenic
O’Malley and coworkers developed a special approach that allows AR activity to be measured directly in any tissue where the transgene cassette is active (Ye et al. 2005). The mouse contains a GAL4-AR fusion protein coupled with a GAL4responsive Luciferase reporter cassette. The GAL4-AR was constructed by replacing the DNA-binding domain of AR with that of GAL4 in a Bac construct bearing the native AR locus. The construct contained a 15-kb region upstream of the gene, most of the 195-kb gene (except where the DNA-binding domain had been swapped) and a 10-kb region downstream of the gene. In vivo BLI showed that the reporter gene was expressed largely in the testis, although pathological and immunohistochemical analyses showed that it was also expressed in skin and cerebellum. The reason for the lack of prostate-specific expression is unknown but the authors speculated that certain transcription regulatory regions might be absent from the Bac clone or were present in the intergenic region between exons 2 and 3, which was swapped out. Nevertheless, the model allowed the authors to cross the animals with TIF2 heterozygous knockout mice and show that this particular AR coactivator was important for AR function in the Sertoli cells of the testis. This is an exciting approach and emphasizes how imaging can be combined with knockout models of mice to study very specific aspects of transcription factor signaling pathways.
6.4
6.4.1
Imaging AR Function During Metastasis and Cancer Progression The sPSA Model
Several other transgenic models have been generated and shown to detect metastatic lesions. Chung’s laboratory developed the sPSA model (Hsieh et al. 2005). The sPSA construct contains the core PSA enhancer linked to a minimal region containing the PSA TATA and surrounding sequences. The sPSA FLuc construct displayed strong ventral prostate expression with lower levels in the dorsolateral prostate. Expression was also noted in the cauda epididymis and a small background level of expression was noted in bone marrow of both male and female littermates. Similar
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to the TSTA and ARR2Pb models, the sPSA model responded well to castration with optical signals reaching a baseline 7 days postcastration in 10-week male mice. In a follow-up study, Chung’s group crossed the sPSA transgenic with TRAMP mice to determine the utility of in vivo imaging for monitoring AR activity during tumor development and metastasis (Hsieh et al. 2007). TRAMP mice contain a minimal AR-responsive Pb promoter linked to SV40 T antigen (Tg). The promoter expresses Tg in the dorsolateral and ventral lobes of the prostate (Gingrich et al. 1996). The mice develop adenocarcinoma and display a pattern of metastasis that parallels in several respects the pattern observed in human PCa. Chung’s group combined imaging with pathological examination to identify two groups of mice with different degrees of pathology. In group 1, the signal intensity was restricted to the prostate over 24-week course of the study and correlated with PIN upon gross pathological examination. Group 2 showed strong signals in the prostate and lower abdomen that spread to the groin lymph node by week 16. Interestingly, the signal appeared to decrease substantially by week 20. Histopathological analysis indicated that the majority of the tumors in group 2 were poorly differentiated and, as cancer progressed, AR expression was apparently lost, which was consistent with the decrease in luminescence signal, although, paradoxically, immunohistochemical analyses showed that the cancer expressed Tg from the AR-dependent Pb promoter. With respect to the lesions, molecular analysis of the lymph node metastasis confirmed the presence of luciferase, which disappeared concomitant with increased dedifferentiation of the tumor cells. Bone metastases were also observed in some animals. This particular study emphasizes the need to combine in vivo imaging with ex vivo imaging and pathological analyses to fully exploit the benefits of imaging metastatic lesions in animal models of cancer. It would be very difficult to conclusively distinguish groin metastasis from the signal in the prostate without ex vivo analysis. The study also reveals an interesting property of the TRAMP model. The AR signal decreases rather rapidly and unexpectedly, suggesting that AR function is diminishing rapidly during cancer progression and cellular dedifferentiation.
6.4.2
The EZC Models
Spencer and coworkers performed perhaps the most thorough studies on AR imaging in transgenic mice (Seethammagari et al. 2006). Spencer’s group developed transgenic lines using three different chimeric constructs based on the triplicated enhancer of the human kallikrein (hK2-E3-P) gene, the duplicated enhancer of PSA (PSA-E2-P), and the ARR2Pb construct. They termed these transgenic models EZC1, EZC2, and EZC3. All three models express luciferase in the prostate as determined by in vivo and ex vivo imaging. EZC2 and 3 display much lower expression in other tissues versus EZC1. In vivo imaging revealed high levels of expression in the lower abdomen, which correlated largely with ventral prostate expression when analyzed ex vivo. Minor levels of expression (0.1–2% vs ventral prostate) were detected in various tissues such as dorsolateral prostate, epididymis,
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and testes. These values contrasted with the higher background signals and wider tissue distribution observed in the EZC1 model. Interestingly, the authors were able to observe weak signals in utero of developing male and female animals. Additionally, the signals allowed in vivo and ex vivo analyses of AR function in numerous tissues during early development up to and past puberty. The luminescence signals like all other models were highly responsive to castration in adult males and recovered in castrated males implanted with testosterone pellets. The signals also responded well to treatments that lower circulating levels of testosterone including a GnRH modulator. The study established the utility of the EZC models in monitoring AR during cancer progression and metastasis through the generation of bigenic cancer models. In one model, EZC was crossed with the JOCK-1 model, which employs a prostatetargeted form of FGFR1 that is attached to a ligand-inducible dimerization domain. Addition of the ligand causes receptor dimerization and enhanced tyrosine kinase activity. This system generates hyperplasia at 2 weeks, PIN at 24 weeks, and widespread adenocarcinoma by 40 weeks. Interestingly, despite tumor progression, the luminescence signal intensity did not increase concomitantly. Indeed, AR activity decreased on a per cell basis, and the authors speculated that this may be due to numerous factors including dedifferentiation, hypoxia, tumor burden, etc. Similarly, studies in the TRAMP model, which developed adenocarcinomas by 12 weeks and metastases by 12–18 weeks, did not reveal significant increases in signal intensity and in some cases showed decreases despite the obvious progression of the cancer. Analysis of the EZC3 mice, which displayed the least amount of extraprostatic background signals, allowed facile detection of metastases. Precise localization of the lesions is quite difficult in vivo but subsequent dissection and ex vivo analysis of the signals revealed high efficiency of metastasis to the pancreas, lymph nodes, kidney, adrenal glands, liver, spleen, and salivary glands. In summary, these animals serve as excellent models to systematically analyze various PCa treatments in a preclinical setting.
7 Conclusions and Future Prospects The emphasis on understanding AR-responsive promoters and signaling pathways in the 1990s has led to the development of tissue-specific vectors that allow the detection of AR-dependent transcriptional activity via BLI. Numerous investigators in the AR field have demonstrated the utility of BLI in understanding AR signaling in xenograft tumors (Adams et al. 2002; Zhang et al. 2003), during mouse development (Ellwood-Yen et al. 2006; Hsieh et al. 2005; Iyer et al. 2005a; Seethammagari et al. 2006) and in mouse bigenic models of PCa (Hsieh et al. 2007; Seethammagari et al. 2006). Studies on subcutaneous and orthotopic PCA xenografts using viral transduction have revealed that AR is indeed transcriptionally active in the AD, recurrent, and metastatic models of PCa (Sato et al. 2005; Zhang et al. 2003). Indeed, in some models, AR seems to be hyperactive in recurrent cancer. Systemic
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injection of viral vectors bearing imaging cassettes has also revealed the ability of these vectors to infect xenograft tumors and metastatic lesions resulting from the xenograft (Adams et al. 2002; Iyer et al. 2006; Sato et al. 2005). Studies on transgenic mice bearing AR-responsive imaging cassettes have shown that AR activity can be tracked in utero and throughout development providing new clues to AR signaling during development (Hsieh et al. 2007; Seethammagari et al. 2006). The models have been validated in terms of their tissue specificity, which varies by the model but is mostly prostate specific. More importantly, the ARmediated light signals in the mouse models respond appropriately to various treatments such as castration and drugs, which include GnRH modulators and nonsteroidal antiandrogens. Studies of bigenic models, particularly TRAMP mice, have revealed the loss of AR function during cancer progression and demonstrated the ability to visualize metastasis (Hsieh et al. 2007; Seethammagari et al. 2006). These studies have also demonstrated the limitations of the imaging methodology and the need for ex vivo imaging and detailed pathological analyses in order to specifically localize the lesions and track the route of dissemination. Future studies will incorporate the various imaging systems to study the effects of new drugs and to study in more detail the role of AR during cancer progression and dedifferentiation. New reporter genes such as red-shifted reporter proteins are already allowing higher resolution localization of light-emitting cells. PET coupled with CT in the micro-PET scanners allows significantly higher resolution than bioluminescence in animal models but is more suited for laboratories with specialized facilities. We demonstrated that the use of AR-responsive promoters to express imaging reporter genes can be an effective means to interrogate AR activity in prostate tumors in living subjects. However, this form of imaging is an indirect measurement of AR function. Recently, PET probes derived from substrates of nuclear receptors that can bind to AR have been synthesized. For instance, F18-DHT has already been successfully employed to monitor metastatic lesions in patients and their response to treatment (Dehdashti et al. 2005). A new generation of probes should permit more specific assessment of various treatments on AR-containing lesions in man.
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Zhang, J., Thomas, T. Z., Kasper, S., and Matusik, R. J. 2000. A small composite probasin promoter confers high levels of prostate-specific gene expression through regulation by androgens and glucocorticoids in vitro and in vivo. Endocrinology 141: 4698–4710. Zhang, L., Adams, J. Y., Billick, E., Ilagan, R., Iyer, M., Le, K., Smallwood, A., Gambhir, S. S., Carey, M., and Wu, L. 2002. Molecular engineering of a two-step transcription amplification (TSTA) system for transgene delivery in prostate cancer. Mol Ther 5: 223–232. Zhang, L., Johnson, M., Le, K. H., Sato, M., Ilagan, R., Iyer, M., Gambhir, S. S., Wu, L., and Carey, M. 2003. Interrogating androgen receptor function in recurrent prostate cancer. Cancer Res 63: 4552–4560.
Increased Expression of Genes Converting Adrenal Androgens to Testosterone in Castration-Recurrent Prostate Cancer Steven P. Balk
Abstract Androgen deprivation is still the standard systemic therapy for locally advanced or metastatic prostate cancer (PCa), but patients invariably relapse with a more aggressive form of PCa that has been termed castration-recurrent PCa (CRPCa). The androgen receptor (AR) is expressed at high levels in most cases of CRPCa, and these tumors resume their expression of multiple AR regulated genes, which indicates that AR transcriptional activity becomes reactivated at this stage of the disease. Mechanisms that may contribute to AR reactivation in CRPCa include increased AR protein expression, AR mutations, increased expression of transcriptional coactivator proteins, and activation of signal transduction pathways that can enhance AR responses to low levels of androgens. Recent data indicate that a further mechanism for AR reactivation in CRPCa cells may be through increased intracellular synthesis of testosterone and 5a-dihydrotestosterone (DHT). The enzymes that mediate androgen synthesis and metabolism in normal prostate and in PCa, and evidence indicating that their increased expression contributes to the development of CRPCa, are outlined in this chapter. The early use of therapies that more aggressively block androgen production may enhance responses to androgen deprivation therapy, and prevent or delay the adaptations that eventually lead to CRPCa.
1 Introduction Androgen receptor (AR) is expressed at low levels in most tissues and its transcriptional activity is stimulated by circulating testosterone, which in males is produced primarily by the testes. AR is also expressed at low levels by a subset of stromal
S.P. Balk Cancer Biology Program, Hematology Oncology Division, Beth Israel Deaconess Medical Center and Harvard Medical School, 330 Brookline Avenue, Boston, MA 02215, USA, E-mail: sbalk@ bidmc.harvard.edu
D. Tindall and J. Mohler (eds.), Androgen Action in Prostate Cancer, DOI: 10.1007/978-0-387-69179-4_5, # Springer Science + Business Media, LLC 2009
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cells in prostate, but is expressed at high levels by the luminal epithelial cells in adult prostate. The majority of prostate cancers (PCa) similarly express high levels of AR, and their growth can be suppressed by androgen-deprivation therapy, which involves removal of testicular androgens (surgical or medical castration) or treatment with an AR antagonist (or a combination of both). Androgen deprivation is still the standard systemic therapy for locally advanced or metastatic PCa, but patients invariably relapse with a more aggressive form of PCa that has been termed hormone-refractory, androgen-independent, or castration-recurrent PCa (CRPCa). Significantly, the AR is expressed at high levels in most cases of CRPCa, and these tumors resume their expression of multiple AR-regulated genes (including TMPRSS2-Ets fusion genes), which indicates that AR transcriptional activity becomes reactivated at this stage of the disease. Moreover, studies in cell line and xenograft models of CRPCa indicate that AR remains essential for tumor growth, which suggests that the reactivated AR provides a therapeutic target in CRPCa. However, the molecular basis for this reactivation remains unclear. Mechanisms that may contribute to AR reactivation in CRPCa include increased AR protein expression, AR mutations (which can enhance AR activation by weak androgens, other steroid hormones, and drugs), increased expression of transcriptional coactivator proteins, and activation of signal transduction pathways that can enhance AR responses to low levels of androgens. Recent data indicate that a further mechanism for AR reactivation in CRPCa cells may be through increased intracellular synthesis of testosterone and 5a-dihydrotestosterone (DHT). The enzymes that mediate androgen synthesis and metabolism in normal prostate and in PCa, and evidence indicating that their increased expression contributes to the development of CRPCa, are outlined in this chapter.
2 Intraprostatic Androgen Production in Normal Prostate and Prostate Cancer The majority of circulating testosterone in healthy adult males is synthesized in the testes, with only about 5–10% being produced by the adrenal glands. However, the adrenal gland in humans is a major source of weak androgens, which include androst-4-ene-3,17 dione (androstenedione), dehydroepiandrosterone (DHEA), and DHEA-sulfate (DHEA-S). These adrenal androgens serve as precursors that can be converted to testosterone and DHT in some peripheral tissues, with the levels of circulating DHEA-S being 100-fold higher than those of testosterone. Interestingly, the adrenal glands of many other species, including mouse, rat, and dog, do not produce significant levels of these weak androgens (Belanger et al. 1989). Prostate is one of the peripheral tissues that can convert DHEA-S and DHEA to androstenedione, and further convert androstenedione to testosterone, although this may not be a major source of intraprostatic testosterone in the setting of normal circulating levels (see later).
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Although testosterone is a high-affinity ligand for AR, AR transcriptional activity in prostate is further stimulated through the intraprostatic reduction of testosterone to the higher-affinity ligand DHT (approximately eightfold higher affinity). This reaction in benign prostate is mediated primarily by the type 2 5a-reductase, and inhibition of this enzyme with the drug finasteride decreases AR transcriptional activity by 50% based on expression of PSA. The prostatic expression of enzymes that regulate testosterone and DHT synthesis and metabolism presumably reflects the need to maintain DHT at levels that are optimal for maximal AR activity.
2.1
Intraprostatic Androgen Levels in Benign Prostate and Untreated Primary PCA
While intraprostatic synthesis may not be a major source of testosterone in men with intact testes and normal circulating testosterone, it does appear to be a substantial source when testicular production is blocked. In one study of healthy men, treatment for one month with a GnRH antagonist caused a 94% decline in serum testosterone, but only a 70–80% decline in prostate tissue levels of testosterone and DHT (Page et al. 2006). Several other studies similarly have found that intraprostatic androgen levels do not decline as markedly as serum levels after androgen-deprivation therapy. One early study found that DHT levels declined by only 75% after castration, and declined by 86% after ketoconazole to suppress both testicular and adrenal androgen production (Geller and Albert 1987). More recent studies also found that prostate tissue levels of DHT in PCa patients undergoing neoadjuvant androgen-deprivation therapy were only 75% lower than control levels (Belanger et al. 1989; Mizokami et al. 2004; Nishiyama et al. 2004). Taken together, these data show that intraprostatic synthesis can be a significant source of testosterone and DHT in benign prostate in the absence of testicular androgens.
2.2
Intraprostatic Androgen Levels in CRPCA
Several studies have examined intraprostatic androgen levels in men with CRPCa and intact prostates. An early study in men with CRPCa found that intraprostatic DHT levels were elevated to >50% of control levels (noncastrated men) in 2/20 men treated with surgical castration and in 4/9 men who received estrogen treatment (Geller et al. 1984; Geller 1985). A more recent larger study was done in 22 castrated men with CRPCa who had tissue obtained from transurethral resections performed to relieve obstruction. Importantly, intraprostatic testosterone levels were not reduced significantly relative to controls with normal serum androgen levels, while DHT levels were reduced to 18% of controls (Mohler et al. 2004; Titus et al. 2005b). Overall, these data indicate that the progression to CRPCa may be
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associated with increased intratumoral accumulation or synthesis of testosterone, while DHT levels appear to be similar in primary PCa shortly after castration and in subsequent CRPCa.
3 Expression of Enzymes Mediating Androgen Synthesis in Normal Prostate Consistent with the relatively high tissue levels of testosterone and DHT in benign prostate after castration, the enzymes that can generate these potent androgens from weak adrenal androgen precursors are expressed in benign prostate. The major enzymes that appear to regulate androgen synthesis and metabolism in benign prostate are outlined in Fig. 1.
3.1
3 b-Hydroxysteroid Dehydrogenase 1 (HSD3B1)
The enzyme HSD3B1 is expressed in prostate basal epithelial cells and converts DHEA to androstenedione, which can then be reduced to testosterone (see later) (Labrie et al. 2000). DHEA-S is the major androgen precursor synthesized by the adrenal gland and circulates at relatively high levels. DHEA-S is converted to DHEA in prostate, so 3b-HSD1 has the potential to provide the prostate with substantial substrate for intraprostatic androgen production. A study using cultured benign prostate epithelial cells (PrEC, which are predominantly basal cells) found that HSD3B1 expression could be induced rapidly by IL-4 and IL-13, which suggests a mechanism by which inflammatory cells may enhance intraprostatic androgen synthesis (Gingras and Simard 1999).
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α
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Fig. 1 Outline of enzymes mediating androgen metabolism described in the text
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Aldo-Keto Reductase Family 1, Member C3 (AKR1C3)
AKR1C3 is the major enzyme in prostate that functions as a 17-ketoreductase to reduce androstenedione to testosterone (Lin et al. 1997; El Alfy et al. 1999; Dufort et al. 1999; Penning et al. 2000; Lin et al. 2004). This enzyme has also been referred to as 17b-hydroxysteroid dehydrogenase type 5 (17b-HSD5) or 3a-hydroxysteroid dehydrogenase type 2. The AKR1C family in humans has four linked and highly homologous (>86% amino acid identity) members, AKR1C1–4, which function primarily as ketosteroid reductases because the reverse oxidative reactions are inhibited strongly by NADPH (Ratnam et al. 1999; Penning et al. 2000; Rizner et al. 2003; Steckelbroeck et al. 2004). AKR1C1 and AKR1C2 are also expressed in prostate, but in contrast to AKR1C3, the AKR1C1 and C2 enzymes function primarily to inactivate DHT (see later). Testosterone synthesis by Leydig cells in the testes is mediated by a distinct enzyme, type 3 17b-hydroxysteroid dehydrogenase (Andersson et al. 1995). In normal prostate, AKR1C3 has been localized to stromal, endothelial, and perineural cells using in situ hybridization and immunohistochemistry (Pelletier et al. 1999; Lin et al. 2004; Fung et al. 2006; Penning et al. 2006). A biological function for AKR1C3 in normal prostate may be to increase testosterone levels in prostate stroma and/or to generate increased testosterone for the epithelia. However, AKR1C3’s importance as a source of testosterone in men with normal testicular function is not clear, and the enzyme may have alternative biological functions in normal prostate. Other reactions catalyzed by AKR1C3 include the reduction of estrone to 17b-estradiol and the reduction of prostaglandin D2 to prostaglandin F2 (Matsuura et al. 1998; Komoto et al. 2004). This latter reaction could decrease the production of prostaglandin J2 ligands for PPARg, which are normally derived from prostaglandin D2 (Desmond et al. 2003). AKR1C3 also can function as a 3-ketosteroid reductase and convert DHT to the weak androgen, 5a-androstane-3a,17b-diol (3a-androstanediol), similar to AKR1C2 (see later), but this activity does not appear significant in vivo (Penning et al. 2000).
3.3
Type 1 and 2 5a-Reductases (Steroid Reductase 5a1 And 5a2, SRD5A1, And SRD5A2)
Both SRD5A1 and SRD5A2 are expressed in normal prostate epithelium and mediate the conversion of testosterone to DHT, which has an eightfold higher affinity for AR (Russell and Wilson 1994). However, the type 2 enzyme is the major form expressed in normal prostate. The importance of this enzyme for prostate function has been clearly established based on the failure of prostate development in men with SRD5A2 deficiency (Wilson et al. 1993). In addition, treatment of healthy men with finasteride, a relatively specific inhibitor of the type 2 enzyme, results in a substantial 50% reduction in expression of the androgenregulated PSA gene and can decrease prostate size (Thompson et al. 2003).
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Moreover, a major study in healthy men showed that PCa incidence decreased significantly in men taking finasteride versus placebo (Thompson et al. 2003).
3.4
Aldo-Keto Reductase Family 1, Member C2 (AKR1C2)
AKR1C2 has also been termed 3a-hydroxysteroid dehydrogenase type 3 (3a-HSD type 3). AKR1C2 is closely related to AKR1C3, but it acts primarily as a 3ketosteroid reductase and reduces DHT to 5a-androstane-3a,17b-diol (3a-androstanediol), which is an extremely weak androgen (Penning et al. 2000; Rizner et al. 2003; Ji et al. 2003). This compound is subsequently glucuronidated to form 3aandrostanediol-glucuronide, which is eliminated into the circulation. In contrast to AKR1C3, AKR1C2 is expressed at comparable or higher levels in the luminal epithelium versus the stroma of benign prostate (Ji et al. 2003; Bauman et al. 2006a). Consistent with a biological role in the inactivation of DHT, expression of AKR1C2 is increased by DHT treatment of LNCaP PCa cells (Ji et al. 2003). Moreover, LNCaP cells expressing increased levels of transfected AKR1C2 have a diminished response to DHT, but not to the synthetic agonist R1881, with similar findings in transfected PC3 and LAPC-4 cells (Rizner et al. 2003; Ji et al. 2003).
3.5
Aldo-Keto Reductase Family 1, Member C1 (AKR1C1)
AKR1C1 is the third member of the AKR1C family that is expressed in normal prostate. Similar to AKR1C2, it can function as a 3-ketosteroid reductase to reduce DHT, and increased expression by transfection in LNCaP cells decreases DHT stimulated growth. However, AKR1C1 is less active as a DHT reductase, and in contrast to AKR1C2, it converts DHT primarily to 5a-androstane-3b,17b-diol (3bandrostanediol) (Steckelbroeck et al. 2004). 3b-androstenediol has been identified as a potential endogenous ligand for the estrogen receptor b, which provides another link between androgen metabolism and estrogens (Weihua et al. 2001; Weihua et al. 2002). AKR1C1 also functions as a 20a-ketosteroid reductase, and converts progesterone to 20a-hydroxyprogesterone, a weak progestin, which has its major activity in breast (Penning et al. 2000).
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Retinol Dehydrogenase like 3a-Hydroxysteroid Dehydrogenase (HSD17B6)
While DHT is synthesized primarily from testosterone, an alternative source is through oxidation of 3a-androstanediol. This is the reverse of the reaction catalyzed
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by AKR1C2, but AKR1C2 does not carry out this reaction efficiently in vivo. Recent studies of enzymes with 3a-HSD activity in human prostate have shown that the major enzyme mediating this reaction is retinol dehydrogenase like 3a-hydroxysteroid dehydrogenase (HSD17B6) (Bauman et al. 2006b). Based on real-time RT-PCR of cultured prostate stromal and epithelial cells, HSD17B6 is expressed primarily in stromal cells and is increased in BPH (Bauman et al. 2006a; Penning et al. 2007).
3.7
UDP Glycosyltransferase 2, B15 (UGT2B15) and UDP Glycosyltransferase 2, B17 (UGT2B17)
Both DHT and 3a-androstanediol can be inactivated irreversibly by glucuronidation. The major metabolite in prostate is generated by addition of a glucuronyl group to the 17-hydroxy position of 3a-androstanediol to generate 5a-androstane3a,17b-diol glucuronide (3a-androstanediol-17 glucuronide) (Belanger et al. 2003). This reaction can be carried out by UGTB15 and UGTB17, which are expressed in human prostate. In situ hybridization and immunohistochemistry indicate that UGT2B15 is expressed primarily in the luminal epithelium of normal prostate, while UGT2B17 is in the basal cells (Chouinard et al. 2004). Both enzymes are expressed in LNCaP PCa cells, but their expression is downregulated by androgen stimulation, suggesting that they do not regulate androgen metabolism by a straightforward negative feedback loop (Guillemette et al. 1996; Chouinard et al. 2006). In contrast, UGT2B15 expression is increased strongly by estrogen in breast cancer cells (Harrington et al. 2006).
4 Enzymes Mediating Androgen Metabolism in Untreated Primary PCA Altered expression of the aldo-keto reductases and 5a-reductases has been reported in several studies of untreated primary PCa.
4.1
Altered Expression of AKR1C2 and AKR1C3
In an initial study using RT-PCR to assess AKR1C family expression in matched normal versus PCa tissue, one group found a variable (approximately 3 to 10-fold) and significant reduction in AKR1C2 message levels, but only small and variable changes in AKR1C1 and AKR1C3 (Ji et al. 2003). In a subsequent study, this group found decreased AKR1C1 expression (Ji et al. 2007). Moreover, they also found that DHT levels were increased in PCa tissue compared to paired benign tissue
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(42% higher) and that metabolism of 3H-DHT was reduced in freshly isolated malignant versus benign tissues. Consistent with this result, an early study also found increased DHT and decreased 3a-androstanediol levels in PCa (Krieg et al. 1979). Other studies using epithelial cells cultured from PCa versus benign prostate have not detected significant changes in AKR1C2, AKR1C1, or AKR1C3 transcript levels, but it is not clear whether expression levels in the cultured cells are similar to those in tumor cells in vivo (Bauman et al. 2006a). Indeed, immunohistochemical studies using a monoclonal antibody against AKR1C3 have shown that AKR1C3 protein is detectable in primary PCa cells, but absent in benign luminal epithelial cells (Lin et al. 2004; Fung et al. 2006). AKR1C3 protein expression was also increased in endothelial cells in PCa. Taken together, these results suggest that DHT levels may be increased in primary PCa through decreased catabolism (due to lower AKR1C2 expression) and increased tumor cell synthesis of testosterone (mediated by AKR1C3).
4.2
Increased Expression of SRD5A1 and Decreased SRD5A2
While type 2 5a-reductase (SRD5A2) is the predominant form in benign prostate, several studies have shown that expression of the type 1 5a-reductase (SRD5A1) is increased in primary PCa based on transcript and protein levels (Bonkhoff et al. 1996; Iehle et al. 1999; Thomas et al. 2003, 2005; Titus et al. 2005a). In contrast, SRD5A2 levels were not increased and were found to be decreased in some studies (Bjelfman et al. 1997; Soderstrom et al. 2001; Luo et al. 2003; Thomas et al. 2005; Titus et al. 2005a). This apparent switch from expression of SRD5A2 in normal prostate to SRD5A1 in PCa may have important clinical implications, as the commonly used 5a-reductase inhibitor (finasteride) is relatively specific for the type 2 enzyme.
5 Enzymes Mediating Androgen Metabolism in CRPCA Two studies of global gene expression in CRPCa using Affymetrix oligonucleotide microarrays have found altered expression of enzymes mediating androgen synthesis and catabolism. The first study found increased expression of multiple enzymes in the cholesterol biosynthetic pathway, which may provide increased cholesterol for de novo steroid biosynthesis (Holzbeierlein et al. 2004). The second found increased expression of enzymes that mediate testosterone and DHT synthesis from adrenal precursors, and that mediate DHT inactivation (Stanbrough et al. 2006). Significantly, both studies showed marked increases in AR message levels and found that expression of AR-regulated genes was restored, consistent with reactivation of AR transcriptional activity in CRPCa.
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Increased Expression of Enzymes Mediating Cholesterol Synthesis
The first study used Affymetrix oligonucleotide microarrays to assess global changes in gene expression in 23 untreated primary PCa, 17 primary PCa after 3 months of androgen-deprivation therapy, six cases of untreated metastatic PCa, and three cases of metastatic CRPCa (Holzbeierlein et al. 2004). Of the 650 genes that were downregulated in the group after 3 months of androgen deprivation, 25% appeared to be direct AR targets. Significantly, the overall pattern of gene expression in the three CRPCa samples was more similar to the untreated tumors than the androgen-deprived tumors, consistent with the reactivation of AR transcriptional activity. A comparison of gene expression in the untreated primary and metastatic PCa versus the metastatic CRPCa revealed 100 genes that were at least threefold differentially expressed in the CRPCa. One of these genes with increased expression in CRPCa was AR, which was increased approximately tenfold. A series of genes whose products regulate cholesterol synthetase were also increased (HMG-CoA synthase, squalene synthtase, squalene monooxygenase, and lanosterol synthase), and may provide increased endogenous substrate for steroid and lipid synthesis. Previous studies have shown that androgens activate both fatty acid and cholesterol biosynthetic pathways by increasing expression of sterol response-element-binding proteins 1 and 2 (SREBP), which are the transcription factors regulating multiple genes that control fatty acid synthesis (such as fatty acid synthetase) and cholesterol synthesis (such as HMG-CoA synthase) (Swinnen et al. 1997; Heemers et al. 2001). Androgens further enhance the activation of SREBP by increasing expression of SREBP cleavage-activating protein (SCAP), which binds to SREBP in the endoplasmic reticulum and facilitates its cleavage and subsequent nuclear translocation (Heemers et al. 2001). Importantly, another study showed that SREBP and SCAP levels were increased in LNCaP xenografts that recurred after castration (Ettinger et al. 2004). Moreover, in clinical samples examined using immunohistochemistry, this study found that SREBP-1 levels decreased after androgen-deprivation therapy, but were increased in CRPCa. Therefore, the apparent increase in cholesterol synthesis in CRPCa is consistent with AR reactivation, and may provide a positive feedback loop for further AR activation by providing cholesterol for de novo androgen synthesis in addition to lipid synthesis.
5.2
Increased Expression of Enzymes Mediating Androgen Synthesis from Adrenal Precursors
Affymetirx oligonucleotide microarrays were used in a larger study of 33 CRPCa bone metastases, which were compared with 22 laser capture microdissected primary PCa from patients who had not received androgen-deprivation therapy
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(and control bone marrow biopsies that did not contain tumor) (Stanbrough et al. 2006). AR message levels were increased consistently, with the average increase being approximately sixfold. Multiple AR-regulated genes were also highly expressed, although their levels were still approximately threefold lower than in the primary tumors, which indicates that AR transcriptional activity was only partially restored. Significant changes also were found in the expression of multiple enzymes mediating androgen metabolism. The largest change was in AKR1C3, which reduces androstenedione to testosterone and was increased >5-fold in the CRPCa samples. This increase was also observed using real-time RT-PCR and immunohistochemistry, with increased immunostaining observed in 11/19 CRPCa samples. Importantly, the increase in AKR1C3 is consistent with data outlined in Sect. 2.2, which showed that intraprostatic testosterone levels are increased in CRPCa (relative to levels shortly after androgen deprivation) and do not differ significantly from the levels in benign prostate with normal systemic androgen levels. An alternative reaction catalyzed by AKR1C3 is the reduction of DHEA to androst-5-ene-3b,17bdiol (5-androstenediol), which has weak AR agonist activity (Miyamoto et al. 1998). This activity may be significant, as a recent study showed that levels of 5-androstenediol in prostate were not decreased by castration (Mizokami et al. 2004). Although 5-androstenediol is 100-fold less potent than DHT as an AR agonist, its potency is markedly increased on the T877A mutant AR expressed by LNCaP cells (Mizokami et al. 2004). This study also found that type 1 5a-reductase expression was increased (approximately twofold) in CRPCa, while expression of the type 2 5a-reductase was reduced to 50% of the levels in primary untreated prostate cancers (Stanbrough et al. 2006). This shift from the type 2 to the type 1 5a-reductase is also observed in primary untreated PCa (Sect. 4.2), and increases in the type 1 protein and activity in CRPCa have been observed in other studies (Thomas et al. 2005; Titus et al. 2005a). Overall, these results indicate that there is a progressive shift to the type 1 5a-reductase that occurs during the development of PCa and continues as the tumors adapt to androgen deprivation to become CRPCa. The selective advantages conferred on the tumor cells by this shift are not clear, but these observations suggest that a dual type 1 and type 2 5a-reductase inhibitor may have additional activity in PCa relative to a type 2 specific inhibitor (Thomas et al. 2007). A final enzyme mediating androgen synthesis that was increased (1.8-fold) in CRPCa was 3b-hydroxysteroid dehydrogenase 2 (HSD3B2), which converts DHEA to androstenedione. In humans there are two 3b-hydroxysteroid dehydrogenase isoforms (HSD3B1 and HSD3B2), and these are also required for the synthesis of all other steroid hormones (Simard et al. 2005). As noted in Sect. 3.1, HSD3B1 is the isoform that normally is expressed in prostate basal cells and in other peripheral tissues such as breast and skin, while HSD3B2 is primarily expressed in adrenal, ovary, and testes (Simard et al. 2005). Therefore, as observed for 5a-reductases, both an increase and a shift to a distinct isoform for HSD3B activity appear to occur in CRPCa.
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Increased Expression of Enzymes Mediating Androgen Inactivation
Surprisingly, expression of AKR1C2 and AKR1C1, which function to inactivate DHT (Sects. 3.4 and 3.5), also was increased significantly in the CRPCa tumors (each approximately threefold) (Stanbrough et al. 2006). Moreover, there was a strong correlation between these increases and the increased AKR1C3 expression in individual tumors. The increases in AKR1C1 and AKR1C2 were in contrast to their decreased expression in untreated primary PCa (Sect. 4.2). One interesting possibility is that this increase in CRPCa is in response to elevated intracellular testosterone and increased DHT synthesis, as AKR1C2 expression is stimulated by DHT in PCa cells (Sect. 3.4). In this case, increased androgen synthesis would represent a primary adaptation to androgen deprivation in CRPCa, with the increases in AKR1C1 and AKR1C2 being secondary. Finally, expression of UDP glycosyltransferase 2, B15 (UGT2B15) was increased 3.5-fold. This enzyme mediates the glucuronidation of DHT metabolites, in conjunction with UGT2B17 (Sect. 3.7). The selective increase in UGT2B15 vs. UGT2B17 may be related to UGT2B15 expression in the luminal epithelia of normal prostate, with UGT2B17 being expressed in basal cells (Sect. 3.7). While the increase in UGT2B15 could be in response to increased androgen synthesis (as suggested for AKR1C2), androgens do not stimulate UGT2B15 in LNCaP cells and instead decrease its expression (Sect. 3.7). Increased expression of another enzyme mediating androgen inactivation, 17bhydroxysteroid dehydrogenase type 2 (HSD17B2), also was observed in another study of CRPCa. This study examined a small group of selected genes using realtime RT-PCR in primary untreated PCa versus 13 CRPCa (the latter tissues were obtained from transurethral resections) and found an increase of approximately fourfold in HSD17B2 in CRPCa (Fromont et al. 2005). The major activity of this enzyme with respect to androgens is inactivation of testosterone by converting it to androstenedione (the reverse of the reaction mediated by AKR1C3). This result contrasts with findings in LNCaP cells, in which HSD17B2 was decreased markedly when the cells progressed to an androgen-independent stage (Harkonen et al. 2003). An early study also found that HSD17B2 activity was decreased in primary PCa compared to BPH (Elo et al. 1996).
5.4
Implications of Increased Androgen Synthesis for AR Antagonists
A poorly understood feature of CRPCa is that it generally does not respond to treatment with AR antagonists, including high-dose (150–200 mg) bicalutamide, which can block the AR when used as single agents for initial hormone therapy
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(Fowler et al. 1995; Scher et al. 1997; Joyce et al. 1998). One explanation may be that bicalutamide and hydroxyflutamide (the active metabolite of flutamide), as relatively weak competitive antagonists, no longer can compete effectively for AR binding due to increased intracellular testosterone and DHT. Moreover, other molecular changes in CRPCa may increase markedly the AR affinity foragonist ligands (‘ ‘hypersensitive’ ’ AR). If this is correct, then the efficacy of AR antagonists in CRPCa may be enhanced by agents that decrease androgen synthesis. However, an alternative general hypothesis is that bicalutamide can compete for AR binding in CRPCa cells, but no longer functions as an antagonist due to increased AR expression or relative increases in coactivator versus corepressor proteins (Shang et al. 2002; Cheng et al. 2002; Masiello et al. 2002; Chen et al. 2004; Zhu et al. 2006; Baek et al. 2006; Hodgson et al. 2007).
6 Summary and Clinical Implications Taken together, these studies indicate that residual weak androgens, which in castrated men are produced primarily by the adrenal gland, may be an important source of ligand for AR in CRPCa. Consistent with this hypothesis, early studies found that about one-third of patients with CRPCa had objective responses (tumor shrinkage) to adrenalectomy or hypophysectomy, and that the majority of patients improved symptomatically (Mahoney and Harrison 1972). This surgical approach was later replaced by treatment with aminoglutethimide or ketoconazole, which both suppress adrenal androgen synthesis. However, responses are generally partial and transient, and it appears clear that even total elimination of adrenal androgen production will have limited efficacy. This may reflect additional sources of androgen precursors or other alternative ligands, including the de novo synthesis of androgens from cholesterol by CRPCa cells, but may also reflect the eventual emergence of tumor cells that are either truly ligand independent or are no longer dependent on AR transcriptional activity. In any case, it is clear that current androgen-deprivation therapies do not ablate androgen synthesis and that a subset of CRPCa may respond to inhibitors that can decrease further intracellular androgen levels. Abiraterone is one such promising inhibitor of adrenal androgen production that is currently in clinical trials, and dutasteride may have some value as a dual type 1 and type 2 5a-reductase inhibitor. Importantly, the efficacy of available competitive AR antagonists also may be enhanced by therapies that decrease intracellular androgen levels. Finally, it is possible that the early use of therapies that more aggressively block androgen production will enhance responses to androgen-deprivation therapy, and prevent or delay the adaptations that eventually lead to CRPCa.
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Ji, Q., Chang, L., VanDenBerg, D., Stanczyk, F.Z., and Stolz, A. (2003). Selective reduction of AKR1C2 in prostate cancer and its role in DHT metabolism. Prostate 54, 275–289. Joyce, R., Fenton, M.A., Rode, P., Constantine, M., Gaynes, L., Kolvenbag, G., DeWolf, W., Balk, S., Taplin, M.E., and Bubley, G.J. (1998). High dose bicalutamide for androgen independent prostate cancer: effect of prior hormonal therapy. J. Urol. 159, 149–153. Komoto, J., Yamada, T., Watanabe, K., and Takusagawa, F. (2004). Crystal structure of human prostaglandin F synthase (AKR1C3). Biochemistry 43, 2188–2198. Krieg, M., Bartsch, W., Janssen, W., and Voigt, K.D. (1979). A comparative study of binding, metabolism and endogenous levels of androgens in normal, hyperplastic and carcinomatous human prostate. J. Steroid Biochem. 11, 615–624. Labrie, F., Luu-The, V., Lin, S.X., Simard, J., and Labrie, C. (2000). Role of 17 beta-hydroxysteroid dehydrogenases in sex steroid formation in peripheral intracrine tissues. Trends Endocrinol. Metab. 11, 421–427. Lin, H.K., Jez, J.M., Schlegel, B.P., Peehl, D.M., Pachter, J.A., and Penning, T.M. (1997). Expression and characterization of recombinant type 2 3 alpha-hydroxysteroid dehydrogenase (HSD) from human prostate: demonstration of bifunctional 3 alpha/17 beta-HSD activity and cellular distribution. Mol. Endocrinol. 11, 1971–1984. Lin, H.K., Steckelbroeck, S., Fung, K.M., Jones, A.N., and Penning, T.M. (2004). Characterization of a monoclonal antibody for human aldo-keto reductase AKR1C3 (type 2 3alpha-hydroxysteroid dehydrogenase/type 5 17beta-hydroxysteroid dehydrogenase); immunohistochemical detection in breast and prostate. Steroids 69, 795–801. Luo, J., Dunn, T.A., Ewing, C.M., Walsh, P.C., and Isaacs, W.B. (2003). Decreased gene expression of steroid 5 alpha-reductase 2 in human prostate cancer: implications for finasteride therapy of prostate carcinoma. Prostate 57, 134–139. Mahoney, E.M. and Harrison, J.H. (1972). Bilateral adrenalectomy for palliative treatment of prostatic cancer. J. Urol. 108, 936–938. Masiello, D., Cheng, S., Bubley, G.J., Lu, M.L., and Balk, S.P. (2002). Bicalutamide functions as an androgen receptor antagonist by assembly of a transcriptionally inactive receptor. J. Biol. Chem. 277, 26321–26326. Matsuura, K., Shiraishi, H., Hara, A., Sato, K., Deyashiki, Y., Ninomiya, M., and Sakai, S. (1998). Identification of a principal mRNA species for human 3alpha-hydroxysteroid dehydrogenase isoform (AKR1C3) that exhibits high prostaglandin D2 11-ketoreductase activity. J. Biochem. 124, 940–946. Miyamoto, H., Yeh, S., Lardy, H., Messing, E., and Chang, C. (1998). Delta5-androstenediol is a natural hormone with androgenic activity in human prostate cancer cells. Proc. Natl. Acad. Sci. U. S. A 95, 11083–11088. Mizokami, A., Koh, E., Fujita, H., Maeda, Y., Egawa, M., Koshida, K., Honma, S., Keller, E.T., and Namiki, M. (2004). The adrenal androgen androstenediol is present in prostate cancer tissue after androgen deprivation therapy and activates mutated androgen receptor. Cancer Res. 64, 765–771. Mohler, J.L., Gregory, C.W., Ford, O.H., III, Kim, D., Weaver, C.M., Petrusz, P., Wilson, E.M., and French, F.S. (2004). The androgen axis in recurrent prostate cancer. Clin. Cancer Res. 10, 440–448. Nishiyama, T., Hashimoto, Y., and Takahashi, K. (2004). The influence of androgen deprivation therapy on dihydrotestosterone levels in the prostatic tissue of patients with prostate cancer. Clin. Cancer Res. 10, 7121–7126. Page, S.T., Lin, D.W., Mostaghel, E.A., Hess, D.L., True, L.D., Amory, J.K., Nelson, P.S., Matsumoto, A.M., and Bremner, W.J. (2006). Persistent intraprostatic androgen concentrations after medical castration in healthy men. J. Clin. Endocrinol. Metab 91, 3850–3856. Pelletier, G., Luu-The, V., Tetu, B., and Labrie, F. (1999). Immunocytochemical localization of type 5 17beta-hydroxysteroid dehydrogenase in human reproductive tissues. J. Histochem. Cytochem. 47, 731–738.
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Androgen-Metabolic Genes in Prostate Cancer Predisposition and Progression Juergen K.V. Reichardt and Ann W. Hsing
Abstract Prostate cancer is the most common nonskin cancer and the second leading cause of cancer deaths among men in most Western countries, including the US and Australia. Despite its high morbidity and mortality, the etiology of prostate cancer remains elusive. Longstanding clinical and compelling laboratory data suggest a role for androgens in prostate carcinogenesis. This chapter reviews the status of research on hormones, particularly androgens, and prostate cancer and focuses first on hormone-related genetic loci in constitutional (‘‘germline’’) DNA. This set of loci has been investigated in a number of studies to date that will undoubtedly expand further. These data provide insights into susceptibility for prostate cancer. This review next explores the emerging field of somatic mutations in tumor (‘‘somatic’’) DNA in androgen-metabolic genes, especially the androgen receptor and the type II steroid 5a-reductase. Integration of these forthcoming data with those on susceptibility may provide novel insights into the etiology and progression of prostate cancer. These lines of investigation may lead to the presymptomatic identification of high-risk individuals for active disease prevention, diagnostic improvements in affected men and personalized treatment. Current and future data on individual markers and genes should be integrated into a comprehensive, pathway-based picture that includes constitutional DNA (for prostate cancer susceptibility) and tumor DNA (for disease progression). These efforts may lead to a comprehensive genetically based risk and progression assessment algorithm.
1 Introduction Prostate cancer is the most common nonskin cancer among American men, with 218,890 new cases expected in 2007 alone, and it ranks second in the number of estimated male cancer deaths, behind only lung cancer (Jemal et al. 2007). Despite the J.K.V. Reichardt(*) Plunkett Chair of Molecular Biology (Medicine), University of Sydney, Bosch Institute, Camperdown, NSW, 2006, Australia, E-mail:
[email protected]
D. Tindall and J. Mohler (eds.), Androgen Action in Prostate Cancer, DOI: 10.1007/978-0-387-69179-4_6, # Springer Science + Business Media, LLC 2009
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magnitude of prostate cancer incidence and mortality, few risk factors have been identified definitively other than age, race, and family history of prostate cancer. Prostate cancer is a hormone-dependent cancer. Longstanding biological and medical evidence supports the fact that androgens play a role in the growth and maintenance of the prostate, that prostate cancer regresses after androgen-deprivation or antiandrogen therapy, and that administration of testosterone (T) induces prostate tumors in laboratory animals (Huggins and Hodges 1941; Noble 1977; Richie 1999). Estrogenic compounds have long been used to control prostate cancer growth, though recognition of their serious cardiac and sexual side effects has diminished their use (Harkonen and Makela 2004). Similarly, strong laboratory evidence shows that vitamin D, another hormone, has strong antiproliferative and proapoptotic effects on prostate cancer (Zhao and Feldman 2001). In addition, obesity, which has been linked to elevated risk of aggressive prostate cancer and prostate cancer death (Freedland et al. 2006), is associated with lower levels of sex-hormone-binding globulin (SHBG) and presumably higher levels of free T (Pasquali et al. 1991). Prostate carcinogenesis, like most if not all cancers, is a multistep process involving multiple genes in both the susceptibility, initiation and progression phases along with environmental factors, including diet (Hsing et al. 2002a). This chapter focuses on the current status and implications of androgen-metabolic genes and their contributions to the susceptibility and progression of prostate cancer.
2 Androgen Metabolism Androgens play a critical role in normal and malignant development of the prostate. Studies of androgens and prostate cancer go back over 60 years (beginning with Huggins and Hodges (1941)) and led to a Nobel Prize for Charles Huggins. Normal prostate development is induced by DHT (5a-dihydrotestosterone or dihydrotestosterone), which is synthesized from T by the enzyme steroid 5a-reductase (e.g., Hsing et al. 2002a, b; Coffey 1993; SRD5A; Fig. 1). T is produced in large amounts by the testes and small amount by the adrenal gland (e.g., Hsing et al. 2002a, b; Coffey 1993). T is irreversibly metabolized intracellularly to DHT (Fig. 1). Intracellularly, DHT is bound by a cytosolic receptor, the androgen receptor (AR; Fig. 1). This complex is translocated to the cell nucleus where it activates transcription of genes with hormone-responsive elements in their promoters (e.g., Hsing et al. 2002a; Coffey 1993). DHT can be inactivated in the prostate by further reduction (Fig. 1) to 3a- or 3b-androstanediol (or ‘‘diol’’; Fig. 1), which circulate as glucuronide conjugates. DHT homeostasis is regulated by its biosynthesis and degradation (see Fig. 1). Both ends involve multiple enzymatic steps. Those of most interest include the reactions catalyzed by the cytochrome P450 17a-hydroxylase (CYP17), 17b-hydroxysteroid dehydrogenase (HSD17B3), SRD5A2, 3b-hydroxysteroid dehydrogenase (HSD3B2), and UGT2B15 (encoding the glucuronidase) gene products (Fig. 1). DHT binds directly to the AR to exert its signal transduction activity (Fig. 1).
6 Androgen-etabolic Genes in Prostate Cancer
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Outside the Prostate
Within the Prostate 3β-Diol G 3α-Diol G
5-androstenediol CYP17 gene
HSD3B1/2/AKR gene
HSD3B1/2 gene
17α-Hydroxylase/ 17,20-Lyase
3β-Hydroxysteroid Dehydrogenase
3β-Hydroxysteroid Dehydrogenase
Preg
DHEA
Androstenedione
T
3β-Diol
HSD3B
T
17β-Hydroxysteroid Dehydrogenase
UGT2B 3α-Diol HSD3A/AKR
DHT Steroid 5α-Reductase
Androgen Receptor AR gene
SRD5A2 gene
HSD17B3 gene
DHT/AR CYP3A4/5 gene Complex OH-T
Transactivation Cell Division
Fig. 1 Androgen metabolism and androgen-metabolic genes 0. The abbreviations used are: preg pregnelone, DHEA dehydroepiandrosterone, DHT dihydrotestosterone, diol androstanediol, diol G androstanediol glucuronide, and OH-T hydroxy-testosterone
Animal studies have shown that androgen, in particular DHT, plays a pivotal role in prostate cancer development (Noble 1977); enzymes modulating DHT levels are usually targeted for the design of antiprostate cancer drugs. Finasteride is the first drug that was used in the chemoprevention of prostate cancer (Thompson et al. 2003). It acts by inhibiting the enzyme steroid 5a-reductase type II (encoded by the SRD5A2 gene; Fig. 1), hence blocking DHT synthesis from testosterone (T; Fig. 1). In the Prostate Cancer Prevention Trial (PCPT) finasteride was tested in healthy men 55 years of age or older. The prevention study resulted in a significant (25%) decrease in prostate cancer incidence over the seven-year study period (Thompson et al. 2003).
3 Androgen-Metabolic Genes Prostate cancer is an androgen-dependent tumor. Therefore, it is likely that genes whose products are involved in androgen metabolism are involved in prostate cancer etiology. Ross et al. (1998) first proposed a polygenic model to help explain the racial/ethnic difference in prostate cancer risk. This model triggered a series of studies that investigated the involvement of genes such as CYP17, aromatase (CYP19), steroid 5a-reductase (SRD5A2), (HSD3B2), and androgen receptor (AR) in prostate cancer (cf. Fig. 1). These candidate genes and their effects in constitutional (‘‘germline’’) DNA on risk are considered first (cf. Table 1). Data on acquired somatic mutations (i.e., de novo mutations in tumor tissue) are also accumulating (cf. Table 1). We will summarize and integrate data on constitutional and somatic DNA in androgen-metabolic genes here into a comprehensive picture of the combined role of these genes in prostate cancer.
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Table 1 Characteristics of androgen-metabolic genes analyzed in prostate cancer Chromosomal Constitutional DNA Somatic Gene Gene product location SNPs mutations Androgen biosynthesis CYP17 Cytochrome p450c17 10q24.3 Reviewed in None reported Chokkalingam et al. (2007) T metabolism SRD5A2 Steroid 5a-reductase
2p23
Reviewed in Chokkalingam et al. (2007)
CYP3A4
7q22.1
Reviewed in Chokkalingam et al. (2007) Reviewed in Chokkalingam et al. (2007)
CYP3A5
cytochrome P450, subfamily 3A, polypeptide 4 cytochrome P450, subfamily 3A, polypeptide 5
7q22.1
DHT metabolism HSD3B2 3B-hydroxysteroid dehydrogenase
1p13
HSD17B3
17B-hydroxysteroid dehydrogenase
9q22
SULT
sulfotransferase
various
UGT2B15
UDPglycosyltransferase
4q13
Androgen signaling AR Androgen receptor
Reviewed in Chokkalingam et al. (2007) Reviewed in Chokkalingam et al. (2007) Reviewed in Culig et al. (1997) Reviewed in Reichardt (2006)
Penning et al. (2004) and Fung et al. (2006) None reported
None reported
None reported
None reported
None reported None reported
Reviewed in Reviewed in Chokkalingam Culig et al. et al. (2007) and (1997) Culig et al. (1997) AIB1 Nuclear receptor 20q12 Reviewed in Culig None reported coactivator et al. (1997) Notes: The numbers in this table indicate references. The presence of constitutional and/or somatic mutations refers to publications referring to or reviewing data on DNA from either lymphocytes (constitutional or ‘‘germline’’ DNA) or prostate tumor tissue (somatic DNA). Absence of publications, e.g., on somatic mutations, may only indicate that this gene has not yet been investigated and hence somatic mutations may be found in the future
3.1
Xq11-12
CYP17
Cytochrome P450c17a hydroxylase, encoded by the CYP17 gene located in chromosome band 10q24.3, catalyzes early steps in the biosynthesis of testosterone (see Fig. 1; Table 1). A single nucleotide polymorphism (SNP), that changes the normal T to the polymorphic C, also known as the A2 allele in the 50 -untranslated region (UTR) of the CYP17 gene, has been linked to male pattern baldness (Carey et al.
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1994), a putative risk factor for prostate cancer. However, the relationship between CYP17 and prostate cancer is inconclusive, which was recently reviewed in (Chokkalingam et al. 2007). These somewhat inconsistent results suggest that the effect of the CYP17 gene on prostate cancer, if any, is likely small. No reports on somatic mutations in this gene are available.
3.2
SRD5A2
The SRD5A2 gene, which encodes the prostatic or type II steroid 5a-reductase enzyme (Fig. 1; Table 1), came to the forefront of prostate cancer research after cross-sectional surveys showed that Western men have higher serum levels of the DHT metabolite, 3a-diol glucuronide (cf. Fig. 1), than native Japanese men (Ross et al. 1992). Serum hormone levels of 3a-diol glucuronide may reflect prostatic steroid 5a-reductase activity. These observations led to the hypothesis that population differences in steroid 5a-reductase activity, encoded by polymorphisms in the SRD5A2 gene, may be related to the development of prostate cancer and may contribute, at least in part, to racial/ethnic differences in risk (Makridakis et al. 1999, 2000). More than 22 mutations, including ten single amino acid missense substitutions, have been reported for the SRD5A2 gene (Makridakis et al. 2000) (Table 1). Four of these SNPs – A49T (substitution of threonine for the normal alanine at codon 49), V89L (replacement of leucine with valine at codon 89), R227Q (arginine-227 to glutamine), and a (TA)n dinucleotide repeat in the 30 UTR- have been investigated for their association with prostate cancer in 18 epidemiologic studies that have produced mixed results: see Chokkalingam et al. (2007) for a recent review. A case series reported that the A49T genotype was associated with more aggressive prostate cancer (Chokkalingam et al. 2007). However, of the 11 studies investigating the A49T marker in the SRD5A2 gene, which increases enzyme activity fivefold in vitro (Makridakis et al. 2000), just one reported a statistically significant association between the higher activity polymorphic variant and prostate cancer, and this association was only significant among African-American and Latino men (Makridakis et al. 1999). The null results of most epidemiologic studies may be due to the low prevalence of the variant T allele (about 1% in most populations), despite the large difference in enzymatic activity that results from this nucleotide change. In general, the results of the studies investigating the prostate cancer associations of the V89L marker (Chokkalingam et al. 2007), which modestly reduces enzyme activity modestly in vitro and in vivo (Makridakis et al. 2000), and the (TA)n repeat length marker (Chokkalingam et al. 2007), which has no known functional relevance, are mixed. The V89L polymorphic LL genotype, which is associated with lower serum levels of 3a-diol glucuronide (Makridakis et al. 2000), occurs in Asian men much more frequently than in African-American and Caucasian men. The R227Q mutation, which is also found in Asian male pseudohermaphrodites and almost abolishes enzyme activity (Makridakis et al. 2000), has been detected only
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in Asians thus far, and the only study investigating its role found no significant association with prostate cancer risk (Chokkalingam et al. 2007). The inconsistent findings in the SRD5A2 constitutional DNA markers in various epidemiologic studies for prostate cancer susceptibility may be due largely to the low frequency of most polymorphic alleles in the SRD5A2 gene. For example, other than the V89L mutation, the frequency of the polymorphic alleles in various markers (including A49T and R227Q) is less than 5%, limiting the power of detection. Nonetheless, a recent meta-analysis by Ntais et al. concluded that the polymorphic T allele of the A49T SNP in the SRD5A2 gene is associated with a modest risk increase, and that, although the evidence is less consistent, the LL genotype of the V89L polymorphism and longer (TA)n repeats may also be associated with increased risk for prostate cancer (Ntais et al. 2003). Although the epidemiologic data are less than convincing, the SRD5A2 gene and its variants are among the best characterized in cancer biology. Enzyme activity varies in vitro by about 200-fold variation, and there is also a 60-fold pharmacogenetic variation for finasteride among SNPs (Makridakis et al. 2000). This extensive variability is likely to have important consequences for the risk of prostate cancer, and it may also be relevant in active, personalized disease prevention, e.g., in the context of the PCPT (Thompson et al. 2003). Finally, detailed in vitro biochemical and pharmacogenetic investigations of SRD5A2 haplotypes (i.e., combinations of multiple SNPs) have been reported (Reichardt 2006). Such studies may become significant in the future as haplotypes move to the forefront of molecular epidemiology and pharmacogenetics. Somatic mutation of the SRD5A2 gene has been investigated in prostate cancer tissue (Akalu et al. 1999; Makridakis et al. 2004) (Table 1). These investigations have uncovered common somatic mutations in the SRD5A2 gene in prostate cancer, and their in vitro biochemical characterization (Makridakis et al. 2004) is consistent with driving tumor growth through increased DHT synthesis. These early encouraging data on somatic DNA remain to be confirmed. Furthermore, these data on somatic mutations should be integrated fully with data on constitutional DNA to develop a comprehensive picture of the role of the SRD5A2 gene in prostate cancer susceptibility and disease progression.
3.3
HSD Genes
Incomplete activation or slow catabolism of DHT within the prostate could lead to the accumulation of DHT and, perhaps, increased androgenic action (cf. Fig. 1). Thus, enzymes that catalyze the activation and inactivation of DHT may be of potential etiologic importance for prostate cancer. As shown in Fig. 1, at least four enzymes – 17b-hydroxysteroid dehydrogenase type I (encoded by the HSD17B1 gene), 17b-hydroxysteroid dehydrogenase type III (encoded by the HSD17B3 gene), 3a-hydroxysteroid dehydrogenase, and the 3b-hydroxysteroid dehydrogenases (encoded by the HSD3B1 and HSD3B2 genes located in chromosome
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band 1p13.1) – may be involved in the catabolism of DHT in the prostate (Table 1). Three studies have examined these genes. One reported a significant association of the G289S polymorphism in the HSD17B3 gene (Margiotti et al. 2002) and another reported an association of the N367T marker within the HSD3B1 gene, but no prostate cancer associations of the C7062T marker in the HSD3B1 gene and the C7159G and C7474T SNPs in HSD3B2 (Chang et al. 2002). These early results are evidence for a role of these genes in prostate cancer risk, but further studies are needed. In contrast, the most recent study, a comprehensive examination of the HSD17B1 gene, examined cases and controls from a consortium of cohort studies (Kraft et al. 2005). A large sample size (>9,000 cases and 9,000 controls), multiple ethnicities investigated, and utilization of haplotype-tagging SNPs rather than single SNPs provide strong evidence for a lack of association of the HSD17B1 gene with prostate cancer risk. Finally, the list of genes involved in DHT inactivation (Fig. 1) may still be incomplete. The Aldo-Keto Reductase (AKR) 1C gene family includes AKR1C1, AKR1C2, AKR1C3, and AKR1C4 (Ji et al. 2007). These four genes share a high sequence homology (>80%). The AKR1C2, AKR1C3, and AKR1C4 genes are also known as 3a-hydroxysteroid dehydrogenase (HSD) III, HSD II, and HSDI, respectively. The gene product of AKR1C2 synthesizes the DHT metabolite, 3 a-diol, which is a more water-soluble metabolite, for elimination from the prostate (Ji et al. 2003; Penning et al. 2004; Fung et al. 2006). Increased expression of AKRC3 in prostate cancer cells has been reported (Fung et al. 2006). Few epidemiologic studies have investigated the role of AKR genes, but molecular studies have shown that expression of AKR1C2 was associated with higher tissue levels of DHT (Penning et al. 2004). Thus, further investigations of the AKR genes are warranted in epidemiologic analyses. Finally, no publications to date have systematically addressed these loci in prostate cancer tissue.
3.4
CYP3A Genes
The CYP3A (cytochrome P450, family 3, subfamily A) subfamily members CYP3A4 and CYP3A5 are located at a single locus in human chromosome band 7q22.1 (Table 1). The CYP3A4 gene product is involved in the oxidative conversion of testosterone to the biologically less active forms 2b-, 6b-, and 15b-hydroxytestosterone (Fig. 1). The variant *1B allele (of the A391G SNP in the 50 UTR of CYP3A4) has been linked to higher prostate cancer stage and grade (Rebbeck et al. 1998). However, while one case-control study showed significant excess risk of aggressive disease with the *1B allele among African-Americans, it did not find this risk among Caucasians (Bangsi et al. 2006). Several other studies, which were reviewed recently in Chokkalingam et al. (2007), reported a modest but nonsignificant reduced risk of prostate cancer in connection with the variant *1B allele. The low frequency of the polymorphic variant in most populations limited the statistical
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power of many of these studies. The CYP3A4 gene product may also be involved in the metabolism of finasteride and hence have relevance for prostate cancer chemoprevention, e.g., via the PCPT (Thompson et al. 2003). The CYP3A5 gene product, which is highly expressed in the prostate, is a major contributor to the metabolic clearance of many CYP3A substrates, including drugs and other xenobiotic compounds, as well as steroid hormones. A number of polymorphic markers have been identified in the CYP3A5 gene, two of which have been studied already in epidemiologic investigations of prostate cancer. The CYP3A5*1 marker, whose variant allele may promote inactivation of testosterone, was found to have a borderline significant association with reduced risk of prostate cancer in one study but not another (see Chokkalingam et al. 2007). The variant allele of the CYP3A5*3 marker, which results in a truncated and presumably less functional protein (reviewed recently in Ntais et al. 2003), supports the observation in a Japanese population that the CYP3A5*3 wild-type allele was associated with reduced prostate cancer risk (reviewed in Chokkalingam et al. 2007). To date, no systematic mutational investigations have been reported of the two CYP3A genes in prostate tumor tissue.
3.5
UGT Genes
Conjugation with UDP (uridinediphosphate) by UGT (UDP-glycosyltransferase) enzymes (i.e., a glucuronidation reaction) is one of the major routes of elimination of endogenous compounds. UGT2 B15 and B17 enzymes are expressed in the prostate, in particular in androgen-sensitive cell lines (Belanger et al. 1998). In vitro, these enzymes conjugate DHT and its metabolites, 3 alpha diol and androsterone, into a less active form (Belanger et al. 1998). The glucuronidation by UGT2B15 and B17 is an irreversible process and an important step in DHT catabolism. Data on the role of UGT2B15 and B17 in prostate cancer are limited, with three recent studies showing that the variant allele of the UGT2B15 gene, which resulted in higher transcription, was associated with a higher risk of prostate cancer (Chouinard et al. 2007; Park et al. 2004, 2006; Gallagher et al. 2007; Dalhoff et al. 2005).
3.6
SULT Genes
Sulfation is an important steroid inactivation process in human tissue. To date, 11 human cytosolic sulfotransferase (SULT) isoforms have been identified (Wilborn et al. 2006), with SULT2A1 being the major enzyme responsible for sulfating dehydroepiandrosterone (DHEA), a precursor of T. A recent series of studies revealed that sulfotransferase SULT2B1b selectively conjugates 3b-hydroxysteroids and is expressed in epithelial cells of normal and cancerous prostate tissues (He et al. 2004; He and Falany 2007; Table 1). Despite the importance of
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these genes in steroid inactivation, data on these genes remain limited. Further studies are warranted, including constitutional DNA and tumor DNA.
4 Androgen-Signaling Genes 4.1
Androgen Receptor
Currently the most studied gene with regard to prostate cancer is that encoding the AR (Table 1). The AR is expressed in all histological types and stages of prostate cancer (Culig et al. 1997). Interestingly much of the focus in the AR has been on somatic mutations. In fact, numerous somatic mutations in the AR gene, located on the long arm of the X chromosome, have been reported in prostate cancer (Table 1). Most of these mutations have been detected in tumor tissue of late stage prostate cancer, with consistent findings showing that somatic mutation of the AR gene is involved in the progression and aggressiveness of prostate cancer (Culig et al. 1997). Most epidemiologic studies of the AR gene in constitutional DNA have focused on two trinucleotide repeat polymorphisms in exon 1, the (CAG)n and (GGN)n (where n stands for any nucleotide) repeats, which encode polyglutamine and polyglycine, respectively (Table 1). The (CAG)n repeat length ranges from 11 to 31 repeats in most men, and experimental data show that shorter (CAG)n repeat lengths are linked to increased AR trans-activation activity (Chamberlain et al. 1994; Edwards et al. 1992). In addition, (CAG)n repeat length has been linked to male pattern baldness (Sawaya and Shalita 1998; Hoffmann and Happle 2000), a medical condition associated with higher DHT levels and prostate cancer risk. Epidemiologic studies show, that in general, shorter (CAG)n repeat lengths are associated with increased risk in many but not all populations (Chokkalingam et al. 2007) (for a review). Evidence of association for the second AR repeat, (GGN)n, is less consistent than that reported for the (CAG)n repeat. The AR protein has a modular structure, consisting primarily of (a) a large amino-terminal transactivation domain, (b) a centrally located DNA-binding domain, and (c) a carboxy-terminal ligand-binding domain that confers high affinity and specificity for androgen binding. AR gene mutations have been reported in prostate cancer at a frequency of 5–50%, with a higher frequency reported in more advanced disease and following androgen-deprivation therapy, reviewed recently in Makridakis et al. (2005). In contrast to the inherited syndrome of androgen insensitivity, where AR somatic mutations result in a loss of receptor function, those detected in prostate cancer occur in distinctly different regions of the receptor, and generally confer promiscuity or increased sensitivity for receptor activation by other steroid hormones and/or by the specific antiandrogen used in clinical management of the disease (Makridakis et al. 2005). These observations led to the concept of ‘‘therapy-mediated selection pressure,’’ which is exemplified by the detection of AR gene mutations that confer enhanced receptor activity in response
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to flutamide but not to other antiandrogens in tumors from patients treated with hydroxyflutamide plus orchidectomy or LHRH agonists/antagonists, as part of a combined androgen blockade strategy (Makridakis et al. 2005). The rapidly evolving evidence that the AR is a key mediator of continued prostate cancer growth during androgen deprivation makes a compelling argument for the development of new strategies that directly target the receptor (Makridakis et al. 2005). Such new therapies are necessary for significant advances in the management of patients with advanced prostate cancer who will ultimately fail androgen-deprivation therapy. Several approaches to achieve this goal have been documented. Reducing the level of AR and suppressing human prostate cancer cell growth in vitro and in vivo used a variety of methods, including double-stranded RNA interference, antisense oligonucleotides, hammerhead ribozymes, and analogs of the ansamycin antibiotics such as 17-allylamino-17-demethoxygeldanamycin (Makridakis et al. 2005). Inhibition of AR function has been achieved with dominant negative AR inhibitors, microinjection of AR antibodies, ‘‘decoy’’ double-stranded DNA fragments containing specific AR response elements, and histone deacetylase inhibitors including suberoylanilide hydroxamic acid and phenylbutyrate (Makridakis et al. 2005). Provided issues such as the mode of delivery and the potential for disruption of multiple signaling pathways can be circumvented, these newer approaches that directly target the AR provide promise as therapeutic options for castration-resistant prostate cancer.
4.2
AR Coactivators
AR coactivators enhance transactivation of the AR several-fold (Yeh and Chang 1996) and therefore may increase the risk of prostate cancer as well. One AR coactivator, encoded by the AIB1 (Amplified in Breast Cancer 1) gene located in chromosome band 20q12, has a (CAG)n/(CAA)n (polyglutamine) trinucleotide repeat. Two epidemiologic studies have investigated the role of this repeat polymorphism in prostate cancer: one found a positive association between AIB1 (CAG)n repeat length and prostate cancer (Hsing et al. 2002b), and the other reported no association (Platz et al. 2000; Table 1). Future studies should be directed at clarifying the combined effects of AR and AR coactivators in prostate cancer risk, because of their importance in modulating the effects of androgenic action.
5 Current Status and Implications for Androgen-Metabolic Genes in Prostate Cancer Much current clinical, epidemiological, and molecular evidence support a central role for androgen-metabolic genes in prostate cancer etiology. Current data are very strong for the AR and fairly supportive on the biosynthetic end of the pathway
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(leading to DHT; Fig. 1). These investigations should be extended to DHT inactivation which is currently underinvestigated. These combined studies (such as those proposed in Ross et al. (1998)) may be useful in identifying at-risk men presymptomatically and for developing personalized chemopreventive strategies. The evidence on somatic mutations is only now beginning to emerge, but it will probably play a significant role in explaining disease progression in the near future. Finally, the heterogeneity (or multifocality) of prostate tumors is an underappreciated and under-reported phenomenon that deserves more attention (Platz et al. 2000).
6 Future Directions Although much progress has been made on androgen-metabolic genes and prostate cancer, some areas deserve more attention in the future. First, DHT inactivation appears understudied to date (cf. Fig. 1). This area is critical to fully assess DHT homeostasis. Second, full functional (e.g., biochemical and pharmacogenetic) analyses of SNPs and especially haplotypes of all androgen-metabolic genes will be useful for rational marker selection. Third, prostatic tumor tissue should be investigated systematically for the emergence of somatic mutations in all androgenmetabolic genes (Fig. 1). Fourth, the full impact of chemopreventive strategies needs to be rigorously assessed molecularly. Finally, data on individual markers and genes must be fully integrated into a comprehensive, pathway-based picture that includes constitutional DNA (for prostate cancer susceptibility) and tumor DNA (for disease progression). This strategy will lead to an overall genetically based risk and progression assessment algorithm. Considerable work remains to achieve this goal. These integrated investigations will lead to a full picture of the multistep nature of prostate cancer development and progression (e.g., Hsing et al. 2002a, b; Ntais et al. 2003; Reichardt 2006; Dalhoff et al. 2005).
Acknowledgments This work was supported in part by NCI grant P01 CA108964 (project 1) to JKVR who is also a Medical Foundation Fellow at the University of Sydney and by NIH Intramural Support (AWH).
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Rebbeck, T. R., Jaffe, J. M., Walker, A. H., Wein, A. J., Malkowicz, S. B.: Modification of clinical presentation of prostate tumors by a novel genetic variant in CYP3A4. Journal of National Cancer Institute, 90: 1225–1229, 1998. Reichardt, J. K. V.: Unexpected biochemical and pharmacogenetic consequences of SNPs and haplotypes: A cautionary tale for human molecular genetics and epidemiology. Genomics, 88: 673–674, 2006. Richie, J. P.: Anti-androgens and other hormonal therapies for prostate cancer. Urology, 54: 15–18, 1999. Ross, R. K., Bernstein, L., Lobo, R. A., Shimizu, H., Stanczyk, F. Z., Pike, M. C. & Henderson, B. E.: 5-alpha-reductase activity and risk of prostate cancer among Japanese and US white and black males. Lancet, 339: 887–889, 1992. Ross, R. K., Pike, M. C., Coetzee, G. A., Reichardt, J. K., Yu, M. C., Feigelson, H., et al: Androgen metabolism and prostate cancer: establishing a model of genetic susceptibility. Cancer Research, 58: 4497–4504, 1998. Sawaya, M. E., Shalita, A. R.: Androgen receptor polymorphisms (CAG repeat lengths) in androgenetic alopecia, hirsutism, and acne. Journal of Cutaneous Medicine and Surgery, 3: 9–15, 1998. Thompson, I. M., Goodman, P. J., Tangen, C. M., Lucia, M. S., Miller, G. J., Ford, L. G., Lieber, M. M., Cespedes, R. D., Atkins, J. N., Lippman, S. M., Carlin, S. M., Ryan, A., Szczepanek, C. M., Crowley, J. J., Coltman, C. A. Jr.: The Influence of Finasteride on the Development of Prostate Cancer, New England Journal of Medicine, 349: 215–224, 2003. Wilborn, T. W., Lang, N. P., Smith, M., Meleth, S., Falany, C. N.: Association of SULT2A1 allelic variants with plasma adrenal androgens and prostate cancer in African American men. The Journal of Steroid Biochemistry and Molecular Biology, 99: 209–214, 2006. Yeh, S., Chang, C.: Cloning and characterization of a specific coactivator, ARA70, for the androgen receptor in human prostate cells. Proceedings of the National Academy of Sciences of the United States of America, 93: 5517–5521, 1996. Zhao, X. Y. and Feldman, D.: The role of vitamin D in prostate cancer. Steroids, 66: 293–300, 2001.
Effect of Steroid 5a-Reductase Inhibitors on Markers of Tumor Regression and Proliferation in Prostate Cancer Lynn N. Thomas and Roger S. Rittmaster
Abstract Intracrine conversion of testosterone to the more potent dihydrotestosterone (DHT) by the enzymes 5a-reductase type 1 (5aR1) and type 2 (5aR2) is important for normal and pathological growth of the prostate. Consequently, inhibitors of 5aR have been developed to treat prostate disease. Finasteride, the first commercially available 5aR inhibitor, targets 5aR2 since this isoenzyme predominates in benign prostate tissue. Finasteride has been shown to cause a 20–30% reduction in prostate volume in men with BPH, through a combination of atrophy and apoptosis. It has further been shown to reduce the incidence of detectable prostate cancer by 25% in the Prostate Cancer Prevention Trial (PCPT), through prevention and/or treatment of subclinical carcinomas. However, development of prostate cancer is accompanied by a decrease in 5aR2 levels and an increase in 5aR1. The dual 5aR inhibitor dutasteride provides greater suppression of DHT formation than finasteride (93–97% vs. 68–86%) and may be even more effective in prevention of prostate cancer. The 4-year Reduction by Dutasteride of Prostate Cancer Events (REDUCE) trial is currently underway to test this hypothesis. Defining the role of 5aR inhibitors in treatment of established prostate cancer requires further investigation. However, the effect of such inhibitors on markers of tumor regression (apoptosis, proliferation, angiogenesis, and tumor histopathology) has been explored, in part. In several studies of men with BPH, who were treated with finasteride for 3 months to 4 years and who subsequently developed prostate cancer, the effect of finasteride on the histopathology of malignant cells was unclear. Atrophic changes such as pyknosis and vacuolization were reported in some studies, and no significant effect of finasteride was observed in others. Finasteride was not shown to have an effect on prostate cancer cell proliferation. Its effect on apoptosis and angiogenesis in prostate cancer has not been studied. In contrast, the dual 5aR inhibitor dutasteride has been shown to cause significant regressive changes in established prostate cancer, although some of the effect is time L.N. Thomas(*) Department of Biochemistry and Molecular Biology, Dalhousie University, 9D Sir Charles Tupper Building, Halifx, NS, Canada B3H 1X5, E-mail:
[email protected]
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dependent. Short-term treatment (6–10 weeks) caused a 40% reduction in tumor volume and resulted in significant increases in apoptosis, number of atrophic glands, and stromal:epithelial ratio. It was also associated with a nonsignificant decrease in microvessel density. After longer treatment (4 months), the regressive changes induced by dutasteride waned, although reductions in tumor volume continued to be evident. In summary, the results from the PCPT demonstrated that inhibition of 5aR may prevent or delay prostate cancer in some patients. From preprostatectomy trials of dutasteride, it is also clear that suppression of DHT formation with a 5aR inhibitor may cause regression of prostatic carcinomas. The fact that regressive changes were not observed to the same extent with finasteride may be for two reasons. First, since finasteride is a 5aR2 selective inhibitor, DHT formation can continue due to the action of 5aR1. Second, the timing of the observations in many of the studies with finasteride may not have been optimal.
1 Introduction In the prostate, normal growth, development, and secretory activity are androgen dependent. Intracrine conversion of circulating testosterone to the more potent androgen dihydrotestosterone (DHT) by the enzyme 5a-reductase (5aR) is a key step in both normal prostate function and development of prostate cancer (George et al. 1989; Imperato-McGinley et al. 1992; Petrow 1986). DHT has approximately a twofold higher intrinsic potency for stimulating proliferation and secretory function in the prostate (Wright et al. 1999), and it also binds the androgen receptor with approximately a tenfold greater affinity, providing a more stable receptorligand complex (Deslypere et al. 1992). Deficiency of 5aR results in male pseudohermaphroditism (Andersson et al. 1991). There are two well-characterized 5aR isoenzymes, designated type 1 (5aR1) and type 2 (5aR2). They are encoded by two distinct genes, SRD5A1 and SRD5A2, which are located on separate chromosomes, and they have differing kinetic properties (Jenkins et al. 1991, 1992; Russell and Wilson 1994; Span et al. 1996). In the normal prostate, 5aR2 is the predominant isoenzyme. It is present at high levels and has a higher binding affinity for testosterone than 5aR1 (Km value 11.8 vs. 1,995 nM) (Span et al. 1996). The 5aR1 isoenzyme was first detected in nonreproductive tissues such as the liver and skin (Normington and Russell 1992; Thigpen et al. 1993). However, studies now have shown that 5aR1 is present in the prostate, and its levels are elevated in prostate cancer (Bonkhoff et al. 1996; Iehle et al. 1999; Span et al. 1999; Thomas et al. 2003). Recent reports have identified a third 5aR isoenzyme, but the clinical significance of this isoenzyme has yet to be elucidated (Titus et al. 2007; Uemura et al. 2007). This chapter will focus on the relationship between 5aR and prostate cancer, with particular emphasis on the effect of inhibition of 5aR on markers of regression and proliferation in prostate cancer.
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2 5a-Reductase Inhibitors and BPH Because DHT formation is crucial for normal and abnormal prostate growth, 5aR inhibitors were developed for treatment of prostate disease. Finasteride was the first commercially available 5aR inhibitor. It targeted 5aR2, since it is the predominant isoenzyme in the benign prostate. It has been widely used for treatment of benign prostatic hyperplasia (BPH), because it suppresses intraprostatic DHT formation by 68–86% (Span et al. 1999; McConnell et al. 1992). This results in decreased prostate volume, improved symptoms and urinary flow rates, and a reduction in the risk of acute urinary retention or BPH-related surgery (McConnell et al. 1998). Prostate volume is decreased by 20–30% through a combination of increased atrophy and apoptosis (Marks et al. 1999; Montironi et al. 1996a; Rittmaster et al. 1996). Finasteride has been shown to cause a 55% decrease in benign epithelial cell content after 6 months of treatment (Marks et al. 1997). Despite its proven benefit, finasteride provides only partial suppression of serum and intraprostatic DHT (Span et al. 1999; Cote et al. 1998; McConnell et al. 1992). Residual DHT formation is likely due to the action of 5aR1. The dual 5aR inhibitor dutasteride provides greater and more consistent suppression of serum DHT than does finasteride (95 and 70%, respectively) (Clark et al. 2004). Like finasteride, it has been shown to be effective in relieving symptoms of BPH (Roehrborn et al. 2002). Studies of the efficacy of 5aR inhibitors in the treatment of prostate cancer are ongoing. The Prostate Cancer Prevention Trial (PCPT) has shown that finasteride can be a useful therapy, which may prevent development of prostate cancer and/or prevent progression of subclinical malignancies to clinically significant prostate cancer (Thompson et al. 2003). Further studies have examined the effect of 5aR inhibition on regression of existing prostate cancer.
3 Androgen-Deprivation Therapy for Treatment of Prostate Cancer Currently, androgen-deprivation therapy (ADT) is widely used as a treatment for prostate cancer. It is accomplished through the use of GnRH agonists/antagonists, alone or in combination with antiandrogens, which bind the androgen receptor. ADT results in characteristic changes in the histopathology of benign, PIN, and prostate cancer tissues. These changes include a reduction in tumor volume, as well as shrunken nucleoli, pyknosis, vacuolated cytoplasm, and smaller tumor glands (Bostwick et al. 2004; Civantos et al. 1996). In addition to these changes, ADT also causes increased apoptosis (Rittmaster et al. 1999), decreased proliferation (Armas et al. 1994), and decreased microvessel density (Marshall and Narayan 1993; Mazzucchelli et al. 2000). Figure 1 shows increased staining for two markers of
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Fig. 1 Apoptosis in prostate adenocarcinoma after ADT. Panels a and b show representative TUNEL staining in untreated prostate cancer (a) and prostate cancer treated by ADT (b). Panel c shows representative tTG staining in untreated prostate cancer, while panel d shows prostate cancer treated by ADT. (Reproduced from Rittmaster et al. 1999, with permission from Elsevier)
apoptosis, TUNEL and tissue transglutaminase (tTG), in prostate cancer tissues after 3 months of neoadjuvant ADT. Although ADT initially is very effective in causing regression of prostate cancer, there are problems associated with this treatment. The first is the high risk of recurrence, since most patients eventually become refractory to androgen withdrawal. The second problem is side effects, which include impotence, hot flashes, depression, osteoporosis, weakness, and fatigue. For these reasons, it is important to discover new treatment options.
4 5a-Reductase Inhibition and Prostate Cancer Studies of nude mice bearing prostate cancer cell xenografts have demonstrated that DHT has a stronger stimulatory effect on prostate tumor growth than testosterone (Xu et al. 2006). A number of studies have shown that inhibition of 5aR can decrease proliferation of prostate cancer cells in vitro (Bologna et al. 1995) or reduce the growth of prostate cancer xenografts in nude mice (Lamb et al. 1992).
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Festuccia et al. (2005) have shown that both the 5aR2 inhibitor finasteride and the 5aR1 inhibitor MK386 decrease proliferation in primary cultures of BPH, PIN, and prostate cancer cells. More recently, these authors have shown that the dual 5aR inhibitor dutasteride decreases proliferation and increases apoptosis to a greater extent and in a higher proportion of prostate cancer cells in primary cultures than either finasteride or MK386. Dutasteride reduced growth in 78% of cases compared to 39% of cases for both finasteride and MK386 (Festuccia et al. 2008). Inhibitors of 5aR also have been shown to reduce the growth of the Dunning R3327 tumor in rats (Lamb et al. 1992; Zaccheo et al. 1998) and to inhibit growth of PC-82 xenografts in mice (Lamb et al. 1992). Finally, finasteride has been shown to prevent progression of prostate cancer from microscopic to macroscopic tumors in rats (Tsukamoto et al. 1998). It has been reported that 5aR activity levels may be correlated with risk of prostate cancer. Asian men, who have a lower risk of developing prostatic malignancies, have decreased levels of androstanediol glucuronide compared to Caucasians, suggesting lower 5aR activity (Lookingbill et al. 1991; Ross et al. 1992). In addition, genetic variants of 5aR with greater intrinsic activity are associated with prostate cancer progression (Makridakis et al. 2004). However, in studies examining the effect of 5aR inhibitors on prostate cancer prevention or treatment, results have been mixed. In one of the earliest studies of 120 men with detectable PSA levels (0.6–10 ng/ml) but no evidence of bone metastases following radical prostatectomy for localized prostate cancer, finasteride treatment did not significantly affect tumor recurrence, as defined by a positive bone scan or biopsy. However, treatment was associated with a delay of 14 months in the time to an increase in serum PSA (Andriole et al. 1995). Similarly, in a study by Cote et al. (1998) of 52 men with elevated PSA (>4) and a negative biopsy for prostate cancer, who received finasteride (n = 27) or no medication (n = 25) for 12 months prior to being rebiopsied, finasteride was found to have no effect on cancer incidence. However, this was a small study that was not appropriately powered to assess this endpoint. In contrast, the Prostate Cancer Prevention Trial (PCPT) was powered to assess prostate cancer incidence and, in this study of 18,882 men with serum PSA values 3 ng/ml, treatment with finasteride was associated with a 24.8% reduction in prostate cancer detection rates. This result has provided significant evidence of a role for 5aR inhibition in the prevention or treatment of prostate cancer (Thompson et al. 2003).
5 Markers of Tumor Regression 5.1
TUNEL
Apoptosis is an important component of prostate regression following ADT. Castration-induced apoptosis in benign human prostate xenografts has been
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shown to account for the loss of nearly 90% of epithelial cells, even though the apoptotic index was relatively low (Staack et al. 2003). In men with prostate cancer, neoadjuvant ADT increased apoptosis in prostate cancer tissue (Rittmaster et al. 1999; Matsushima et al. 1999). However, sensitivity and specificity of apoptotic measurements can be problematic, since marker expression is dependent upon the stage of apoptosis. TUNEL staining, a widely accepted method for detecting apoptosis, identifies fragmentation of DNA, which is a hallmark of apoptosis (Gavrieli et al. 1992). However, TUNEL staining is evanescent, and many apoptotic cells are not labeled at any given time.
5.2
Tissue Transglutaminase
Tissue transglutaminase is an alternate marker for apoptosis (Piacentini et al. 1991), which persists longer than TUNEL, thereby providing more information regarding cumulative levels of apoptosis. Although it has not been as widely used as TUNEL, there is sufficient evidence to conclude that it is a useful marker of apoptosis. In a study in which dexamethasone, which induces apoptosis in the thymus, was administered to tTG knock-out mice and wild-type mice, cell viability was decreased to a greater extent in the knock-out mice, even though there was no difference in the rate of apoptosis. This suggests that there was increased clearing of apoptotic cells in the knock-out mice, which in turn suggests that tTG may be involved in stabilizing apoptotic cells before clearance (Nanda et al. 2001). Tissue transglutaminase may have more than one function in the cascade of events during apoptosis. Cells that overexpress tTG have been shown to have ultrastructural changes associated with hyperpolarization of the mitochondria and increased production of reactive oxygen species during staurosporine-induced apoptosis. Tissue transglutaminase may have a role in sensitizing cells for apoptosis through modification of mitochondrial homeostasis (Piacentini et al. 2002). However, tTG is not necessarily specific for apoptosis, since it is involved in other cellular processes, such as signal transduction, cell adhesion, and wound healing. Due to the limitations of these methods for evaluating apoptosis, it is prudent to assess both.
5.3
Microvessel Density and Vascular Endothelial Growth Factor
Angiogenesis is a potent prognostic indicator for prostate cancer patients, since it is a crucial factor for the growth of solid tumors beyond 2 cm2 (Margolin 2002). Since studies have shown that DHT may be necessary for induction of angiogenesis (Pan et al. 2003), microvessel density (MVD) or vascular endothelial growth factor (VEGF) may be useful markers of tumor regression. MVD is progressively
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increased in BPH, PIN, and prostate cancer, compared to normal prostate tissue (Bigler et al. 1993; Brawer et al. 1994; Montironi et al. 1996b), and it is increased in high-grade prostate cancer compared to low-grade prostate cancer (Bettencourt et al. 1998; Mydlo et al. 1998). Staining for vascular endothelial growth factor (VEGF) is decreased in benign, PIN, and prostate cancer tissues after 3 months of androgen ablation (Mazzucchelli et al. 2000).
5.4
Histopathology
Since atrophy is also a component of regression (Rittmaster et al. 1995, 1996), histopathological evaluation of tissues for characteristic changes of atrophy is valuable. Such an evaluation should include morphometrics measurements of epithelial cell height, duct width, and nuclear area, as well as assessment of mean tumor volume, stroma:gland ratio, and Gleason score.
6 Effect of 5a-Reductase Inhibitors on Prostate Cancer Histology 6.1
Finasteride
The earliest observations of the effect of 5aR on prostate cancer histopathology produced conflicting results. The studies were conducted in men who were treated with finasteride for BPH and subsequently developed cancer. In a study of cancer containing biopsy specimens from the Proscar Long-Term Efficacy and Safety Study (PLESS), which assessed the efficacy of finasteride in the treatment of benign prostatic hyperplasia (BPH), finasteride (4 years) was not shown to have an effect on the histopathological features of prostate cancer tissue compared to placebo (Yang et al. 1999). Likewise, in the study by Cote et al. (1998) described earlier, the authors reported no change in PIN and no change in the proliferation index, as assessed by proliferating cell nuclear antigen (PCNA) staining after treatment with finasteride for 1 year. Finally, Carver et al. evaluated prostate cancer tissue from men who received finasteride for at least 6 months in order to treat lower urinary tract problems and subsequently developed prostate cancer, which was treated by radical prostatectomy. Their aim was to determine whether finasteride caused sufficient change in tumor histopathology to prevent accurate Gleason-grade assignments, and they concluded that it did not (Carver et al. 2005). In contrast, Civantos et al. examined radical prostatectomy specimens from five men who had received finasteride for 3–24 months prior to being diagnosed with prostate cancer. They reported that finasteride induced similar, but less pronounced histopathological changes to androgen ablation. These changes included atrophy, vacuolization,
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pyknosis, and smaller tumor glands. However, these changes were less common and milder with finasteride compared to androgen ablation (Civantos et al. 1997). Finasteride also has been used in preprostatectomy studies of men with prostate cancer, designed to assess the impact of 5aR inhibition on markers of regression in prostatic adenocarcinoma. In one such study, the effect of at least 6 months of finasteride treatment on prostate cancer morphology and clinical outcome was compared to 3 months’ androgen ablation with an LHRH agonist. Fifty-six patients received finasteride, 56 patients received surgery only, and 44 patients received an LHRH agonist. After radical prostatectomy, the prostate tissues were scored in a blinded manner on a scale of 0–2, in which 0 was assigned when there was no apparent effect, 1 was assigned when the tissue looked suspicious for hormonal effect, and 2 was assigned when the histopathological features were highly suggestive of hormonal treatment. The morphologic changes in the finasteride and surgery-only groups were comparable (mean scores of 0.5 and 0.4, respectively), whereas the mean score for the LHRHa group was fourfold higher (1.6). Likewise, clinical outcomes were similar for finasteride and control groups. (Figure 2 shows the distribution of scores in control, finasteride, and LHRHa groups.) (Rubin et al. 2005).
6.2
Dutasteride
Because there are two well-characterized 5aR isoenzymes, and finasteride at clinical doses inhibits only 5aR2, it is possible that dual inhibition of 5aR may
Fig. 2 Distribution of treatment score in biopsy and prostatectomy specimens from control, finasteride, and LHRHa treatment groups. No hormonal effect was evident in the biopsy specimens. However, in prostatectomy specimens, approximately 80% of LHRH patients showed suspicious or strong treatment effects, compared to 20% of control patients and 26% of finasteride patients. (Reproduced from Rubin et al. 2005, with permission from Elsevier)
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have greater efficacy in treatment of early stage prostate cancer. There is increasing evidence that 5aR1 expression is greater in prostate cancer compared with benign tissue at both the messenger RNA (mRNA) level (Iehle et al. 1999; Thomas et al. 2005) and at the protein level (Bonkhoff et al. 1996; Thomas et al. 2003), whereas 5aR2 expression and activity are decreased (Thomas et al. 2005; Bjelfman et al. 1997; Luo et al. 2003; Soderstrom et al. 2001; Titus et al. 2005). The potential role of 5aR1 in prostate pathology is supported clinically by evidence that dual inhibition with dutasteride provides greater and more complete suppression of serum DHT levels than finasteride in men with BPH (Clark et al. 2004). Furthermore, dutasteride inhibits growth of prostate cancer cells in vitro (Lazier et al. 2004) and growth of the Dunning R-3327H tumor in rats (Xu et al. 2006) to a greater extent than finasteride. Preprostatectomy trials to assess the effect of dutasteride on markers of regression in prostate cancer were undertaken. Two studies were conducted, both of which will be discussed in depth. In the first, dutasteride was administered for 6–10 weeks prior to prostatectomy, and in the second, dutasteride was administered for 4 months prior to prostatectomy. In both studies, the decision to undergo a prostatectomy was made independent of participation in the study.
6.3
ARIA2003 (Preradical Prostatectomy Study with Dutasteride)
ARIA2003 was a study designed to determine whether dutasteride causes regressive changes in prostate cancer in vivo. In a double-blind, multicenter trial, 46 men with clinically staged T1 or T2 prostate cancer were randomized to receive 5 mg/ day of dutasteride or placebo for 6–10 weeks prior to radical prostatectomy. Resected tissues were analyzed to determine the effect of dutasteride on intraprostatic androgen levels, and indices of apoptosis and MVD in malignant tissue, as well as degree of atrophy in benign tissue (Andriole et al. 2004a). Surgical margins, extraprostatic extension, stroma:gland ratio, percent atrophic epithelium, and PIN and tumor volumes were also evaluated (Iczkowski et al. 2005). Serum DHT decreased by 96.5% from 296 to 10.3 pg/ml after dutasteride treatment. Treatment with dutasteride also caused a 97.0% reduction in intraprostatic DHT – from 6,178.8 pg/g in the dutasteride group to 177.2 pg/g in the placebo group. There was no significant change in serum testosterone levels, but intraprostatic testosterone levels increased from 124.5 pg/g in the placebo group to 2,502 pg/g in the dutasteride group. The combined T and DHT concentrations in the prostate were 60% lower in the dutasteride group compared to placebo. TUNEL staining (number of positive cells/mm2 of tumor) showed a nonsignificant increase of 64% from 32.5 7.9 in the placebo group to 53.3 11.9 in the dutasteride group. Staining for tTG, which was measured using image analysis software, was doubled in the dutasteride group compared to placebo. When the men were grouped according to duration of treatment (<45 days or 45 days), tTG staining was quadrupled in the longer-term treatment group (P = 0.036).
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Fig. 3 Apoptosis in prostate adenocarcinoma after treatment with dutasteride. Panels a and b show representative TUNEL staining in prostate cancer tissues from patients treated with placebo and dutasteride, respectively. Panels c and d show representative tTG staining in prostate cancer tissues from patients treated with placebo and dutasteride, respectively. (Reproduced from Andriole et al. 2004a, with permission from Elsevier)
Figure 3 shows representative staining for TUNEL and tTG in placebo and dutasteride-treated subjects. MVD was assessed by staining for the vascular endothelial antigen CD34. While there was no statistical difference overall between the two groups, MVD was decreased by 45% from 6.6 0.9 in the placebo group to 3.6 0.8 in men treated with dutasteride for 45 days. In addition there was a trend toward reduced MVD over time (Fig. 4). Histopathological assessment of cancerous tissue showed that tumor volume was decreased by 40%, PIN volume was decreased by 50%, stroma:gland ratio was doubled, and the percent of atrophic epithelium was doubled. The treatment alteration score, which was determined by a pathologist based on cytological and architectural changes in the cancer tissue, was also doubled in the dutasteride group compared to placebo (Table 1). The number of patients with positive surgical margins was not statistically different between the two groups, but the mean extent of margin involvement
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CD34 (% of tumor area staining positively)
30
25
20
15
10
5
0 30
35
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45
50
55
60
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70
75
80
Duration of Treatment Fig. 4 Trend toward reduced MVD over time in prostate cancer after finasteride treatment. This graph shows the negative correlation between duration of dutasteride treatment (up to 10 weeks) and MVD. (Reproduced from Andriole et al. 2004a, with permission from Elsevier) Table 1 Histopathological features of prostate cancer after treatment with dutasteride Placebo Dutasteride No. of subjects 19 18 Mean margin involvement 11.7 4.7 4.2 0.9 Mean extraprostatic extension 15.4 7.9 4.3 1.8 Mean PIN volume 0.67 0.16 0.31 0.08 (P = 0.052) Tumor volume:prostate weight ratio 0.09 0.02 0.06 0.02 Gleason score 6.9 0.75 6.9 0.9 % of specimen involved by cancer 24.2 12.4 15.2 12.6 (P = 0.025) Treatment alteration score 1.6 2.2 3.2 2.5 (P = 0.055) % of atrophic epithelium 26.7 31.3 52.9 38.9 (P = 0.041) Stroma:gland ratio 0.26 0.15 0.52 0.56 (P = 0.046)
was decreased from 11.7 4.7 in the placebo group to 4.2 0.9 in the dutasteride group. Similarly, the mean extent of extraprostatic extension was decreased from 15.4 7.9 to 4.3 1.8 in the placebo and dutasteride groups, respectively (Table 1).
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Benign epithelial cell width decreased by 18% from 43.0 1.4 in the placebo group to 35.4 1.0 in the dutasteride group (P < 0.0001). The decrease was evident in both the transition zone (where BPH usually develops) and the peripheral zone (where the majority of prostate cancers arise). In this study, dutasteride significantly increased apoptosis in men treated for 45 days or more and resulted in a trend toward reduced microvessel density. It also caused significant histopathological changes in both benign and cancer tissues, which included increased atrophy, increased stroma:gland ratio, and decreased tumor volume. Clinically, there was a nonsignificant decrease in surgical margin involvement and mean extent of extraprostatic extension, although no reduction in incidence, in men taking dutasteride. These findings, in combination with the almost complete suppression of intraprostatic DHT by dutasteride, support a role for this compound in prevention or early stage treatment of prostate cancer.
6.4
ARI40010 (4-Month Preradical Prostatectomy Study with Dutasteride)
The follow-up study to ARIA2003 was ARI40010. In this study, 81 men with clinically localized prostate cancer were randomized to receive no treatment or dutasteride at a dose of 0.5 or 3.5 mg/day for 4 months prior to radical prostatectomy. Resected tissues were analyzed for intraprostatic androgen concentrations; indices of apoptosis and proliferation; MVD and histopathological features such as prostate volume, tumor volume, atrophy, Gleason grade, stroma:epithelial ratio, and nuclear/architectural changes (treatment alteration score). Serum PSA measurements were also made (Gleave et al. 2006). The results demonstrated that intraprostatic DHT was significantly reduced by 93.1% in the 0.5-mg dutasteride group and 98.8% in the 3.5-mg dutasteride group compared to the surgery-only group. Intraprostatic testosterone levels were significantly higher in both dutasteride groups compared to the surgery-alone group. In benign tissue, there was no significant effect of dutasteride on apoptosis, MVD, or stroma:epithelial ratio. The proportion of atrophic epithelium was higher in dutasteride-treated subjects, in both the transition and the peripheral zones. In malignant tissues, median tumor volume was decreased by 41 and 46% in the 0.5- and 3.5-mg dutasteride groups, respectively. Unexpectedly, TUNEL staining was decreased in the dutasteride groups, but tTG staining was nonsignificantly increased by a factor of 3.6 in the dutasteride-treated subjects as a whole. The trend was similar in both dutasteride groups. The results for MVD and proliferation measurements were opposite to the expected results. MVD was greater in the dutasteride groups compared to the surgery-alone group. The increase reached statistical significance in the 0.5-mg dutasteride group (P = 0.031). Similarly, Ki67 staining for cell proliferation was increased in both dutasteride groups, and the increase was significant in the 0.5-mg group compared to the surgery-alone group (P = 0.038).
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Table 2 Time-dependent changes in markers of tumor regression after treatment with dutasteride Fold change <7 weeks 7 weeks 16 weeks TUNEL 1.64 1.65 0.51 tTG 1.92 3.75 3.63 MVD 1.0 0.55 1.22
Treatment alteration scores were nonsignificantly greater in tissues from treated men. Mean serum PSA concentrations were 47.1 19.2% lower in men receiving 0.5 mg/day dutasteride and 58.0 17.9% lower in men receiving 3.5 mg/day dutasteride, compared to untreated men. Dutasteride treatment resulted in almost total suppression of intraprostatic DHT formation and a decrease in tumor volume. Serum PSA values were reduced to a similar extent as in men with BPH treated with 0.5 mg dutasteride for 6 months or longer (Andriole et al. 2006), suggesting that dutasteride suppressed PSA from both benign and cancer tissue. Taken together, these data indicate that 4 months of treatment with dutasteride was effective in causing regression of prostate cancer. Cumulative levels of apoptosis, as assessed by tTG staining, were nonsignificantly increased, and there was a trend toward decreased levels of vascular endothelial growth factor (VEGF), an important growth factor for promoting angiogenesis. Proliferation and MVD were increased after dutasteride treatment and the rate of apoptosis, as assessed by TUNEL, was decreased. Histopathological features such as proportion of atrophy and treatment alteration score were not significantly altered by dutasteride treatment. The apparent disparity in the results of ARIA2003 and ARI40010 may be because the remodeling induced by treatment with dutasteride occurs early after onset of treatment and levels off thereafter. By 4 months, the remodeling process may be near complete, and the cells that are left are demonstrating a reaction to androgen deprivation. This theory is supported by studies of ADT showing that apoptosis in prostate cancer tissues, as visualized by TUNEL, is increased in the first few days after initiation of treatment, and then returns to baseline (Staack et al. 2003; Ohlson et al. 2005). This phenomenon has also been shown to occur in benign prostate tissue after treatment with finasteride. In men treated for 6–18 days, mean TUNEL staining (counts/mm2) was increased from 0.4 0.2 in the placebo group to 2.8 0.9 (P = 0.0026). After treatment for 23–73 days, TUNEL staining was 1.7 0.5 (P = 0.10) and returned to near baseline levels (0.7 0.5) after 3 months of treatment. Staining for tTG, which has a longer duration of staining than TUNEL, peaked in men treated for 23–73 days and returned to baseline thereafter (Rittmaster et al. 1996). A similar time-dependent effect has been documented for Ki67 staining. A decrease in proliferation for a few days is followed by an increase to levels greater than baseline (Ohlson et al. 2005). The same may be true for MVD, since a trend toward a decrease in MVD was observed in ARIA2003. This is supported by the observation of a trend toward a decrease in VEGF in the dutasteride group in ARI40010. Alternately, treatment with a 5aR inhibitor may have a neutral or slight
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effect on MVD, which is offset by the reductions in prostate and tumor volumes caused by inhibition of 5aR. The changes in markers of tumor regression over time are outlined in Table 2. It is clear from these studies that timing is crucial in any assessment of the effect of traditional ADT or 5aR inhibition on regression of prostate cancer. The likely explanation for the time dependence of regressive changes in growth characteristics is that androgen-sensitive cells respond early to treatment through increases in apoptosis and decreases in proliferation and angiogenesis. The treatment effect subsequently may wane when cells resistant to androgen deprivation emerge, as an adaptive or selective mechanism in response to low androgen levels.
7 Effect of 5a-Reductase Inhibitors on Gleason Scores Gleason scoring has been used for decades as a prognostic indicator in prostate cancer. In the PCPT, although finasteride reduced the overall incidence of prostate cancer, there were more Gleason 7–10 (high grade) tumors in the finasteride arm. Current evidence suggests that this was due partly to an ascertainment bias and partly to reduced efficacy of finasteride in shrinking high-grade tumors. However, some investigators have hypothesized that either finasteride-induced androgen deprivation made tumors look higher grade or that finasteride actually induced development of high-grade tumors. In the 4-month preradical prostatectomy study (ARI40010), the rate of Gleason upgrading between the initial biopsy and the radical prostatectomy was lower in the two dutasteride arms than in the control arm (Fig. 5). This result suggests that dutasteride does not lead to Gleason upgrading, either through creating a pathological artifact or actual induction of more aggressive tumors.
8 Conclusion Taken cumulatively, the data from these studies provide evidence that apoptosis and atrophy are important processes during androgen-deprivation induced regression of prostate cancer. Changes in proliferation and MVD may also be factors, but likely have a smaller impact on regression. The consensus of evidence indicates that finasteride treatment causes changes in prostate cancer histopathology that are similar to, but less pronounced than ADT. That such changes were not observed in all studies is likely because the timing of the observations was not optimal in some of the studies. It is also likely that changes could not be detected in the studies of biopsy specimens due to the incomplete and heterogeneous pattern of the changes induced by finasteride. Finasteride has been shown to have little impact
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Fig. 5 Rate of Gleason upgrading between initial biopsy and radical prostatectomy. This graph shows the percentage of prostate cancers with a decreased, same, or increased Gleason score between biopsy and radical prostatectomy. (Permission obtained)
on cancer cell proliferation in vivo, and its effect on apoptosis has not been studied in prostate cancer. Although finasteride has not been proven to have a significant impact on regression of clinically apparent prostate cancer, the PCPT has demonstrated that finasteride can reduce prostate cancer detection rates through prevention and/or through treatment of early stage, subclinical carcinomas. Since detection rates diverged early in the study, it seems likely that the latter possibility can account for at least part of the reduction in prostate cancer prevalence. Dutasteride has been shown to have an effect on markers of regression in prostate cancer, although some of the effect is time dependent. Treatment for 6–10 weeks is associated with an increase in apoptosis, a nonsignificant decrease in MVD, an increase in the proportion of atrophic tumor glands, an increase in stroma:gland ratio, and reductions in prostate and tumor volumes. Despite these results, the role of dutasteride in the treatment of prostate cancer has not yet been defined, as some of the effects were diminished after a longer duration of treatment. With regard to prevention or treatment of subclinical prostate cancer, it is likely that dutasteride may be even more effective than finasteride. The results from the two preprostatectomy trials, in combination with increased expression of 5aR1 in prostate cancer and the almost complete suppression of intraprostatic DHT formation by dutasteride, provide support for this hypothesis. The 4-year Reduction by Dutasteride of Prostate Cancer Events (REDUCE) trial is currently underway to test this hypothesis (Andriole et al. 2004b).
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5a-Reductase Isozymes in Castration-Recurrent Prostate Cancer Mark A. Titus and James L. Mohler
Abstract Intracrine biosynthesis of dihydrotestosterone is the final step in anabolic androgen metabolism in the prostate. The NADPH-dependent steroid 5a-reductase isozymes irreversibly catalyze 5a-reduction of intracellular testosterone to dihydrotestosterone. In castration-recurrent prostate cancer mean mRNA levels suggest relative gene expression gradients of 5a-reductase-3 > 5a-reductase-1 5a-reductase-2. Furthermore, sufficient levels of testosterone and dihydrotestosterone were observed in castration-recurrent prostate cancer to activate androgen receptor signaling pathway. In intact and recurrent CWR22 human xenografts, persistent dihydrotestosterone formation was observed after pretreatment with dutasteride. Improved inhibitors that target 5a-reductase-1, 2 and 3 isozymes may stop intraprostatic DHT biosynthesis and prevent the development of clinical prostate cancer or its progression.
1 Introduction The first androgen, androsterone, a 5a-reduced 19-carbon steroid, was isolated from human male urine in 1929 by Nobel Laureate, Adolf Butenandt. Androsterone was considered the active human male hormone until 1935 when testosterone (T) was purified from several tons of bull testes (Fieser and Fieser 1959). This discovery suggested that the 5a-reduced androgens, androsterone, dihydrotestosterone (DHT), and 5a-androstane-3a,17b-diol (Fig. 1) were inactive catabolic metabolites of T (Fieser and Fieser 1959). 5a-Reductase was discovered by incubating desoxycorticosterone with rat liver slices and characterizing the major metabolite, 5a-pregnan-3a,21-diol-3,20-dione (Schneider and Horstmann 1951). It was proposed that 5a-reduction directed steroids toward catabolic 3-ketone reduction, phase II sulfation and glucuronidation, and excretion. Further studies showed that
M.A. Titus(*) Department of Urologic Oncology, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA, E-mail:
[email protected]
D. Tindall and J. Mohler (eds.), Androgen Action in Prostate Cancer, DOI: 10.1007/978-0-387-69179-4_8, # Springer Science + Business Media, LLC 2009
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Fig. 1 Testosterone is irreversibly metabolized to the more potent androgen, dihydrotestosterone, and dihydrotestosterone catabolites, 3a-androstanediol and androsterone. 5a-Reductase isozymes (SRD5A) and aldo–keto reductase isozymes (AKR1C)
5a-reductase catalyzed the irreversible reduction of C19 and C21 steroids (epitestosterone, androstenedione, progesterone, 17a-hydroxy-progesterone, 20a-hydroxy-progesterone), which suggested steroid 5a-reductase might be a regulatory enzyme (Tomkins 1957). The enzymatic reduction is NADPH dependent and catalyzes trans addition of a hydride ion at carbon-5 and a proton from water at carbon-4 (Wilton and Ringold 1968). Reversal of this reaction was not observed using mammalian enzymes due to the high energy barrier for hydride ion abstraction from carbon-5 (Wilton and Ringold 1968). Bjorkhem (1969) showed that the pro-S-hydrogen from NADPH is delivered stereospecifically to the less hindered alpha face of 7a-hydroxycholest-4-en-3-one in rat liver microsomes. The same mechanism was proven for the reduction of T to DHT using rat seminal vesicles (Suzuki and Tamaoki 1974). The importance of T to DHT metabolism was recognized first when rats were injected with radiolabeled T, and it was found that DHT, not T, was the predominant androgen isolated from prostate nuclei (Anderson and Liao 1968; Bruchovsky and Wilson 1968). The detection of DHT in the nucleus within 5–10 min of T injection led to complete revision of classical androgen action and promoted DHT to the status of primary AR ligand in prostate physiology. The 5a-reduction of T to the more potent androgen, DHT, suggested that T, the major circulating androgen, acts as a prohormone in androgen prostate cells, which express steroid 5a-reductase enzymes. Furthermore, Bruchovsky et al. (1971) showed that androgen target tissues convert 3a-androstanediol and androsterone back to DHT. 5a-reduction of the D4–5 double bond in T creates the planar and less polar DHT with a trans A–B ring system and decreased 3-keto enolization. Structure–function analysis by Liao and Fang (1969) concluded that the more planar DHT fit more tightly than T to the AR-ligand binding domain. Thus, most of the enhanced affinity is due to the decreased dissociation rate of DHT from the AR–DHT complex (Grino et al. 1990).
2 5a-Reductase Isozyme Molecular Genetics The differential role of T and DHT in androgen signaling was determined initially during clinical studies of androgen insensitivity, an X-linked recessive genetic disorder. Simpson et al. (1971) described a family with three affected brothers
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who had 46 XY karyotype ambiguous genitalia as children and masculinized at puberty. However, inheritance of this reproductive disorder is autosomal recessive, and T and estrogen levels are normal in affected children. Walsh et al. (1974) described these patients as pseudohermaphrodites (type 2) and suggested that DHT biosynthesis was defective. Further clinical evidence was documented when Imperato-McGinley et al. (1974) studied 24 male pseudohermaphrodites in the Dominican Republic. The affected males present with ambiguous genitalia at birth, attain normal male secondary characteristics and spermatogenesis at puberty but suffer decreased fertility. These men have decreased acne, facial hair, impaired conversion of T to DHT, and small seminal vesicles and prostates. The primary sequence of human 5a-reductase (SRD5A, EC 1.3.99.5) isozymes was determined using cDNA libraries constructed from surgical specimens of prostate tissue. 5a-Reductase 1 was the first isozyme to be cloned and characterized biochemically (Anderson and Russell 1990). The 5a-reductase 2 isozyme was cloned in 1992 and distinguished from 5a-reductase 1 using biochemical and pharmacological assays (Jenkins et al. 1992). 5a-Reductase 1 and 2 are 259 and 254 amino acids in length with unique enzyme activity pH optima of 8.0 and 5.0, respectively. The predicted molecular weight of 5a-reductase is 28,000–29,000 Da. The sequence identity between the two proteins is 47%, and over one-third of amino acids are classified as hydrophobic (Anderson et al. 1991). 5a-Reductase functional domains were studied by identifying mutations in the 5a-reductase 2 gene (Anderson et al. 1991; Thigpen et al. 1992a; Thigpen et al. 1992b). One set of mutations affected the ability of the enzyme to bind the substrate T, and the other set decreased affinity for the cofactor, NADPH (Russell et al. 1994). Amino acid substitutions near the N or C terminal ends of 5a-reductase 1 and 2 increase the Km for T binding. To confirm substrate-binding location, a chimeric rat N-terminus/ human C-terminus steroid 5a-reductase 1 protein demonstrated rat-like finasteride sensitivity. In contrast, amino acid substitutions in the carboxylic half of the protein reduced NADPH cofactor binding (Thigpen, Davis, Gautier et al., 1992). In addition, Bhattacharyya et al. (1995) isolated and sequenced an NADPH photoaffinity-labeled carboxy terminal peptide from rat steroid 5a-reductase 1. The sequence consisted of 11 amino acids and corresponded to carboxy terminal residues 170–180.
3 5a-Reductase Isozymes in Prostate Cancer 5a-reductase 1 and 2 are located in androgen-responsive tissues, such as prostate, and exhibit distinct biochemical parameters (Russell and Wilson 1994). Both 5areductase 1 and 2 are active in androgen-stimulated benign prostate and are expressed at higher levels in high grade compared to low-grade clinically localized prostate cancer (CaP) (Thomas et al. 2008). However, compared to 5a-reductase 2, 5a-reductase 1 protein (Bonkhoff et al. 1996; Titus et al. 2005a) and gene expression (Iehle et al. 1999) increase in CaP versus androgen-stimulated benign prostate. Two inhibitors have been used in CaP chemoprevention, finasteride (in the PCPT clinical trial) (Thompson et al. 2003) and dutasteride (in the REDUCE
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clinical trial) (Andriole et al. 2004). Finasteride selectively inhibits 5a-reductase 2 (IC50 = 69 nM) and dutasteride inhibits both 5a-reductase 1 (IC50 = 6 nM) and 2 (IC50 = 7 nM) (Frye 2006). The PCPT study was terminated early due to a 25% reduction in CaP occurrence over a 7-year period, although the proportion of highgrade CaP cases was found to be increased. Thorpe et al. (2007) suggested the increased high-grade CaP is due to small prostate volume and biopsy sampling error. However, Cussenot et al. (2007) and Li et al. (2003) suggested that the most common 5a-reductase 2 V89L polymorphism, which decreases 5a-reductase 2 activity (Makridakis et al. 2000), is associated with more aggressive CaP risk and may explain the increase in high-grade CaP associated with finasteride use. Hsing et al. (2001) and Boger-Megiddo et al. (2008) advise caution in focusing on a single polymorphism and suggest that the 5a-reductase 2 V89L variant may be part of a set of enzymatic polymorphisms, which act together to increase CaP risk. Xu et al. (2006) hypothesize that dutasteride will reduce CaP growth only if used in combination with medical or surgical castration, because 5a-reductase inhibition will increase T levels that will activate AR and stimulate growth.
4 5a-Reductase Isozymes in Castration-Recurrent Prostate Cancer Men who fail curative therapy of clinically localized CaP or advanced disease usually receive androgen-deprivation therapy (ADT) that causes regression of androgen-dependent CaP through programmed cell death (Denmeade et al. 1996). However, ADT is palliative (Huggins, 1941) since CaP almost always recurs and causes death. A molecular role for androgen receptor in the transition from androgen-stimulated CaP to castration-recurrent CaP is supported by androgen receptor expression (Culig et al. 1998; de Vere White, 1997; Mohler et al. 2004) and expression of androgen-regulated genes (Gregory et al. 1998; Mousses et al. 2001) in castration-recurrent CaP. Many laboratories have focused upon mechanisms for androgen receptor transactivation despite castrate levels of circulating testicular androgens, such as androgen receptor amplification (Visakorpi et al. 1995), gene mutations (Culig et al. 2000), promiscuous ligand binding (Tan et al. 1997), and/or post-translational modification through peptide growth factor signaling (Arnold and Isaacs 2002; Feldman and Feldman 2001). However, Mohler et al. (2004) and Titus et al. (2005b) and others (Belanger et al. 1989; Geller et al. 1979) have demonstrated that androgen-stimulated CaP and castration-recurrent CaP exhibit tissue levels of DHT sufficient for activation of even wild-type androgen receptor. Castration-recurrent CaP must synthesize tissue T from weak circulating adrenal androgens, androstenedione and DHEA, or cholesterol and metabolize T to DHT at levels sufficient for androgen receptor activation. Limited clinical experience has not demonstrated efficacy for finasteride added to ADT for treatment of advanced CaP (Fleshner and Trachtenberg 1995) or
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bispecific 5a-reductase inhibitor (LY320236) for secondary treatment of castrationrecurrent CaP (Eisenberger et al. 2004). Additionally, dutasteride was shown to decrease serum DHT to 95% of normal levels (Clark et al. 2004) and decrease intraprostatic DHT by 94% in human androgen-stimulated benign prostate (Wurzel et al. 2007) compared to placebo controls. But DHT persists at measurable levels despite high doses of bispecific 5a-reductase inhibition. These clinical observations led to speculation about the existence of a third 5areductase enzyme when the Human Genome Project identified a sequence consistent with a 5a-reductase isozyme that mapped to chromosome 4q12 (Ota et al. 2004; Strausberg et al. 2002). 5a-Reductase 1, 2, and 3 (Mohler et al. 2006) mRNA levels were quantified in androgen-stimulated benign prostate, androgen-stimulated CaP, and castration-recurrent CaP. Mean 5a-reductase 1 mRNA levels decreased 30% in androgen-stimulated CaP and 75% in castration-recurrent CaP normalized to androgen-stimulated benign prostate. 5a-Reductase 2 gene expression was negligible in androgen-stimulated CaP and was not detected in castration-recurrent CaP. 5aReductase 3 mRNA levels decreased 30% in androgen-stimulated CaP and 45% in castration-recurrent CaP versus androgen-stimulated benign prostate. Moreover, Uemura et al. (2008) observed upregulation of 5a-reductase 3 mRNA in castrationrecurrent CaP. These results suggested a relative gene expression gradient of 5areductase 1 5a-reductase 3 5a-reductase 2 in androgen-stimulated CaP specimens. The mean mRNA levels in castration-recurrent CaP specimens suggested an expression gradient of 5a-reductase 3 > 5a-reductase 1 5a-reductase 2 (Titus et al. 2007). The relative 5a-reductase isozyme mRNA contribution suggested that (1) 5a-reductase 1 and 5a-reductase 2 mRNA correlated with 5a-reductase isozyme protein levels and enzymatic activities (Titus et al. 2005a) and (2) 5a-reductase 3 mRNA expression levels were similar in castration-recurrent CaP and androgenstimulated CaP, and maintained at higher levels than either 5a-reductase 1 or 5areductase 2 in castration-recurrent CaP. EST analysis also demonstrated 5a-reductase 3 upregulation in CaP compared to normal prostate controls (Table 1, Unigene: Hs.552, 5a-reductase 1; Hs.458345, 5a-reductase 2; 5a-reductase 3, Hs.39311). 5a-Reductase 3 ESTs were not observed in cancer of the adrenal gland or bladder. To study 5a-reductase 3 enzyme activity the androgen-dependent CWR22 human CaP xenograft model was used. Persistent DHT formation was observed using TLC analysis after dutasteride (5 nM) pretreatment of protein lysates from CWR22 intact or recurrent tumors. Incubations at pH 7.0 using 1 mM labeled (1 mCi-3H) androstenedione and CWR22 intact or CWR22 recurrent protein lysates Table 1 5a-Reductase 1, 2, and 3 (5aR) express sequence tags (EST) measured as transcripts per million (TMP) in cancer and normal tissues Cancer tissue TPM Normal tissue TPM 5aR1 5aR2 5aR3 5aR1 5aR2 5aR3 Prostate 20 0 48 Prostate 21 48 31 Skin 40 0 64 Skin 47 0 47 Adrenal 78 0 0 Adrenal 90 0 0 Bladder 0 – 0 Bladder 0 0 0
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produced DHT at 19% (intact) or 17% (recurrent), and 5a-ASD at 15% (intact) or 19% (recurrent) of controls in absence of DHT, respectively (Titus et al. 2007). 5aReductase 3 purified recombinant protein expressed in CHO cells also exhibited T conversion to DHT by a tandem mass spectrometry assay. Hence, 5a-reductase 3 can convert both androstenedione and T to 5a-reduced androgens, 5a-androstanedione and DHT, and the bispecific inhibitor, dutasteride, does not block 5a-reductase 3 activity. This unique 5a-reductase may play a significant role in the persistent DHT levels observed in castration-recurrent CaP and should be inhibited to block completely DHT biosynthesis.
5 5a-Reductase Intracellular Location in Castration-Recurrent Prostate Cancer 5a-reductase isozyme location and biochemical properties are as important as expression levels in determining intracellular CaP DHT concentration. Moore and Wilson (1972) used rat prostate organelle fractionation to localize 5a-reductase enzyme activity to the nuclear membrane, and Houston et al. showed 5a-reductase activity was located in human prostate epithelial nuclei. Savory et al. (1995) immunolocalized 5a-reductase 1 to the nuclear periphery in rat prostate, and Aumu¨ller et al. (1996) observed 5a-reductase 1 in prostate epithelial and stromal nuclei by immunohistochemistry. 5a-Reductase 2 was immunolocalized to granular endoplasmic reticulum in prostate tissue (Eicheler et al. 1994); however, 5areductase 2 mRNA and enzyme activity were decreased in CaP specimens (Luo et al. 2003; Soderstrom et al. 2001). In castration-recurrent CaP, the majority of 5areductase 1 immunostaining was perinuclear, whereas 5a-reductase 2 immunostaining was decreased and dispersed in the cytoplasm (Titus et al. 2005a). 5a-Reductase 3 immunostaining was equally distributed between nuclei and cytoplasm in castration-recurrent CaP using a monoclonal antibody. The overall 5a-reductase isozyme distribution and expression suggest that cytoplasmic AR, bound to heat shock proteins, will compete with 5a-reductase 1, 2, and 3 for intracellular T in castration-recurrent CaP cells. The AR equilibrium dissociation constant (KD) for T is 0.49 nM (Wilson and French 1976), and the apparent 5a-reductase 1 and 2 dissociation constant (Km) (Fersht 1985) is 6.3 mM and 6.3 nM (Frye 2006), respectively (Km 6¼ KD, comparison is to illustrate concept). 5a-Reductase 3 kinetic parameters are unknown but may be similar to 5a-reductase 1 and 2. Hence, increased T levels in castration-recurrent CaP (Mohler et al. 2004; Titus et al. 2005b) should interact primarily with AR and then 5a-reductase 1 and 3. AR nuclear translocation will deliver T to the nucleus and concentrate T. Bruchovsky et al. (1975) suggested androgen levels can approach 200–250 nM. More recently, Gioeli et al. (2006) proposed that AR ligand-binding cycle occurs in the nucleus as well as the cytoplasm. Therefore, concentrated intranuclear T may be converted to DHT by nuclear 5a-reductase 1 and 3, and displace T as DHT binds to AR to promote
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stable AR gene activation. Taken together, the actual nuclear DHT level may be higher than levels measured in whole tissue using ELISA or tandem mass spectrometry assays.
6 Conclusion Clinical and laboratory observations support the existence of a novel third 5a-reductase enzyme in castration-recurrent CaP. The persistent DHT levels observed in serum and prostate specimens of men receiving dutasteride (5a-reductase 1 and 5a-reductase 2 dual inhibitor) suggest that 5a-reductase 3 in peripheral and androgen-responsive tissue metabolize T to DHT. Both the adrenal androgens and/or cholesterol may be precursors for intracellular DHT formation. Moreover, AR nuclear translocation may concentrate the low DHT levels observed in prostate specimens to the cancer epithelial nuclei and promote AR gene expression. The inability of finasteride or dutasteride to eliminate DHT suggests that 5a-reductase 3 is an important target in CaP, and possibly acne and male pattern baldness. Thus, a trispecific inhibitor may be required to block completely biosynthesis of DHT in CaP and castration-recurrent CaP.
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Wurzel, R., Ray, P., Major-Walker, K., Shannon, J., & Rittmaster, R. (2007). The effect of dutasteride on intraprostatic dihydrotestosterone concentrations in men with benign prostatic hyperplasia. Prostate Cancer Prostatic Dis, 10(2), 149–154. Xu, Y., Dalrymple, S. L., Becker, R. E., Denmeade, S. R., & Isaacs, J. T. (2006). Pharmacologic basis for the enhanced efficacy of dutasteride against prostatic cancers. Clin Cancer Res, 12(13), 4072–4079.
Improving Intermittent Androgen-Deprivation Therapy: OFF Cycle and the Role of Steroid 5a-Reductase Inhibitors Shubham Gupta, Daniel Shevrin, and Zhou Wang
Abstract Intermittent androgen-deprivation therapy (IADT) consists of cycles of androgen-deprivation (ON cycle) and treatment-free periods (OFF cycle), and is under investigation as a means to improve quality of life and retard progression to castration-recurrent prostate cancer in patients with advanced prostate cancer. IADT appears to be as effective as continuous androgen-deprivation therapy, while improving the side-effect profile. During the OFF cycle of IADT, the androgen-deprived prostate cancer cells are re-exposed to androgens. This milieu of androgen-induced regrowth provides a unique window of opportunity for designing OFF cycle-specific interventions. Testosterone, relative to dihydrotestosterone (DHT), is more potent in the induction of growth suppressive androgen-response genes during regrowth of regressed prostate. These findings suggest that during the OFF cycle of IADT, steroid 5a-reductase (SRD5A) inhibitors can be used to enhance the expression of tumor suppressive androgen-response genes and retard tumor growth by blocking conversion of testosterone to DHT. This chapter discusses how addition of SRD5A inhibitors during the OFF cycle of IADT could improve survival under certain clinical conditions. The molecular changes that accompany treatment suggest that prolongation of the OFF cycle may not be advisable.
1 Introduction Prostate cancer survival has improved consistently over the past three decades. Prostate cancer is the most common noncutaneous malignancy in United States (US) men and accounts for about 30% of all such neoplasms (American Cancer Z. Wang(*) University of Pittsburgh School of Medicine, 5200 Centre Ave, Ste G03, Pittsburgh, PA, 15232, USA, E-mail:
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Society 2007). Prostate cancer is second only to lung cancer as a cause of cancer-specific mortality and accounts for 9% of cancer deaths. The 5-year survival was 69% during 1975–1977, 76% during 1984–1986, and 100% during 1996–2002. Current data suggest that while one man in six will be diagnosed with prostate cancer during his lifetime, only 1 in 35 will die of it (American Cancer Society 2007). The present survival figures are attributable to increased diagnosis and aggressive treatment in both the preprostate-specific antigen (PSA) and PSA era (Klein et al. 2007). Given the long natural history of prostate cancer, whether PSA screening by itself has reduced prostate cancer mortality remains unclear (Thompson and Ankerst 2007). However, PSA screening has led to significant clinical and pathological stage migration; more and more cancers are being diagnosed at early stages (Newcomer et al. 1997; Thompson and Ankerst 2007). The high incidence and the long natural history of prostate cancer make it a disease with enormous social and economic burdens. There are close to 2 million men living with prostate cancer in the US, and this number is expected to increase (American Cancer Society 2007). The medical expenditure for prostate cancer was $1.3 billion in 2000 (Penson and Chan 2007). In addition, the indirect costs due to premature mortality equal the direct medical costs of prostate cancer (Max et al. 2002). Prostate cancer is primarily a disease of old age. A multitude of factors including androgens, genetic predisposition, inflammation pathways, and inactivation of caretaker genes influence the development of prostate cancer. The role of androgens in prostate carcinogenesis is critical but incompletely understood. Androgens are required for the development of the prostate, and exposure to androgens over a long period of time is required for prostate carcinogenesis (Klein et al. 2007). In the PSA era, more than 90% of new prostate cancer cases are diagnosed when the cancer is confined locally or regionally (American Cancer Society 2007). Several options are available for managing these cases, which include watchful waiting, active surveillance, radical prostatectomy, external beam radiotherapy, androgen-deprivation therapy (ADT); and also newer treatments like cyroablation, radiofrequency ablation, and high-intensity focused ultrasound. Locally advanced prostate cancer may require combinations of two or three modalities. Recurrence after potentially curative therapy and metastatic disease at presentation are managed usually with ADT. The response of prostate cancer to ADT is consistent and dramatic. Most patients exhibit a strong and prolonged response manifested by improvement in symptoms and objective measures of disease response. Ever since Huggins’ seminal papers in 1941 (Huggins and Hodges 1941; Huggins et al. 1941), few cancer interventions have come close to ADT in terms of simplicity of concept and reliability of response. Options for ADT have evolved from Huggins’ orchiectomy to maximal androgen blockade using gonadotropin-releasing hormone (GnRH) agonists and antiandrogens. Shahinian et al. (2005) reported that the use of ADT has increased dramatically for all stages and grades of prostate cancer; being used even in circumstances where ADT has not been shown to be of benefit. For instance, in men over 80 years of age with localized stage and low- to moderate-grade tumors, primary GnRH use increased 9-fold from 3.7% in 1991 to 30.9% in 1999.
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An analysis of Medicare claims data estimated that one-third of all patients diagnosed with prostate cancer underwent ADT (Krupski et al. 2006). The annual costs for prostate cancer related costs are highest in patients treated with ADT (Wilson et al. 2007); men on ADT cost the health care system almost twice as much as those not on ADT (Krupski et al. 2007). In 2001, the Medicare reimbursements for GnRH agonists exceeded 1 billion dollars, which ranked as the second highest part B drug expenditure (Shahinian et al. 2005). Costs of ADT extend beyond the drug costs. Complications of ADT include osteoporosis, hot flashes, sexual dysfunction, cognitive dysfunction, anemia, gynecomastia, and changes in body habitus. The most dreaded and inevitable eventuality of ADT is progression to castration-recurrent prostate cancer, where the tumor continues to grow despite castrate levels of circulating testicular androgens. ADT has limitations, and strategies to reduce the economic burden, improve the side-effect profile, and delay progression to castration-recurrent prostate cancer would be embraced quickly.
2 Intermittent Androgen-Deprivation Therapy ADT as currently practiced is usually continuous and lifelong. Intermittent androgendeprivation therapy (IADT) has been proposed as a viable alternative for selected patients. The patients are treated cyclically, wherein periods of ADT (On treatment time, or ON cycle) are alternated with treatment-free periods (Time OFF period, or OFF cycle) when androgen levels recover (Fig. 1). For each cycle, ADT is continued until the PSA drops below a pre‐established threshold. Sometimes, ADT continues for a specified time period after the PSA drops below the threshold. Patients are then observed without treatment, and ADT is restarted after serum PSA reaches a preset arbitrary level which varies among protocols. Patients are no longer eligible for OFF cycle periods if PSA continues to increase, or if other evidence of disease progression develops despite ADT. In the Shionogi androgen-dependent mouse mammary carcinoma model, castration induces tumor regression, but also leads to androgen independence (Bruchovsky et al. 1990). Bruchovsky’s group expanded on these ideas to develop IADT. They proposed that the replacement of androgens at the end of a period of apoptotic regression might result in the regeneration of differentiated tumor cells with further apoptotic potential. IADT would promote tumor cytoreduction in the treatment period and the expansion of androgen-sensitive clones in the OFF treatment period (Akakura et al. 1993). These androgen-dependent cells, by competing with androgen-independent cells, would prevent occupation of the tumor by androgenindependent elements and would sustain androgen dependence for longer periods (Rennie et al. 1990; Akakura et al. 1993; Sato et al. 1996). These laboratory studies and the presumed benefits of OFF cycle periods, which include improved quality of life (QOL) and reduced costs, made a strong case for clinical trial of IADT. The notion that IADT would lead to improvement in overall QOL is based on the fact that many of the adverse effects of ADT are related to the lack of
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Fig. 1 Schematic illustration of the phases of intermittent androgen-deprivation therapy (IADT) and the expected cycling of prostate-specific antigen (PSA) and testosterone. One full cycle of IADT consists of 1 ‘‘ON Cycle’’ + 1 ‘‘OFF Cycle.’’ Note the rapid fall in PSA and testosterone levels upon androgen deprivation, and the more gradual rise upon cessation of ADT. The graph is representative of the expected changes in serum PSA and testosterone levels, and does not depict any reported dataset. The OFF cycle of IADT provides the opportunity to design interventions that target androgen-deprived prostate cancer cells that are re-exposed to androgens. The inhibition of testosterone to DHT conversion by the addition of a steroid 5a-reductase (SRD5A) inhibitor (such as finasteride or dutasteride) during the OFF cycle may selectively enhance the expression of tumor suppressive genes and improve survival
androgens and are reversible upon androgen replenishment. During the OFF cycles of IADT, testosterone levels recover and side effects are alleviated (Albrecht et al. 2003; Mottet et al. 2005). IADT appears sound theoretically and firmly grounded on preclinical data; however, insufficient clinical data prevent/preclude unconditional endorsement. Klotz et al. (1986) reported the first clinical use of IADT using diethylstilbestrol (DES) and flutamide in 20 patients. Subsequently, several phase II (Goldenberg et al. 1995; Higano et al. 1996; Oliver et al. 1997; Crook et al. 1999; Strum et al. 2000; Sato et al. 2004; Peyromaure et al. 2005; Bruchovsky et al. 2006; BocconGibod et al. 2007) studies have evaluated IADT, and benefits appear more definitive on side effects than overall survival (OS). In a study of 250 men with locally advanced or metastatic prostate cancer treated with IADT, QOL was assessed every 3 months for 30 months using the European Organization for Research and Treatment of Cancer QOL Questionnaire (EORTC
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QLQ-C30) and EORTC prostate cancer module (QLQ-PR25) (Spry et al. 2006). Testosterone suppression led to a significant reduction in global health-related QOL (HrQOL) and deterioration in most function and symptom scales. During the OFF cycles, a trend of progressive improvement in HrQOL paralleled testosterone recovery. Maximum recovery of HrQOL occurred most frequently by months 9–12. Testosterone recovery was slower and less complete in older men, and led to concomitant poorer HrQOL recovery. However, Bouchot et al. (2000) reported no difference in QOL function assessed using EORTC QLQ-C30 between ON and OFF cycles in 43 patients on IADT. IADT appears beneficial for sexual function. Klotz et al. (1986) reported that 90% of the patients who were rendered impotent after initiation of ADT resumed sexual activity within 3 months of stopping treatment. Malone et al. (2005) reported the recovery of sexual function in 47% of the 38 IADT cycles that were evaluated. A phase III study by Da Silva et al. comparing IADT to continuous ADT showed that at 12 months, 29% of men receiving IADT reported sexual activity in the previous month, compared with 9% of men on continuous ADT (Boccon-Gibod et al. 2007). Among the other side effects of ADT, the loss of bone mineral density is reversible in part during the OFF cycle of IADT (Higano et al. 2004). Bruchovsky et al. (2007) reported the recovery of blood hemoglobin to normal levels during the OFF cycles of IADT. Increasing evidence supports that ADT leads to metabolic syndrome (Braga-Basaria et al. 2006), and cardiovascular mortality is more prevalent in patients on ADT (Tsai et al. 2007). Patients managed with IADT may experience fewer of these complications. A recent Cochrane review compared IADT and continuous ADT using data from five randomized studies involving 1,382 patients (Conti et al. 2007). All included studies involved men with advanced prostate cancer, had relatively small populations, and were of short duration. No data were available that allowed assessment of the relative effectiveness of IADT versus continuous ADT on overall survival, disease-specific survival, or disease progression. There was a trend toward lower adverse events in IADT groups versus continuous ADT groups, but accurate interpretation was limited due to inadequate sample sizes. The authors concluded that IADT may have reduced slightly adverse events, and that IADT was superior to continuous ADT for erectile function during the OFF cycle periods (Conti et al. 2007). Miller et al. (2007) reported on 335 patients with advanced prostate cancer who were randomized to continuous ADT or IADT. The median time to progression was longer in the intermittent arm (16.6 months) compared with the continuous arm (11.5 months), although the difference was not statistically significant (log rank test, p = 0.1758). Safety was similar and QOL indices were more favorable in the intermittent arm. The authors concluded that IADT was safe and feasible. Currently, several phase III trials that compare IADT with continuous ADT are ongoing, in the next few years, as the results from these trial accrue, (will be reported- this needs to be deleted) questions regarding time to progression and survival and the true efficacy of IADT should be resolved. The currently available data have established the safety of IADT. Notably, tumor ‘‘flare’’ was not identified after ADT was stopped. However, the trigger points for stopping and reinstituting ADT remain unclear.
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In addition to the issues of efficacy, safety, and economics, IADT poses several unique questions. During the ON cycle, ADT leads to decreased cell proliferation and increased apoptosis. During the OFF cycle, the cancer cells are exposed to recovering androgens in most cases. The molecular pathways operational during this androgeninduced regrowth have not been fully illuminated and provide an important area for future research. The elucidation of these molecular mechanisms may also help settle the issue of when to start and stop the ON and OFF cycles. The androgen-deprived cancer cells at the onset of the OFF treatment cycle differ from ADT naı¨ve cancer cells, thereby allowing the possibility of designing interventions targeting the OFF Cycle to further enhance survival and slow progression to the lethal phenotype or castration-recurrent prostate cancer. An ongoing trial (NCT00283803) is evaluating the use of exisulind, a sulindac derivative, that inhibits cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE) and promotes apoptosis, to extend the duration of the OFF cycle of IADT. Another candidate for OFF cycle manipulation is the enzyme, steroid 5a-reductase (SRD5A), which has two established isoenzymes, SRD5A1 and SRD5A2. SRD5A catalyzes the conversion of testosterone to dihydrotestosterone (DHT), and an understanding of differential androgen function can suggest ways to improve IADT.
3 Differential Actions of Testosterone and Dihydrotestosterone Testosterone is the principal circulating androgen in men. Testosterone is synthesized by the Leydig cells in the testes and released into the systemic circulation (Snyder 2006). About 98% of the circulating testosterone is bound to steroid hormone-binding globulin (SHBG) or albumin. Testosterone is converted by the enzyme SRD5A to dihydrotestosterone (DHT) in many target tissues, which include the male reproductive organs and skin (Snyder 2006). There are two wellcharacterized synthetic inhibitors of SRD5A – finasteride, an inhibitor of SRD5A2; and dutasteride, which inhibits both SRD5A1 and SRD5A2 (Thomas et al. 2008). Testosterone can be converted by aromatase to estradiol at other sites such as liver, adipose tissue, central nervous system (Snyder 2006). Both testosterone and DHT are androgens and act via the androgen receptor (AR) (Liao et al. 1973; Wilson and French 1976; Grino et al. 1990). Using Chinese hamster ovary (CHO) cells that have low levels of SRD5A, Deslypere et al. (1992) showed that testosterone can induce an MMTV-CAT promoter to the same extent as DHT, albeit at a concentration that was 10-fold higher than the latter. DHT, relative to testosterone, exhibits a slower dissociation rate for the AR and achieves greater protection of AR against degradation (Grino et al. 1990; Zhou et al. 1995). It has been often proposed that testosterone and DHT differ in their potency, conversion of testosterone to DHT possibly being a metabolic amplification step (George 1997). However, in addition to being a substrate for the formation of DHT and estradiol in specific tissues, testosterone has several independent and unique functions. During embryogenesis, the development of male external genitalia, prostate, and seminal vesicles depends on the availability of DHT, while the development of the
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wolffian system depends on testosterone (Snyder 2006). In addition, androgendependent increase in muscle mass is affected by testosterone (Kumar et al. 1992; Snyder 2006). Similarly, DHT specific effects include the ability to turn down the production of Interleukin 4 (IL-4), Interleukin 5 (IL-5), and Interferon-g (IFN-g) from mouse T-cell hybridomas (Araneo et al. 1991), and the ability to induce the expression of its own biosynthetic enzyme – SRD5A (George et al. 1991). These observations make the case that the difference between testosterone and DHT goes beyond the notion of ligand preference, and has led many researchers to search for more areas of differential activity. Lin and Chang (1997) demonstrated that TDD5/N-myc downstream-regulated gene 1 (NDRG1) is suppressed differentially by testosterone and DHT. In the prostate, Avila et al. (1998) identified several genes that were modulated differently by castration (loss of testosterone and DHT) and finasteride treatment (loss of DHT). From the patterns of gene expression variations, the authors concluded that for some genes, testosterone and DHT exert fundamentally different effects. The differential effects of testosterone and DHT should be explored during prostate regrowth after castration. Under normal physiologic conditions in the adult, the presence of androgens maintains the architecture and turnover of the prostate but does not cause prostatic epithelial proliferation (Bruchovsky et al. 1975). After androgen deprivation, the prostate cells undergo massive apoptosis and prostate size and weight are reduced. Upon reintroduction of androgens, the regressed prostate cells undergo vigorous proliferation, and re-establish the precastration steady state (Bruchovsky et al. 1975). Clearly, the response of the regressed prostate cells to androgens is different from that of the cells in the intact prostate. A panel of over 20 androgen-response genes was identified using a polymerase chain reaction (PCR)-based complementary deoxyribonucleic acid (cDNA) subtraction assay based on upregulation during androgen-stimulated regrowth after castration in rat ventral prostate (Wang et al. 1997). Later, the difference between testosterone and DHT-induced gene expression was investigated in the rat prostate (Dadras et al. 2001). in intact animals, finasteride inhibited the conversion of testosterone to DHT and decreased the wet weight of the prostate (Fig 2). At the transcriptome level, the expression of only a few androgen- response genes was reduced slightly, a finding that was in agreement with previous reports (Rittmaster et al. 1991; Shao et al. 1993). Expectedly, the addition of finasteride decreased the wet weight of the regrowing prostate, which underlined the importance of DHT for the proliferation of prostate cells from the regressed state (Fig. 2). However, the patterns of gene expression during prostate regrowth were remarkable – several of the previously identified androgen-response genes showed higher expression with testosterone than with DHT during prostate regrowth after castration. This differential expression was in marked contrast to that seen in intact animals, where the addition of finasteride produced little effect on the gene expression profile. The most significant outcome was that many of the genes that were selectively enhanced by testosterone were markers of differentiation (prostatein C3) or
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Fig. 2 Effects of hormonal manipulations on the prostate. Inhibition of SRD5A by finasteride or dutasteride leads to a modest decline in the size and weight of the prostate. Castration produces the most rapid and maximum decrease in the weight and size of the prostate. Androgen replacement with DHT (either directly with DHT or with testosterone, allowing for endogenous conversion to DHT by SRD5A) to the regressed prostate is strongly mitogenic; weight and size increase to precastrate levels. Replacement with testosterone (testosterone given with finasteride or dutasteride to prevent the endogenous synthesis of DHT) elicits a much weaker mitogenic response, and the enhancement in size/weight is retarded. During this phase of regrowth from a regressed state, selective exposure to testosterone preferentially enhances the expression of several androgen-response genes that have growth suppressive properties. This selective enhancement of growth suppressive/differentiation genes was not observed under any other conditions of hormonal manipulation
were growth inhibitory (calreticulin, adrenomedullin, human aci-reductone dioxygenase, ELL associated factor 2/upregulated 19 (EAF2/U19)) (Dadras et al. 2001). This phenomenon provides a strategy to preferentially enhance the expression of growth suppressive androgen-response genes, which may improve the efficacy of prostate cancer treatment.
4 SRD5A Inhibition During the OFF Cycle The differential regulation of androgen-response genes by testosterone and DHT during prostate regrowth provides an opportunity to improve IADT for advanced prostate cancer by OFF cycle inhibition of SRD5A. Huggins’ ‘‘biological syllogism’’ (Huggins et al. 1941) equated malignant epithelia to normal prostatic epithelium with
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respect to response to castration. During the regrowth of prostate cancer after a period of ADT, selective exposure to testosterone may enhance the upregulation of markers of differentiation and growth-suppressive genes. The clinical analog of this circumstance is met in IADT, where after a period of ADT (On Cycle), the treatment is discontinued and androgen levels are allowed to return to normal (OFF Cycle). During this OFF cycle, inhibition of SRD5A (by finasteride or dutasteride) should upregulate ‘‘good genes’’ – genes associated with differentiation and growth suppression, and drive the tumor phenotype toward a more differentiated one, and eventually improve survival (Fig. 1). Since the QOL benefits of the OFF Cycle are most often mediated by testosterone, QOL should improve also. Follow-up experiments have shown that addition of finasteride during the OFF cycle of IADT improved survival in vivo (Eggener et al. 2006). Nude mice with subcutaneously implanted androgen-sensitive LNCaP tumors were randomized to continuous ADT, IADT, continuous ADT + finasteride, and IADT + finasteride (finasteride was added during the OFF cycle). After one cycle of therapy, the IADT + finasteride group had significantly less tumor growth than the other treatment groups (Fig. 3). Mice treated with IADT + finasteride also had the longest survival (Fig. 4). The mice in the IADT + finasteride group were cycled similarly to the IADT group, i.e., the duration of ON and OFF cycles was similar relative to standard IADT. Given the upregulation of growth suppressive genes by testosterone during prostate regrowth, and the survival benefit of IADT + finasteride in a murine xenograft tumor model, the same genes may be upregulated during the OFF cycle in IADT upon inhibition of SRD5A. Indeed, SRD5A inhibition (by both finasteride and dutasteride) during the OFF cycle enhances the expression of EAF2/U19 (unpublished data). EAF2/U19 is an androgen-regulated tumor suppressor that frequently exhibits allelic loss and downregulation in high-grade prostate cancer (Xiao et al. 2003). Whether the selective upregulation of EAF2/U19 is indeed the effector of the better survival in the IADT + finasteride group, or the incidental side effect of other mechanisms, remains to be established. In addition, the transcriptome changes that occur during the OFF cycle of IADT, show temporal variation. Finasteride-induced differential gene expression changed during regrowth of the rat prostate (Dadras et al. 2001). In addition, the upregulation of EAF2/U19 in LNCaP tumors with OFF cycle finasteride/duatsteride (see earlier) was observed after 3 days of OFF Cycle intervention. Selective exposure to testosterone during the OFF cycle may lead to an initial enhanced expression of growth suppressive genes, and with time, the expression of these genes subsides to become similar to that in cases where there is no SRD5A inhibition during the OFF cycle. Therefore, the benefit of OFF cycle SRD5A inhibition will be present only while there is selective enhancement of tumor suppressive genes. After the gene expression returns to baseline, no advantages will accrue from continuation of the OFF cycle. In order to use SRD5A inhibitors during the IADT OFF Cycle, the issue of OFF Cycle duration must be addressed. Does OFF cycle duration correlate with prognosis? Does SRD5A inhibition affect the duration of the OFF cycle? Theoretically, the OFF cycle duration and QOL benefits should be correlated, since longer periods of high testosterone levels translate into better QOL function. However, no clear
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Fig. 3 Effect of finasteride maintenance during the OFF cycle of intermittent ADT on the growth of androgen-sensitive LNCaP xenograft tumors. Nude mice with established LNCaP xenograft tumors were castrated and then randomized into following four different treatment groups: (1) continuous ADT (ADT); (2) continuous ADT + finasteride (ADT + F); (3) intermittent ADT (IADT); and (4) intermittent ADT with finasteride during the OFF cycle (IADT + F). More than 40% of the mice treated with finasteride during the OFF cycle of intermittent ADT (IADT + finasteride) showed no increase in tumor volume after one cycle of treatment (a). Tumor volume after 14 days of treatment increased the least in mice treated with IADT + F (b). Asterisk denotes statistical significance. From Eggener et al. (2006) (Reprinted with permission of John Wiley & Sons, Inc.)
correlation between progression to castration-recurrent prostate cancer and OFF cycle duration has been established. In a meta-analysis of 1446 patients, who underwent IADT, Shaw et al. (2007) reported that being OFF treatment for 2 or 3 years was associated with reduced overall mortality and a lower hazard of
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Fig. 4 Effect of finasteride maintenance during the OFF cycle of intermittent ADT on the survival of nude mice bearing androgen-sensitive LNCaP xenograft tumors. The experimental groups were the same as described in Fig. 3. (a) Kaplan–Meier survival curves for different treatments. Mice treated with IADT + F had the longest survival (p = 0.048). Mice treated with IADT + F were 3–5 times more likely alive 70 days after treatment initiation compared to other treatment regimens (b). From Eggener et al. (2006) (Reprinted with permission of John Wiley & Sons, Inc.)
castration-recurrent prostate cancer. Being able to stay at OFF treatment for 2 or 3 years may reflect less aggressive disease characteristics, and thus appear indicate delayed progression to castration-recurrent prostate cancer and improved overall survival. In a retrospective analysis of data from 101 patients treated with IADT with or without finasteride during the OFF cycle, Scholz et al. (2006) found that the addition of finasteride doubled the OFF cycle duration even after correcting for the expected 50% reduction in PSA levels due to finasteride. However, progression to castration-recurrent prostate cancer occurred with similar frequency in both treatment arms. The only variable that affected the progression to castration-recurrent prostate cancer in their analysis was clinical stage. While they did not perform any
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QOL/adverse effect analyses, the authors surmised that increased time OFF treatment should lead to better QOL, and that OFF cycle finasteride may accomplish that. Even with the limitations of a retrospective study, these results suggest the potential for SRD5A inhibitors in prolongation of the OFF cycle duration. Currently, the use of dutasteride to prolong the OFF treatment period in IADT is being explored in two prospective phase II randomized trials (NCT00516815, NCT00553878). The next logical question would be whether there are any survival benefits or risks to extending the OFF cycle duration using SRD5A inhibition. No a priori assumptions should be made about the survival effects of OFF cycle prolongation by SRD5A inhibition because the effect of SRD5A inhibition on prostate cancer biology during IADT remains unclear. Serum PSA, which is used to guide decisions on timing in IADT, may have some problems. PSA is far from the ideal biomarker for prostate cancer detection (Bensalah et al. 2008), and performs less than perfectly as a predictor of disease progression and recurrence (Noguchi et al. 2000; Stamey 2001; Stamey et al. 2004; Chun et al. 2006; Mitchell et al. 2006; Steuber et al. 2006). Neoplastic cells produce less PSA than benign prostatic hyperplasia cells (Roehrborn et al. 1999a, b, 2000), and PSA expression may decrease with advancing Gleason grade (Aihara et al. 1994). In addition, the determination of PSA levels at the systemic level (in the serum) means that changes occurring in PSA-secreting tumor cells must be sustained and strong enough to be detected systemically. This introduces a selection bias and fails to address the issue of tumor heterogeneity. For instance, cells that do not produce a lot of PSA do not contribute substantially to the treatment decisions based on serum PSA levels, although they may be more aggressive than cells that produce more PSA. These limitations of serum PSA must be considered while evaluating the role of SRD5A inhibitors during the OFF cycle of IADT.
5 Challenges and Future Directions Prostate cancer can be targeted during the OFF cycle of IADT in several ways. Questions regarding safety and efficacy must be resolved before such an approach is practiced clinically. The current evidence allows several inferences and shows the path for exploring the answers to other issues. First, the issue of safety – finasteride and dutasteride are frequently used for indications other than prostate cancer, and are fairly safe drugs. Available data suggest that this safety profile is maintained during the OFF cycle. Questions regarding efficacy are trickier. Efficacy will depend on how the drugs are administered, how the response is measured, and what benchmarks are adopted for success. The final goal of any new intervention should be a survival advantage over previously established treatments, and the optimization of OFF cycle duration and the use of SRD5A inhibitors should be done with progression and survival results foremost in mind. Eggener et al. (2006) showed that OFF cycle finasteride can enhance survival in vivo when the OFF cycle duration remains similar to standard IADT; Scholz et al. (2006) showed that OFF cycle finasteride can double the OFF cycle duration
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without influencing progression to castration-recurrent prostate cancer. Evidently, there is a difference in outcome depending on how finasteride is administered – it seems that the survival benefit of OFF cycle finasteride in IADT is sustainable only when the OFF cycle durations remain similar in the IADT and IADT + finasteride groups. Finasteride is capable of increasing the OFF cycle duration; however, when the OFF cycle duration is increased, the survival advantage of IADT + finasteride over IADT is lost. This seems to suggest that one should not aim for an increase in the duration of the OFF cycle when using OFF cycle SRD5A inhibitors. The rationale for these observations may lie in the temporal expression of certain genes that are differentially regulated by testosterone and DHT. The molecular mechanisms that improve survival appear to have a different time course from those that are currently used to determine OFF cycle duration, i.e., PSA levels. Overall, OFF cycle SRD5A inhibitors may influence serum PSA differently than they do the androgen-response tumor suppressive genes at the cellular level in prostate cancer; by the time serum PSA levels increase to the threshold where androgen suppression is restarted, the enhancement in the expression of the tumor suppressive genes may have concluded. Therefore, the OFF cycle duration should be matched to the expression profile – the peaking and return to baseline – of growth suppressive genes like EAF2/U19. Of course, the need to follow gene expression profile in tumor cells necessitates repeated sampling of prostate cancer foci, which may not be possible in most patients. This obstacle may be overcome by tying the gene expression profiles to more readily measurable events and markers. One such candidate is serum testosterone. Since the preferential enhancement of tumor suppressive genes due to OFF cycle SRD5A inhibition is mediated by selective exposure to testosterone during the initial phase of regrowth, the OFF cycle durations could be tailored to testosterone levels. The time course of testosteroneinduced gene expression will help define the duration of the OFF cycle. When the enhanced expression of tumor suppressive genes subsides, further exposure to testosterone would be redundant. Therefore, testosterone levels need to be in the eugonadic range only for such period of time during which growth suppressive genes are elevated, and then androgen suppression could be reinitiated. The question of timing – when to start and stop the ON and OFF cycles – is crucial, and the answer will be critical to the success of SRD5A inhibition during the OFF treatment phase. OFF cycle SRD5A targeting is nascent yet exciting, and the near future may include SRD5A inhibition as an important component of prostate cancer IADT.
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Acknowledgments We thank Minh Nguyen for critical reading, William H. Blair and Russell Gould for insightful discussion, and support from Department of Defense Prostate Cancer Research Program, DAMD 17-02-1-0113, National Institute of Health, R01 DK51193, and National Institute of Health Prostate Cancer Specialized Program of Research Excellence (SPORE), CA90386.
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Insights from AR Gene Mutations Grant Buchanan, Eleanor F. Need, Tina Bianco-Miotto, Norman M. Greenberg, Howard I. Scher, Margaret M. Centenera, Lisa M. Butler, Diane M. Robins, and Wayne D. Tilley
Abstrat The accumulation of somatic mutations on the background of natural germline variation is one of the fundamental mechanisms underpinning both disease and the development and progression of perhaps all tumors. For example, germline inactivating mutations in one allele of a tumor suppressor gene (TSG) (e. g., p53 or BRCA1) predisposes an individual to a lifetime of increased risk of cancer in particular tissues. In those cases, tumors commonly arise after somatic inactivation of the second TSG allele, and are often characterized by a younger age of onset and a more aggressive phenotype than tumors arising in the same tissues without a common or dominant genetic predisposition. A small proportion of prostate cancers do indeed occur in a hereditary manner, but it has been difficult to attribute inheritance and risk to a single gene. Even for the monoallelic androgen receptor (AR) gene on the X chromosome, which exhibits variously traits of an atrisk allele, oncogene, and mediator of prostate cancer progression and therapy resistance, inactivating germline variation in the AR is essentially nonexistent in this disease. Instead, germline AR variants often underpin the relatively common inherited syndrome of androgen insensitivity (AIS). In contrast, however, somatic variation of the AR is potentially a frequent event during tumor progression. As a consequence of these complexities, unraveling the precise role of the AR at each stage of prostate cancer progression, and indeed in different prostatic compartments or populations of tumor cells is challenging. In this chapter, we detail how the identification and characterization of somatic AR variants arising in prostate cancer has provided crucial information on (1) the role of the receptor throughout disease etiology and the emergence of castrate-recurrent disease, (2) the
W.D. Tilley(*) Dame Roma Mitchell Cancer Research Laboratory, School of Medicine, University of Adelaide/ Hanson Institute, PO Box 14, Rundle Mall, SA 5000, Australia, E-mail:
[email protected]
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fine functional subdomain structure of the AR, (3) the oncogenic potential of aberrant AR signaling, and (4) new approaches to targeting AR function in disease management.
1 AR and Prostate Cancer A functional androgen signaling axis is essential for the development, growth, and homeostasis of the normal prostate gland, and has been linked to all stages of prostate tumorigenesis including predisposition and progression (Culig et al. 2000; Feldman and Feldman 2001; Scher et al. 2004). The critical mediator of these effects is AR, a member of the superfamily of nuclear receptor transcription factors that regulates diverse cellular functions in the prostate. The requirement for sustained androgen signaling in prostate cancer underpins hormonal strategies [e.g., androgen-deprivation therapy (ADT) and/or AR antagonists] in disease treatment (Fig. 1, Androgen-deprivation therapy). While virtually all prostate cancers initially respond to ADT, the majority relapse and the disease progresses. Whereas initial studies using the Dunning animal model and with the human prostate cancer cell lines, DU145 and PC-3, suggested that loss of AR gene expression could be a mechanism for failure of ADT (Quarmby et al. 1990; Tilley et al. 1990), subsequent immunohistochemical studies of clinical prostate cancer have demonstrated that the AR is expressed in essentially all metastatic tumors, including those that continue to grow during ADT (Culig et al. 1998). Additionally, tissue levels of prostate-specific antigen (PSA) and other androgen responsive genes increase during castrationrecurrent tumor regrowth (Bentel and Tilley 1996; Culig et al. 1998; Hobisch et al. 1995). The failure of ADT strategies can be explained in many cases by selection for continued androgen and/or AR signaling in the castrate environment (Scher et al. 2004). The contributing mechanisms include increased AR levels, crosstalk between the AR and other signaling pathways, increased response to or level of AR coregulators, and the selection of permissive AR mutations (Buchanan et al. 2001b; Feldman and Feldman 2001; Scher et al. 2004).
2 Mutations in the AR Gene in Prostate Cancer Approximately 130 individual somatic missense or nonsense mutations have been reported in the AR gene in human prostate cancer, several more in latent disease at autopsy, and a significant number in cellular and animal models of the disease (Gottlieb et al. 2004; Scher et al. 2004). Whereas AR gene mutations have been found only rarely in early-stage or primary disease, the reported incidence increases with the stage of disease analyzed, and particularly for those tumors regrowing in a castrate setting (Buchanan et al. 2001b). Moreover, almost overwhelming evidence suggests that the majority of these mutations do not result in a loss of function, but
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Fig. 1 AR signaling and points of therapeutic intervention in prostate cancer. A schematic of AR signaling in a prostate cancer cell highlights known and predicted effects of somatic AR variants detected in clinical disease and animal models (green boxes), and new and traditional approaches to therapeutic intervention (red boxes). After synthesis in a normal cell, the AR is maintained in equilibrium between immature and active states through association/dissociation with a complex that includes heat-shock proteins, p23 and a tetratricopeptide (TPR)-containing protein. The active state is available for binding high-affinity androgenic ligands, such as 5a-dihydrotestosterone (DHT). Ligand binding results in the dissociation of heat-shock proteins, dimerization and phosphorylation, nuclear transport, DNA binding, recruitment of the transcription machinery and cofactors, and activation of downstream gene pathways. AR activity also can be stimulated in the presence and absence of ligand by membrane-bound tyrosine kinase receptors, such as HER2/neu, and by signaling molecules, growth factors, and cytokines. Mutation effects: Promiscuous activation, broadened specificity or sensitivity to noncanonical ligands (e.g., V730M, H874Y, T877A [LNCaP]); Decreased control, altered equilibrium interaction with the chaperone complex leading to multiple modes of activation (e.g., altered interaction with the TPR proteins aSGT of FKBP52); Stimulation, altered phosphorylation status (e.g., creation, disruption, or hyperstimulation of a phosphorylation site (e.g., S515G)); Sensitization, altered physical stability or receptor turnover (e.g., polyQ2L or C619Y); Constitutive bypass, truncated AR variants circumvent the requirement for ligand and are constitutively active (e.g., Q640STOP); others exhibit leaky or increased basal activity (e.g., T877A, E236G); activation of the wild-type receptor in the absence of ligand by growth factor, cytokine or phosphorylation kinase signaling can mimic constitutive bypass mutations; Differential cofactor recruitment, altered ability to recruit coregulator molecules (e.g., I673T, E236G), Differential gene activation, altered capacity to activate downstream genes (e.g., E236G). Therapeutic intervention: Androgen-deprivation therapy,
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can be ascribed one of a range of phenotypes capable of maintaining AR signaling during ADT (Fig. 1). These observations support the theory of ‘‘Therapy-mediated selection pressure’’ for prostate cancer (Scher et al. 2004), which holds that the specific treatment strategy employed against the disease will select for tumor cells containing changes permissive for continued survival and growth during that treatment and/or in the castrate environment. We discuss later how the study of AR gene mutations from prostate cancer samples has provided key insights not only into the role of the receptor in this disease, but also into the function of each of its domains and how we might better target the AR signaling pathway for disease management.
3 The AR-Ligand-Binding Domain The AR is a member of a small number of steroid-activated nuclear transcription factors defined by conserved domains for DNA (DBD) and steroid-ligand binding (LBD) that are separated by a divergent hinge region, and a poorly conserved and highly variable amino-terminal domain (NTD) thought to direct transcriptional response and competency (Fig. 2a). Much of the initial understanding of AR function stems from studies of AIS, in which genetic XY individuals exhibit a phenotype ranging from ambiguous external genitalia and/or impaired masculinization to complete feminization (Quigley et al. 1995). Commonly, germline mutations in the AR-LBD that result in a partial or complete loss of steroid-binding capacity and an inactive receptor are the underlying cause of this syndrome. For this reason, and perhaps additionally for technical or logistical limitations, the vast majority of studies for AR mutations in prostate cancer have been performed only on the LBD, which comprises barely a quarter of the receptor’s coding sequence. Nonetheless, the intensive analysis of this relatively small domain has been particularly instructive in the dissection of AR functional subdomain structure, protein– protein interactions, and the molecular mechanisms of AR activation. In particular, more than 85% of missense mutations detected in clinical prostate tumors were Fig. 1 (Continued) traditional systemic ADT; Antagonists & SARMS, competitively inhibit ligand binding and slow movement of the receptor into and through the nucleus, Enzymatic inhibitors, e.g., 5a-reductase inhibitors such as finasteride or dutasteride prevent conversion of testosterone to the more potent DHT; Antisense, shRNA, and HDACIs, antisense AR oligonucleotides, short hairpin RNAs directed at the AR and histone deacetylase inhibitors reduce AR levels; Ansamycins and HDACIs, Ansamycin antibiotics (e.g., 17-allylamino-geldanamycin) and HDACIs act to disrupt Hsp90 activity or chaperone functioning; Ansamycins, act to inhibit chaperone-mediated trafficking; ARIs, AR inhibitors or dominant negative AR constructs act to prevent receptor dimerization and/or recruitment of coregulators; Response blockers, specific response-elementbinding proteins (e.g., polyamides, decoy response elements) block or compete with AR interactions on DAN; Antibodies, specific for AR to block activity directly or for growth factor receptors (e.g., Herceptin) to prevent crosstalk; Inhibitors, directed at MAPK, JAK-STAT, or Akt pathways to prevent receptor crosstalk; Recruitment inhibitors, act to prevent coactivator recruitment to the receptor (See Color Insert)
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Fig. 2 Collocation of AR gene mutations. Schematic of the human AR (top) delineating aminoterminal (NTD), DNA-binding (DBD) and ligand-binding (LBD) domains. Green shaded areas represent regions where mutations collocate in clinical prostate cancer (see later and Fig. 2). Collocation of mutations in the LBD is shown in greater detail; the position and frequency of missense mutations identified in the inherited from of androgen insensitivity (AIS; upper) and human prostate cancer (hPCa; lower) were used to calculate a proportional frequency distribution of mutations. Vertical bars represent the percentage of mutations identified in the AR-LBD in either hPCa or AIS within a nine-amino acid region centered on individual codons. A cut-off of 4% was applied such that bars represent at least two mutations from prostate cancer within each nineamino-acid region. Areas of collocation in AIS (blue boxes) and hPCa (green boxes) were determined by delineating the actual position of mutations occurring in regions defined by the frequency distribution and required a minimum of four independent mutations. The amino acid residues encompassing these regions are indicated (See Color Insert)
located in five discrete regions of the LBD that collectively comprised less than 10% of the AR coding sequence (Fig. 2b) (Buchanan et al. 2001a; Scher et al. 2004). Of critical importance was the concomitant finding that inactivating mutations in the AR identified in AIS collocated to almost completely distinct regions (Fig. 2b) (Buchanan et al. 2001a). Each of the LBD regions where mutations collocate in prostate cancer has been ascribed to modulation of ligand-binding specificity, cofactor responses, and transactivation capacity of the receptor, thereby facilitating increased AR function and a survival advantage for prostate cancer
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cells. The insights gained from these studies have led to new directions in both defining AR function and investigating better ways to modulate its behavior.
3.1
Collocation of AR Gene Mutations at 874–911
Agonist binding by the AR results in conformational changes in the LBD and the formation of activation function 2 (AF-2), which is a conserved protein–protein binding site on the surface of the receptor formed by the juxtaposition of several distinct structural elements along the length of the LBD (Matias et al. 2000; Sack et al. 2001).The agonist-induced AF-2 surface is responsible for mediating interaction with both the NTD (the so-called amino-carboxyl [i.e., N/C] interaction) and with a set of essential transcriptional coregulators that include the three p160 family members, NCOA1/SRC-1/RIP160, NCOA2/GRIP1/TIF2/SRC-2, and NCOA3/ AIB1/TRAM-1/ACTR/RAC-3/SRC-3. The N/C interaction is thought essential for AR function; its abolition results in a receptor incapable of activating chromatin-integrated targets in vivo (Li et al. 2006). Mutations between residues 874 and 911 identified in prostate cancer lie proximal to the core sequence of AF-2 (amino acids 893–899) and typically result in a receptor that can be inappropriately activated by a range of nonclassical ligands and/or receptor antagonists (Fig. 1, Promiscuous activation). None of these mutations are located within the AF-2 core where missense mutations abolish N/C and/or p160 interactions and result in an inactive receptor (Berrevoets et al. 1998). The prototypical example of a permissive mutation is the AR-T877A variant identified in clinical prostate tumors and in the human prostate cancer cell line LNCaP, which exhibits increased basal transactivation activity and response to progesterone, 17b-estradiol, adrenal androgens, and hydroxyflutamide compared to wild-type AR (wtAR) (Veldscholte et al. 1992). The T877A AR variant inappropriately adopts an AF-2 surface permissive for N/C and p160 coactivator interaction for each of these noncanonical ligands (Kemppainen et al. 1999). Structural analysis has revealed that the T877A mutation increases the volume of the ligand-binding pocket, thereby facilitating accommodation of bulkier ligands and inappropriate formation of a correct AF-2 surface (Han et al. 2001). The selection for AR variants able to utilize endogenous noncanonical ligands somewhat negates the potential for disease treatment by enzymatic inhibitors of androgen biosynthesis such as 5a-reductase (Fig. 1, Enzymatic inhibitors). For other mutations in the vicinity of AF-2, detailed functional and crystal structure analysis has revealed a distinct mechanism of promiscuous activity (Askew et al. 2007; Bohl et al. 2007; He et al. 2006). These mutations slow dissociation of bound ligand, and increase receptor stabilization and recruitment of p160 or other coactivators (Fig. 1, Differential cofactor recruitment). The study of those mutations revealed that AR transcriptional potency depends on the interchange between structural states promoted by ligand and coactivator interactions (He et al. 2006).
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Collocation of AR Gene Mutations at 715–730
One of the most characterized group of prostate-cancer-derived AR-LBD mutations lie between residues 715 and 730, and span the highly conserved signature sequence region of the steroid receptors that is responsible for ligand recognition and interaction, and contributes to the AF-2 surface (Wurtz et al. 1996). Critically, none of the residues mutated in prostate cancer corresponds to those known to be essential for direct contact with the ligand (Matias et al. 2000). Instead, these mutations result in an inappropriate AR response to estrogens, progestins, adrenal androgens, and other nonclassical ligands (Buchanan et al. 2001a, b). In AIS, the loss of ligand binding commonly is associated with mutations that collocate to codons 702–714 and make direct contact with the ligand (Gottlieb et al. 2004).
3.3
Collocation of AR Gene Mutations at 670–678
Mutations in amino acids 670–678 at the boundary of hinge and LBD, which have been found in both clinical prostate cancer and in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model, exhibit a two- to fourfold greater transactivation activity in response to 5-a-dihydrotestosterone (DHT), nonclassical ligands, and hydroxyflutamide compared to wtAR (Buchanan et al. 2000). Unlike mutations in the signature sequence or AF-2, enhanced activity of the 670–678 variants occurred in the absence of changes to ligand-binding kinetics, receptor levels, or DNA-binding capacity. These findings argued that the 670–678 AR variants altered receptor interaction with a coactivator or corepressor protein; the hypothesis is supported by the identification of a putative coregulator-binding site using molecular modeling (Buchanan et al. 2000). Recent crystal studies provide direct evidence that the 670–678 region is contained within a coactivator interaction site distinct from AF-2, but where binding of proteins and other agents weakens the structural integrity of AF-2 and receptor interactions with p160 coactivators (Estebanez-Perpina et al. 2007). Attenuation of this allosteric effect by these prostate cancer mutations should accentuate coregulator function at AF-2, which provides a mechanism for increased function relevant to prostate cancer, particularly in response to increasing levels of p160s or other transcriptional coregulators that occur during progression (Chmelar et al. 2007). These findings have precipitated the search for coregulators that interact with the 670–678 region rather than AF-2 (see Sect. 4.6) (Buchanan et al. 2007), and for compounds that bind to this site and disrupt cofactor interactions here and at AF-2 (Fig. 1, Recruitment inhibitors) (Estebanez-Perpina et al. 2007).
3.4
Collocation of AR Gene Mutations at 741–757 and 791–798
Whereas much less is known about the two regions consisting of residues 741–757 and 791–798, the former is known from AIS to affect the N/C interaction and
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recruitment of transcriptional coactivators to AF-2. The consequences of mutations in these regions remains poorly understood for prostate cancer.
3.5
SARMS and the Syndrome of Antiandrogen Withdrawal
The latent promiscuous potential of the AR-LBD has prompted the search for better antagonists and/or selective AR modulators (Fig. 1, Antagonists & SARMS); the latter being compounds that exert agonist and/or antagonist effects on the AR in a tissue or target gene-specific manner. However, all clinically utilized AR antagonists (e.g., flutamide, nilutamide, and bicalutamide), as well as other hormonal therapies for prostate cancer (e.g., estrogens such as diethylstilbestrol, and progestational agents such as megestrol acetate) have been associated with the syndrome of steroid-hormone and anti-antiandrogen withdrawal (Kelly et al. 1997). This syndrome is characterized by tumor regression and decreasing serum levels of PSA when treatment with antiandrogens is discontinued at the time of clinical progression (Kelly et al. 1997). In many cases of antiandrogen withdrawal, tumors have been found to harbor somatic AR gene mutations permissive for receptor activation by the particular hormonal therapy received by the patient (Kelly et al. 1997). Due to the evolutionary diversity of ligands known to bind with high affinity to the conserved LBD of the steroid and nuclear-receptor superfamily, mediated in most cases by relatively minor differences in primary LBD sequence, permissive AR mutations are likely to arise for all new-generation SARMS. Nonetheless, broadening the arsenal of AR antagonists will facilitate cyclic or effective second and third-tier hormonal treatment options for better overall disease management (Scher et al. 2004).
3.6
Cochaperones as Critical Mediators of AR Function in Prostate Cancer
Molecular chaperones are essential for the activity of diverse signaling molecules, which include steroid and tyrosine kinase receptors, p53 and telomerase, and are emerging as critical players in the pathogenesis of human disease (Auluck et al. 2002; Cheung and Smith 2000; Fonte et al. 2002; Waza et al. 2005; Whitesell and Lindquist 2005). Whereas the Hsp70/Hsp90 chaperone system traditionally has been ascribed to folding and stabilization of these molecules, compelling evidence suggests additional roles in movement in both the cytoplasm and nucleus, signal and/or transcriptional competence after activation, and for the biologically diverse actions mediated by structurally related proteins (Cheung and Smith 2000; Pratt et al. 2004; Pratt and Toft 1997). Indeed, recent data suggest that the Hsp90 chaperone machinery contributes not only to maturation of the steroid receptors,
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but also to receptor movement through the cytoplasm, across the nuclear pore complex, in the nucleus, and cycling between active sites of transcription and nucleoplasmic stores of cofactors (Pratt et al. 2004). An example of how collocation of AR mutations identified in prostate cancer has facilitated a greater understanding of normal receptor function as it relates to chaperones comes from studies of the putative coregulator-binding region encompassing mutations in amino acids 670–678 at the boundary of the hinge and LBD. Previous studies utilized the isolated but complete AR-LBD as bait in the presence of high levels of ligand to identify AR coregulators. In addition to biasing the outcome to the highly structured ligand-dependent AF-2 site, modeling studies and the more recently generated crystal structures of this region indicate that the putative coregulatorbinding site spanning the mutated 670–678 residues might not be fully recapitulated using the LBD alone (Buchanan et al. 2000; Estebanez-Perpina et al. 2007). Consequently, AR residues 618–754 that encompass the hinge and the amino-terminal third of the LBD were used to identify and characterize the evolutionarily conserved Hsp70/Hsp90 cochaperone, small glutamine-rich tetratricopeptide repeat containing protein alpha (aSGT/SGTA), as a critical AR-binding partner in prostate cancer. Distinct from the nuclear transcriptional coregulators identified by most studies, aSGT/SGTA acts in the cytoplasm to maintain precise control of AR sensitivity, specificity, and degree of activation, and is downregulated in advanced prostate cancer (Buchanan et al. 2007). The loss of aSGT/SGTA results in decreased control of AR function (Fig. 1, Decreased Control), and mimics the functional consequences on AR activity as mutations between residues 670 and 678 (see Sect. 4.3). Along with increased expression of the AR, each of these mechanisms is implicated in the sensitization of metastatic prostate tumor cells to androgen signaling in a castrate environment (Fig. 1, Sensitization) (Buchanan et al. 2007). aSGT/SGTA belongs to a small group of steroid-receptor interacting chaperones defined by a conserved tetratricopeptide repeat (TPR) that includes the FK506binding proteins, FKBP51 and FKPB52; the immunophilin, Cyp-40; and the protein phosphatase, PP5. The importance of these TPR proteins to AR signaling is highlighted by the phenotype of FKBP52 knockout mice, which exhibit severe defects in male reproductive tissues (Cheung-Flynn et al. 2005; Yong et al. 2007).
3.7
Ansamycin Antibiotics to Target AR Signaling in Prostate Cancer
Geldanamycin and its derivatives are a class of ansamycin antibiotic that target the ATPase activity of Hsp90 to prevent the energy-dependent maturation of client proteins that include AR, p53, and cell surface receptors such as Her2, and promote their subsequent degradation by the 26S proteasome (Segnitz and Gehring 1997; Solit et al. 2002, 2003). Consequently, at least four compelling reasons warrant evaluation of these agents as a ‘‘double-whammy’’ treatment option for prostate
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cancer (Fig. 1, Ansamycins). First, maturation of the AR is thought essential for activity of both wild-type and mutant ARs. Second, inhibition of a range of Hsp90 client proteins may inhibit direct growth-promoting effects of other signaling molecules in prostate cancer cells, as well as target the stimulation of AR and its coregulators and/or the sensitization of receptor by cytokines, growth factors, and tyrosine kinase receptors (Fig. 1, Stimulation and Sensitization) (Powers and Workman 2006; Saporita et al. 2007). These factors have also been implicated in mediating constitutive bypass activation of the AR in the absence of ligand (Fig. 1, Constitutive bypass; see Sect. 6.6) (Feldman and Feldman 2001; Scher et al. 2004). Third, gain-of-function mutations in signaling molecules such as v-src, bcr-abl, c-kit, and p53 can increase their dependence on Hsp90 compared to their wild-type counterparts and make them more sensitive to degradation by 17-AAG (An et al. 2000; Blagosklonny et al. 1996; Fumo et al. 2004; Grbovic et al. 2006; Schulte et al. 1998; Whitesell and Lindquist 2005). Finally, cancer cells have a highly primed and sensitized Hsp90 chaperone system that allows more rapid responses to insult, buffers cellular signaling pathways against genetic instability, and enhances sensitivity to extracellular signaling, but which makes them particularly susceptible to inhibitors such as geldanamycin and its derivatives (Kamal et al. 2003; Whitesell and Lindquist 2005). To investigate the first of those hypotheses, the geldanamycin derivative 17-allylamino-17-demethoxygeldanamycin (17-AAG) was tested against a range of AR variants with distinct phenotypes (Fig. 3a). MostAR variants tested were equally or more sensitive to inhibition by 17-AAG in a dose-dependent manner compared to the wild-type receptor, and none exhibited resistance (Fig. 3b). In support of a use for these agents, geldanamycin and 17-AAG are able to reduce the levels of AR and suppress the proliferation of human prostate cancer cells both in vitro and in vivo (Solit et al. 2002, 2003). Phase II clinical trials with 17-AAG in prostate cancer are ongoing (Heath et al. 2005; Lattouf et al. 2006), but clinical breast cancer studies suggest that the responses will not recapitulate those found for tumors growing in mice (Eiseman et al. 2007), and that there may be a subclass of AR variants resistant to these inhibitors (see Sect. 4.8).
3.8
Truncated Receptors and the Constitutive Bypass Mechanism
Historical in vitro studies established that truncation of the AR in the hinge or LBD before amino-acid 710 results in a receptor that is constitutively active on classic androgen responsive elements (ARE) (Jenster et al. 1991). However, only recently a somatic AR gene mutation leading to a truncated receptor (Q640STOP) was identified in human prostate cancer (Ceraline et al. 2004). By circumventing completely the requirement for ligand activation, this truncated receptor provides a direct mechanism for constitutive bypass growth of prostate cancer and escape
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Fig. 3 Ansamycin antibiotics effectively block activity of multiple classes of AR variants identified in prostate cancer. (a) Schematic of the human AR showing relative position of a subset of AR variants identified in prostate cancer. (b) Effect of increasing concentrations of 17allylamino-geldanamycin on activity of AR variants shown in A. Human prostate cancer PC-3 cells seeded in 96-well plates at 1.5 104 cells/well for 24 h were transiently transfected for 4 h with AR-wt or variant AR expression vectors (2.5 ng) and with the minimal androgen responsive probasin luciferase reporter construct (tk81-PB3) (100 ng) After transfection, cells were treated for 36 h with 1 nM DHT and increasing concentrations of 17-AAG. Gray shading represents the normalized AR-wt luciferase activity (sem) from each of the component experiments. Luciferase activity of each AR variant is expressed as a percentage of AR-wt activity measured in the same experiment. Data represent the mean (sem) activity measured in six replicate wells (See Color Insert)
from hormonal control (Fig. 1, Constitutive bypass). Signaling by this variant may also impart a paracrine response that promotes constitutive activity of wild-type AR in adjacent cells (Monge et al. 2006). The activity of constitutive truncated AR variants is not inhibited by the ansamycin antibiotics unlike the wtAR or all other mutant ARs analyzed (Buchanan, Butler & Tilley, unpublished observations). The constitutive response of truncated ARs may stem from decreased Hsp90 chaperone control (see Sect. 4.6), probably via amino acids 704–758 (Pratt and Toft 1997). This mechanism may also be related to the inability of the truncated ARs to adopt an N/C interaction, the release of distinct transcriptional domains in the NTD (see Sect. 6.1), constitutively nuclear localization, and more rapid cycling on and off response elements compared to the wild-type receptor (Farla et al. 2004; Tepper
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et al. 2002). Truncation of the AR after amino acid 718 (i.e., retaining the Hsp90-binding site) results in an inactive receptor variant that supports an argument for chaperone involvement (Gottlieb et al. 2004). The options to target constitutive truncated ARs likely are limited to those that act directly on receptor synthesis to reduce AR levels in the cell (see Sect. 4.9).
3.9
A New Wave: Agents Targeting AR Synthesis
As a consequence of the diverse ways the AR protein appears able to circumvent inhibition by specific agents, and to maintain sufficient signaling for continued survival and/or growth of prostate cancer cells, it has long been argued that a better approach to preventing AR signaling may be to inhibit receptor synthesis (Scher et al. 2004). Three approaches have been shown somewhat effective: antisense oligonucleotides, short hairpin RNAs, and histone deacetylase inhibitors (Fig. 1, Antisense, shRNA & HDACIs). Prostate-restricted expression of an AR short hairpin RNA (shRNA) using a lentiviral vector inhibited prostate tumor growth in vivo and delayed tumor progression following castration (Cheng et al. 2006). Similarly, AR antisense oligonucleotides reduced AR level and expression of the androgen-regulated PSA gene in vitro and in vivo (Ko et al. 2004). The histone deacetylase inhibitors (HDACIs) suberoylanilide hydroxamic acid (SAHA/vorinostat) and phenylbutyrate suppressed the growth of prostate cancer cells in vitro and in vivo (Butler et al. 2000; Carducci et al. 2001; Gore and Carducci 2000; Kelly et al. 2003; Marks et al. 2001), with efficacy partially stemming from reduced AR expression and activity (Marrocco et al. 2007). Although it is not yet known whether these agents will perform equally well against known AR variants or select for new ones under treatment conditions, successful application of these compounds clinically depends on developing more suitable means of introducing and maintaining them in prostate cells.
4 The DNA-Binding Domain 4.1
Collocation of AR Gene Mutations at 574–586
The AR-DBD typically is ascribed only the function of binding to specific response elements in DNA, but it has also been implicated in receptor dimerization, forms a putative protein–protein interaction surface, and was found to interact with several coregulators in a ligand-dependent manner (Blanco et al. 1998; Moilanen et al. 1998, 1999; Poujol et al. 1997). A total of five somatic missense mutations were identified in the AR-DBD in clinical prostate cancer that collocate to the 12 amino
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acids 574–586 at the carboxyl-terminal end of the first zinc finger motif (Marcelli et al. 2000; Tilley et al. 1996). None of these residues has been reported to contain mutations that cause receptor inactivation in AIS. Highlighting the potential of these variants to mediate prostate cancer growth, substitution in the DBD of T575A in conjunction with the LNCaP T877A variant results in an AR promiscuous for ligand binding and transactivation at non-canonical-binding sites (Monge et al. 2006). This supports previous studies that demonstrated that mutations in the AR DBD can affect selectively AR transactivation and transrepression functions on different promoters despite reduced DNA-binding ability (Aarnisalo et al. 1999; Bruggenwirth et al. 1998). In addition, it has been suggested that mutations in the DBD might result in AR variants that bind to response elements normally specific for other nuclear receptors, thereby leading to inappropriate activation or repression of growth-regulatory pathways (Poujol et al. 1997). Mutations in the DBD also might alter the affinity of the AR for particular response elements, either directly or via modification of specific coregulator interactions, to produce both qualitative and quantitative differences in the regulation of its target genes. In a converse finding that supports this hypothesis, AR response elements differing by only a single nucleotide were found to exert different allosteric effects on AR structure that may relate to transcriptional capacity (Lai et al. 2007).
4.2
Targeting DNA Binding and Response Elements
Blocking the interaction of the receptor with its specific response elements, or preventing the generation of an active transcription complex on DNA, provides an alternative means of inhibiting AR function. The former approach has been tested by transfecting double-stranded DNA ‘‘decoys,’’ which consist of an androgen response element (Kuratsukuri et al. 1999), and synthetic polyamide molecules designed to target the consensus AR-binding site throughout the genome (Nickols and Dervan 2007) (Fig. 1, Response element blockers). The latter approach has been attempted using dominant negative AR inhibitors (Fig. 1, ARIs) (Butler et al. 2006; Palvimo et al. 1993). Whereas each of these approaches has blocked AR activity on known response elements, slowed growth of prostate cancer cells in culture and/or induced apoptosis (Kuratsukuri et al. 1999; Nickols and Dervan 2007; Palvimo et al. 1993), two conceptual challenges limit further development. First, difficulties associated with delivering these types of molecules to whole animals have not been sufficiently resolved. Second, recent genome-wide AR-binding studies (ChIP-chip) demonstrate that (1) the AR interacts with DNA directly via response elements and indirectly via other factors, and (2) the full complement of AR-binding sites or the determinants of DNA binding remain unknown (Bolton et al. 2007; Takayama et al. 2007; Wang et al. 2007) (Buchanan G, Coetzee GA & Frenkel B, unpublished data). In particular, those studies had difficulty in defining a consensus ARE and suggest that direct binding to DNA is mediated only loosely by the response element, but is stabilized in a cell-specific manner by collaborating factors binding to neighboring sites.
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5 The AR Amino-Terminal Domain Transcriptional regulation by the AR and other nuclear receptors stems from the recruitment of a unique complement of cofactors that mediate nuclear-cytoplasmic movement, response element recognition, chromatin remodeling, and ultimately recruitment of basal transcription factors. Whereas in many nuclear receptors this process is mediated through the conserved AF-2 surface in the LBD (see Sect. 4.1), the structural constraints of high-affinity hormone binding have restricted evolutionary changes in AF-2, and limited interactions with this surface to a few common proteins (He et al. 2004b). Consequently, the evolution of the different steroid receptors for enhancer/promoter and cell-specific regulation has necessitated divergence and strengthening of the NTD for distinct cofactor interactions (Han et al. 2005; He et al. 2004a; Thornton and Kelley 1998). Nonetheless, the nature of the NTDs as large, evolutionarily divergent, and structurally disordered domains has meant that they have received less attention than the domains involved in DNA and ligand binding. Mutations identified in the AR-NTD in prostate cancer have demonstrated in vitro and in vivo the critical roles played by intrinsic and induced structural order of this domain for normal receptor function, provided insight into functional subdomain structures, and have challenged the paradigm of AR involvement in prostate cancer development and progression.
5.1
AR NTD Structure and Activation Functions
For most steroid receptors, the conformational changes wrought in the LBD by agonist binding results in the formation of the primary p160 coactivator interaction surface, AF-2. The binding of p160s to this highly structured and conserved surface via LxxLL-containing peptides in turn mediates receptor transactivation capacity by both direct and indirect mechanisms (He and Wilson 2003). AR activation is distinct from this generalized model in two related ways. First, the AR AF-2 surface has been modified evolutionarily to preferentially interact with the 23FQNLF27 peptide of the AR-NTD in the N/C interaction, thereby displacing p160 interactions to the AR-NTD (Chang and McDonnell 2002). Second, AR transactivation capacity is determined almost exclusively by the NTD (Jenster et al. 1995). Consequently, the AF-2 surface makes a smaller direct contribution to AR transactivation activity than the analogous region of the other steroid receptors, whereas the AR-NTD plays a far more critical role. Early deletion mapping broadly categorized the AR-NTD as containing two transactivation units (TAU; Fig. 4a). TAU-1, which consists of amino acids 111–361, was defined as the minimal region sufficient for normal ligand-dependent activation of the AR, and deletion of TAU-1 completely abrogates receptor transactivation. TAU-5, defined as amino acids 352–537, was found to be sufficient and
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Fig. 4 Structure of the AR amino-terminal domain (AR-NTD) and consequences of somatic NTD mutations in human prostate cancer. (a) Schematic to-scale structure of the AR-NTD delineating activation functions TAU-1 and TAU-5, secondary a-helical structure elements (H) determined by chemical and/or in silico analysis (Reid et al. 2002) and recognized subdomain structures within the NTD including homopolymeric repeat sequences [polyglutamine, (Q)n; polyglycine, (G)n; polyproline, (P)n] and peptides implicated in interdomain communication (N/C interaction) and cofactor binding (i.e., 23FQNLF27 and 433WHTLF437). SIGNATURE refers to the evolutionarily conserved NTD signature sequences 234–247. (b) Amino acid conservation across species in the
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necessary for the constitutive activity of ARs truncated of the LBD (Callewaert et al. 2006; Chamberlain et al. 1996; Jenster et al. 1995). Further experiments are required to determine whether the two TAUs exhibit functional redundancy or are completely independent, and how this relationship changes with the cellular and environmental context. However, the relative contribution of TAU-1 and TAU-5 to AR activity depends on the LBD, which suggests that the N/C interaction is important. Chemical analysis revealed that the AR-NTD has only a small number of predicted secondary structure elements, adopts a disordered but flexible structure typical of transactivation domains, and exhibits increased structural order following recruitment of the transcriptional machinery and other transcriptional coregulators (Kumar et al. 2004; Reid et al. 2003). More recently, core sequences of the NTD have been found to exist in collapsed disordered conformation or molten globulelike state that has distinct implications for AR transcriptional function (Lavery and McEwan 2008a, b).
Fig. 4 (Continued) AR-NTD and relationship to somatic AR mutations in prostate cancer. The five most evolutionarily conserved regions of the AR-NTD (Thornton and Kelley 1998) are shown as blue bars. Orange inset represents the AR-NTD signature sequence, which is almost 100% conserved among all AR species, delineated by alignment of 47 full-length AR-NTD sequences from 42 different animals (note that several fish species have been found to have alpha, beta, and gamma AR isoforms) using the Clustal W algorithm in Vector NTI (Invitrogen). The position of each somatic AR mutation identified in the AR-NTD in prostate cancer is shown as a vertical bar, with length proportional to the number of individual reports of mutations at that residue. Numbers and boxing indicate regions to which mutations cluster/collocate. (c) AR polyQ length frequency data from control populations. The human AR-NTD contains a polymorphic uninterrupted glutamine tract (polyQ) of 6–39 residues with a median of 19–20 in African American, 21–22 in Caucasian, 22–23 in Asian, and 23 in Hispanic populations (Edwards et al. 1992; Irvine et al. 1995; Spurdle et al. 1999), with 91–99% of all alleles within the range Q16–Q29. In addition to germlinelength polymorphisms, recent data suggest that the AR-CAG tract is a target for somatic alterations in clinical prostate cancer and animal models of the disease, which include contractions in repeat length and missense mutations (Buchanan et al. 2004; Hyytinen et al. 2002; Wallen et al. 1999; Watanabe et al. 1997). (d) A novel dual missense mutation identified in human prostate cancer results in disruption of the polyglutamine repeat of the receptor with two leucine (L) residues as indicated (i.e., polyQ2L). In silico modeling and analysis of calculated energy suggest that the polyQ2L variation forces a more rigid structure on the AR-NTD (ribbon diagrams). This conclusion is supported by secondary structure prediction [NNpredict algorithm of Kneller et al. (1990)], which indicates an increase in the a-helical (H) content of the region. (e) The AR-NTD signature sequence. Multiple sequence alignment of ARs from different species was performed using the Clustal W algorithm. The region between residues 234–247 (human numbering) is the most highly conserved in all known AR species. This region contains the only six residues conserved in all 47 known full-length AR NTDs. Blue, identical; Yellow, conserved; White, nonhomologous. (f) Overlays of ten independent 3D molecular dynamic model structures of AR-wt and AR-E326G signature sequence peptides, with each solution colored according to positional fluctuation (blue, minimal displacement; red, maximum displacement). The lower average positional fluctuation (p < 0.05; Root Mean Square Deviation, RMSD) of the E236G compared to AR-wt peptide suggests a more stable secondary structure (See Color Insert)
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AR Gene Mutations in the NTD
The study of the AR-NTD is complicated by the presence of two long GC-rich polymorphic trinucleotide repeat sequences. As a consequence, and due to the relative length of the NTD and other factors, very few studies have sequenced the AR-NTD in prostate cancer. Nonetheless, emerging evidence suggests a role of AR mutations in this domain in disease etiology, including risk, the oncogenic potential of the receptor, and survival of tumor cells during ADT. In particular, three studies suggest that surgical and/or medical castration results in the preferential accumulation of mutations in the AR NTD. This was first documented in the autochthonous transgenic adenocarcinoma of the mouse prostate (TRAMP) model (Han et al. 2001). In that study, 6/8 AR gene mutations identified in tumors from intact TRAMP mice at 24–28 weeks of age were located in the AR-LBD. In mice castrated at 12 weeks, 7/7 AR gene mutations identified in recurrent tumors at 24–28 weeks of age were located in the AR-NTD. In clinical prostate cancer, two independent studies have shown that AR gene mutations predominantly arise in the NTD in patients treated with combined androgen blockade, and moreover are located almost exclusively within the same 40 amino acid region at the C-terminal of TAU-5 (Hyytinen et al. 2002; Scher et al. 2004). A similar incidence and/or pattern has not been observed in primary disease or in patients treated with monotherapy (Scher et al. 2004). Until recently, insufficient AR-NTD mutations had been identified in human prostate cancer to formally test their collocation in this domain. However, those now listed in the AR gene mutations database (http://androgendb.mcgill.ca) and identified recently by us and others (Scher et al. 2004) (unpublished) demarcate four clusters of mutations in the AR-NTD, include two with sufficient numbers to be formally defined as ‘‘hotspots’’ for mutations (Fig. 4b). Three of the five short NTD sequences conserved during evolution (Thornton and Kelley 1998) almost precisely overlap with mutation clusters at 221–269, 433–455, and 497– 537 (Fig. 4b).
5.3
Collocation of AR Gene Mutations at 43–90
The first area of collocation in the AR-NTD is between amino acids 43 and 90, a region with poor evolutionary conservation that contains the polyglutamine tract (polyQ) (Fig. 4b). The AR-polyQ consists of a string of glutamine residues ranging in length from 7 to 40 repeats in healthy individuals, with a median length of 20–21 repeats in African American, 22–23 in Caucasian, 23–24 in Asian, and 24 in Hispanic populations (Balic et al. 2002; Bennett et al. 2002; Edwards et al. 1992; Giovannucci et al. 1997; Hsing et al. 2000; Jin et al. 2000; Platz et al. 2000; Sartor et al. 1999) (Fig. 4c). More than 91–99% of AR alleles across all racial-ethnic groups encode a polyglutamine tract within the range of Q16–Q29 (Buchanan et al.
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2004). Variation within the normal range has been associated with prostate cancer risk, age of onset and/or risk of advanced disease at diagnosis (Beilin et al. 2000; Giovannucci et al. 1997; Hsing et al. 2000; Stanford et al. 1997), and an extraordinary array of human diseases that include breast and endometrial cancers, schizophrenia, infertility, polycystic ovarian syndrome, and cryptorchidism (Palazzolo et al. 2008). The AR-polyQ tract is a target for somatic contraction or missense mutation in clinical prostate cancer and animal models of the disease (Fig. 4b) (Buchanan et al. 2001b; Hyytinen et al. 2002; Schoenberg et al. 1994; Wallen et al. 1999; Watanabe et al. 1997). Because ARs with shorter polyQ tracts are more active (Buchanan et al. 2004), most believe that the relationship between AR polyQ length and disease relates simply to a gradient of AR transcriptional competence. This simple interpretation has been challenged by the study of a dual somatic missense mutation identified in prostate cancer that disrupts the pure AR-polyQ tract with two leucine residues [i.e., from wtAR(Q)23 to AR-polyQ2L = AR(Q)12L(Q)6L(Q)3] (Fig. 4d) (Buchanan et al. 2004). The AR-polyQ2L variant has the following properties compared to wtAR: (1) twofold to fourfold greater ability to transactivate target genes despite decreased protein stability; (2) increased response to the p160 coactivators; (3) increased structural order and less flexibility across the polyQ repeat region (Fig. 4d); and (4) 50% reduced capacity to undergo N/C interaction (Buchanan et al. 2004). Increased p160 coactivator levels during prostate cancer progression (Chmelar et al. 2007; Gregory et al. 2001) provide an obvious means by which AR-polyQ2L might maintain prostate cancer cell growth following ADT. The N/C acts in a temporal and spatial manner to protect the receptor from inappropriate binding and timing of cofactor interactions (van Royen et al. 2007), the polyQ2L variant probably alters. Alternatively, because the dynamic relationship of the AR with N/C interaction and p160 recruitment. The polyQ2L AR variant is consequently predicted to respond quite differently to the cellular milieu of cofactors, which may translate to activation of distinct gene pathways. These studies provided both a novel mechanism for AR-mediated survival of prostate cancer cells during ADT, and a model for the relationship between transactivation capacity, NTD flexibility, and the vital in vivo N/C interaction. These findings also precipitated a more detailed analysis of wtAR N/C interaction and polyQ length, which revealed that maximal N/C is sustained only for 16–29 glutamine repeats (Buchanan et al. 2004). In addition to providing an evolutionary reason why AR-polyQ length has been maintained mostly within this same range across populations (Fig. 4c), these findings suggest that functionality of AR with a polyQ length either shorter or longer than the critical range of 16–29 residues may be a more important mediator of disease phenotype than a stepwise reduction in activity with increasing polyQ length across the entire range (Buchanan and Tilley 2000). In this model, ARs with 16–29 glutamines in the polyQ tract could be considered functionally equivalent, and those with either shorter or longer repeats as distinct. As most population studies have historically used the median AR-polyQ repeat length as a cutpoint, many relationships between AR genotype and disease may well have been missed.
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Clustering of AR Gene Mutations at 221–269. The AR NTD-Signature Sequence
The NTD is the least conserved of the steroid receptor domains, sharing as little as 20% amino acid homology between even the closely related 3-ketosteroid receptors for, progestins, glucocorticoids, and mineralocorticoids (i.e., PR, GR, and MR, respectively). In comparison, these receptors share approximately 70–80% homology in the LBD and DBD regions. The AR NTD itself has diverged in sequence during evolution at almost twice the rate of the NTDs of the other steroid receptors, and only five small regions of the NTD have been conserved (Fig. 4b) (Thornton and Kelley 1998). Alignment of 47 informative full-length AR-NTD sequences from 42 different animals (data not shown) confirmed previous observations (Han et al. 2001; Thornton and Kelley 1998) that the most dramatically conserved region of the NTD resides between amino acids 234 and 247 (Fig. 4b, e). This small region, which our group has coined the AR-NTD signature, has no sequence homology to any other protein, and contains the only six amino acids conserved in all known AR-NTD sequences (Fig. 4e). Mutations have been identified recently proximal to the AR-NTD signature in clinical prostate cancers (DM Robins, unpublished) and TRAMP tumors (i.e., A234K & E236G, Fig. 4E) (Han et al. 2001). In silico modeling suggested that A234K and E236G substitutions stabilize the secondary structure about the NTD signature sequence, which may confer greater order to the disordered NTD (Fig. 4f). In support of this hypothesis, each of these AR variants was found to confer greater basal activity (i.e., in the absence of ligand), and increased response to androgenic and nonandrogenic ligands compared to wtAR (Fig. 5a). The basal activity of AR-E236G consistently was higher than wtAR, but cell and experimental variation in the comparative response of AR-E236G to ligand (Fig. 5b) suggests altered response or interaction with a variable pool of coregulators (Buchanan et al. 2004). AR-E236G exhibits a greater response to coactivators ARA70 (Fig. 5b) and ARA160 (Han et al. 2001), and more than a 40% reduced interaction with the C-terminal of Hsp70-interacting protein (CHIP) chaperone that acts as a negative regulator or AR activity (He et al. 2004a). The prostate and epithelial cell-specific probasin promoter was used to generate three transgenic mouse lines overexpressing either wtAR, the promiscuous ART877A LNCaP variant (see Sect. 4.1), or AR-E236G variant as a model of altered AR function (Han et al. 2005). Whereas transgenic mice overexpressing wtAR and AR-T877A had no discernable phenotype, 100% of mice expressing AR-E236G developed lesions similar to prostatic intraepithelial neoplasia (PIN) at 12 weeks of age, and advanced prostate cancer with lung metastases by 50 weeks of age (Fig. 5c) (Han et al. 2005). Unlike the TRAMP model, AR-E236G mice do not rely on overexpression of viral oncoproteins or supraphysiological levels of steroids to initiate tumorigenesis. Instead, these findings highlight the functional significance of mutations in the AR NTD, and argue that mutations resulting in aberrant AR function (in the case of E236G via altered interaction with coregulators), can turn
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Fig. 5 Mice expressing AR-E236G develop spontaneous prostate cancer with distant lung metastases. (a) Transactivation profiling of AR-wt and receptor variants identified in clinical (LNCaP T877A variant) prostate cancer and animal models (A234K and E236G) of the disease. Data show mean (sem) transactivation capacity of AR-wt and AR variants normalized for b-galactosidase activity in transfected PC-3 cells in the absence or presence of different steroids. (b) AR-E236G transactivation capacity is cell type and/or ligand dependent, and more responsive to cofactors compared to AR-wt. Transactivation analysis was performed in ultraresponsive PC-3 cells (Buchanan et al. 2004) under different conditions as A (left-hand panel), or in the absence or
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the AR into an oncogene sufficient to trigger tumorigenesis in the prostate and promote metastatic progression. An important hypothesis arising from these observations is that a change in the level or composition of the AR cofactor milieu in a prostate cancer cell may be sufficient to promote the receptors oncogenic potential in the absence of structural changes to the receptor itself. Affymetrix 430 2.0 microarray analysis on prostate RNA from wtAR, ART877A, and AR-E236G overexpressing mice early in disease etiology has revealed two distinct facts: (1) overexpression of all three ARs resulted in increased or decreased expression of a unique subset of target genes; and (2) the greatest number of differentially expressed genes was observed in the comparison of wtAR with E236G, and the least for E236G with T875A (Fig. 5d). A subset of AR variants may promote cancer initiation and/or progression by qualitatively or quantitatively affecting expression of a unique complement of target genes (Fig. 1, Differential gene activation). The analysis of these particular AR variants (such as E236G) may allow identification of the pivotal genes capable of mediating oncogenic transformation of the prostate well before prostate cancer arises. This is a paradigm shift from defining those genes expressed at later stages of tumorigenesis as a consequence of malignant transformation.
5.5
Clustering of AR Gene Mutations at 431–455
The clustering of mutations at residues 431–455 intersects three documented structural and/or functional elements in the AR-NTD; TAU-5, and the polyglycine repeat (polyG) and 433WHTLF437 sequences encompassed by it (Fig. 4a). Whereas the individual mutations have not been investigated in detail, their clustering supports an emerging body of evidence that TAU-5 is a critical mediator of normal AR activity (Callewaert et al. 2006; Funderburk et al. 2008) (EF Need and Fig. 5 (Continued) presence of specific receptor coregulators as indicated (right-hand panel). (c) Pathobiology of prostate samples and tumors arising in transgenic mice overexpressing AR-E236G. Representative histological sections (5 um) were prepared from AR-wt and AR-E236G mice at 12 and 50 weeks of age, and analyzed as follows: H&E staining of 12-week-old ventral prostate samples demonstrates (i) normal architecture in AR-wt overexpressing mice, but (ii) the presence of PIN-like lesions in mice overexpressing AR-E236G. Mice overexpressing AR-T877A were indistinguishable from those overexpressing AR-wt (not shown). (iii–vi) Staining of locally advanced prostate cancers in AR-E236G mice at 50 weeks with H&E, and HA, AR and E-cadherin antibodies, respectively. (vii-ix) Staining of lung metastasis from AR-E236G mice at 50 weeks of age with H&E, HA, and NKX3.1 antibodies, respectively. Mice overexpressing the LNCaP AR variant, AR-T877A, exhibited similar phenotype to wtAR overexpressing mice. (d) Differential gene expression in prostate samples from AR-wt, AR-T877A, and AR-E236G overexpressing mice. For each mutant mouse line, RNA pooled from the ventral prostates of three animals at 12 weeks of age was analyzed using Affymetrix mouse genome 430 2.0 arrays. Shown on clock diagrams are the number of genes with significantly increased or decreased gene expression among animal lines. These data suggest that overexpression of each AR is affecting a unique subset of genes, with the largest difference between AR-wt and AR-E236G (See Color Insert)
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G Buchanan, unpublished). In particular, TAU-5 appears to be the primary mediator of transactivation by an AR lacking the LBD or activated by cytokine or growth factor pathways. In addition, TAU-5 has been implicated in DNA binding and as the primary NTD interaction site of the p160 coactivators, the transcriptional coregulator p300/CBP, and the corepressor SMRT (Alen et al. 1999; Berrevoets et al. 1998; Brodie and McEwan 2005; Callewaert et al. 2006; Chamberlain et al. 1996; Dotzlaw et al. 2002; Fronsdal et al. 1998; Ikonen et al. 1997; Ma et al. 1999). Of particular importance is the potential for p160s to form a bridge between the AR-NTD and LBD, thereby stabilizing the receptor for maximal activity (Ma et al. 1999; Shen et al. 2005). In certain stages of AR activation, this bridge may circumvent the N/C interaction and alter AR function (Shen et al. 2005). While a model that incorporates each of these effects is still lacking, the body of evidence argues that TAU-5 functions to ‘‘fine-tune’’ transcriptional response of the AR based on the cellular complement of coregulators. AR N/C interaction is determined primarily by interaction of the 23FQNLF27 sequence with the AF-2 surface in the LBD after agonist ligand binding and has at least three key effects: slowing the rate of ligand dissociation, excluding coregulator interactions from AF-2, and allowing activation of chromatin-integrated target genes (He et al. 2000, 2001; Li et al. 2006; Toumazou et al. 2007). However, N/C also depends partially on the structurally similar 433WHTLF437 peptide in TAU-5, which co-ordinates with 23FQNLF27 to slow ligand dissociation but interacts with the LBD in a ligand and AF-2 independent manner (He et al. 2000). Our unpublished data suggest that deletion of 433WHTLF437 leads to increased interaction of the AR-NTD with both the p160 coactivator, GRIP1 and the corepressor, SMRT (EF Need and G Buchanan, unpublished data). Blocking N/C also increases AR interaction with the p160 coactivators (van Royen et al. 2007). Missense mutations identified in or flanking the 433WHTLF437 peptide in clinical prostate cancer (Fig. 4b) are likely to have significant consequences for AR function and modulate N/C and/or coregulator interactions at either TAU-5 or AF-2. The AR polyglycine (polyG) repeat at amino acids 449–472 in TAU-5 exhibits polymorphic tendencies and ranges 10–27 repeats in the normal population, but its length is biased to just four common alleles of 19–20 (5.5%) or 23–24 (87.5%) residues (Platz et al. 1998). This bias may reflect a biological/functional requirement for a precise polyG length in the protein or greater replication fidelity of the encoding sequence. Nonetheless, longer repeats have been associated with increased AR activity, penile hypospadias, and cryptorchidism in men, and higher-grade endometrial cancers in women (Radpour et al. 2007; Rodriguez et al. 2006; Werner et al. 2006), while men with AR polyG repeat lengths shorter or longer than 23 residues may have a slight decreased risk of prostate cancer (Platz et al. 1998). The effect of polyG length variation may depend on sequences within the AR hinge (Werner et al. 2006), which suggests a link between mutations in this region and communication among AR domains. Four somatic missense mutations have been identified in the polyG tract that result in the missense replacement of glycine residues within the repeat (Sanchez et al. 2006), but the precise consequences for receptor function remain unknown.
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Constitutive Bypass Via Ligand-Independent Activation
Growth factors, cytokines, protein kinase-A and overexpression of HER2/neu have been found to enhance AR transactivation capacity, an effect generally thought to stem from altered/induced states of receptor phosphorylation, and/or phosphorylation of AR cofactors (Fig. 1, Stimulation) (Craft et al. 1999; Culig et al. 2002; Gregory et al. 2004; Nazareth and Weigel 1996; Rowan et al. 2000a, b; Ueda et al. 2002a; b; Yeh et al. 1999). For example, high Her2/neu expression exacerbates the impact of high AR levels on disease progression and metastatic potential in clinical prostate cancer (Ricciardelli et al. 2008). Similar mechanisms appear to promote sufficient AR function in the absence of androgens in vitro or in vivo (Craft et al. 1999; Culig et al. 1994; Gregory et al. 2004; Grossmann et al. 2001; Nazareth and Weigel 1996; Ueda et al. 2002b) for activation of androgen-regulated genes and proliferation and survival of prostate cancer cells (Wen et al. 2000; Yeh et al. 1999). These constitutive bypass mechanisms do not involve an AR truncation mutation. Whereas a direct link between the phosphorylation status of particular AR or cofactor residues and effects on downstream receptor signaling have been difficult to demonstrate conclusively, the AR-NTD appears pivotal. Of the approximately 13 putative AR phosphorylation sites, 11 reside in the NTD and only one in each of the LBD and hinge regions (Gioeli et al. 2002, 2006; Guo et al. 2006; Lin et al. 2001; Mahajan et al. 2007; Weigel and Moore 2007). While none of the individual phosphorylation sites has been identified as mutated in prostate cancer, several lie within areas of mutation clustering and have been implicated in similar transcriptional functions. In particular, S256 and Y267 phosphorylation sites lie with mutations clustered about the NTD signature at residues 221–269; S81 and S94 sites are proximal to collocation with the polyQ at 43–90; S515 and Y534 are located within the hotspot for mutations in TAU-5 at residues 497–537. Mutation of the two NTD Erk2 phosphorylation sites in TAU-5 to alanine (i.e., S425A and S515A) impaired AR activity, decreased nuclear localization, and caused cytoplasmic aggregation of the receptor (Funderburk et al. 2008), which suggests an important link between AR phosphorylation and normal receptor function. Whether receptor NTD mutations identified in prostate cancer affect interaction with, or response of, the AR to kinase signaling has not been addressed specifically. Alternatively, AR activation by androgens initiates a rapid nongenomic phosphorylation cascade that has been implicated in a multitude of downstream consequences for AR activity and growth of prostate cancer cells. For example, Hsp27 is phosphorylated via MAPK14/p38 within minutes of androgen binding to the AR, displaces Hsp90 in the AR complex, and chaperones the receptor into the nucleus and onto AREs (Zoubeidi et al. 2007). Specific AR mutations may affect these events. Recognition of the importance of phosphorylation cascades in AR signaling should lead to the study of specific inhibitors (Fig. 1, Inhibitors), or the use of antibodies to cell-surface receptors that impact these processes (Fig. 1, Antibodies). AR antibodies, which have been shown to suppress AR activity and proliferation of androgen-independent prostate cancer cells, have the potential to circumvent both classical AR activity and nongenomic AR signaling (Zegarra-Moro et al. 2002).
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Collocation of AR Gene Mutations at 497–537
The second area of collocation in TAU-5 lies at the far C-terminus of the NTD between amino acids 497 and 537, in one of only three small areas of high amino acid sequence identify between species (Fig. 4b). Remarkably, more than 25% of all mutations detected in the AR-NTD in prostate cancer have been detected in the 497–537 region (Fig. 4b), with the majority identified in prostate tumors from men following complete androgen blockade (Need, Tilley, Buchanan, unpublished) (Hyytinen et al. 2002). Preliminary investigation of the 497–537 region, which lies at the far C-terminus of TAU-5, has revealed two dramatic results. First, several mutations identified in prostate cancer between residues 497 and 537 result in increased response to all three of the p160 coactivators compared to wtAR (EF Need, G Buchanan and WD Tilley, unpublished), which is consistent with previous studies that demonstrate a role for his region in maximal p160 response (Callewaert et al. 2006). Enhancement of AR response to coregulators, analogous to increased levels of the p160s in prostate cancer (Culig et al. 2004; Gregory et al. 2001), likely creates a permissive environment for AR activation in the low androgen environment or in response to nonclassical agonists. Second, deletion of residues 497–537 only moderately affected transcriptional response of the AR, but completely abolished receptor N/C and p160 interactions as determined using the mammalian two-hybrid system (EF Need, G Buchanan and WD Tilley, unpublished). Whether these two events are mutually exclusive and/or exhibit a spatial and temporal relationship with the AR remains to be defined. Nonetheless, as regions primarily responsible for p160 and N/C interactions lie elsewhere in the NTD, these findings highlight a structural or indirect functional requirement for residues 497–537 in normal AR function. Clustering of mutations to the 431–455 and 497–537 regions of TAU-5 thus constitutes two examples of where mutations in prostate cancer disrupt inter- or intramolecular communication between AR domains or cofactor interactions. Like other regions of mutation clustering in the NTD, these changes may be permissive for continued AR signaling during ADT and/or alter, AR function sufficiently to promote oncogenic transformation and disease progression as exemplified by the E236G mutation.
6 Conclusions There are several compelling genetic and functional reasons why germline variations in the AR that relate to disease are tolerated, and why the receptor itself appears to be particularly vulnerable to somatic mutations in prostate cancer. (1) The AR resides on the X chromosome and is present as a single allele in men, and as a single active allele in women due to X inactivation. Thus, somatic and germline alterations in the AR are more likely to exhibit functional consequences. (2) The AR itself is not essential for life but exhibits diverse functional roles from
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apoptosis to growth stimulation, and impacts, or is impacted on, by a vast array of cellular signaling systems. This provides the necessary functional leeway by which interrupted androgen signaling can be tolerated and ultimately adapted to the particular needs of a prostate cancer cell, for example. (3) PolyQ and polyG repeat sequence expansion in higher-order mammals appears to represent a trade-off between the capacity for transcriptional activation and fine-tuning of functional control. Unfortunately, the nature of the encoding DNA sequence makes these repeats particularly susceptible to both germline and somatic variation. (4) By aggressively targeting the AR pathway in a multifocal prostate cancer, and particularly after the disease has acquired the necessary genetic heterogeneity for metastatic spread, mutant ARs that serendipitously confer resistance to treatment (or are stimulated by them) are selected. Given the protracted development and progression of prostate cancer, and that combinations of local and systemic therapies are now administered earlier in the course of disease, the characteristics of prostate cancer have changed. (5) Transcriptional regulation by the AR involves interaction with a unique complement of coregulators that mediate nuclear-cytoplasmic movement, recognition of response elements, chromatin remodeling, interdomain communication, and recruitment of basal transcription factors. As detailed in this chapter, perturbation of this finely balanced cascade of events is achieved easily by a broad range of single point mutations that can have dire functional consequences, which are exemplified by the AR-E236G variant. The collocation of AR gene mutations in prostate cancer has been instructive in defining the role of the receptor in disease etiology, but few mutations have been detected outside of the canonical LBD until recently. Sufficient mutations have now been identified in the AR-NTD to assign four regions of clustering/collocation to that domain, three of which coincide with the limited evolutionarily conserved NTD sequence. A unifying theme is emerging from the vast body of functional data on mutations that cluster/collocate throughout the AR in prostate cancer. In almost every case, the direct or indirect consequence is the disruption of AR-cofactor interactions or interdomain communication. Continued analysis of these AR variants allows delineation of the subdomain structure of the receptor and key coregulatorbinding sites; provides functional data to support and/or initiate structural analysis; defines the biology underpinning ARs role in the development, progression, and treatment resistance of prostate cancer; and suggests new ways of targeting the receptor for treatment. Acknowledgments The authors acknowledge the contributions of Drs. Rene Chmelar and Guangzhou Han (Fred Hutchinson Cancer Research Center, University of Washington) and technical assistance of Ms. Joanna Treloar. The authors are supported by the Prostate Cancer Foundation of Australia (GB, ID#Y102), the National Health and Medical Research Council of Australia (WDT, ID#453662), The United States Department of Defense (WDT, ID#PC060443), and a SPORE Grant from the National Cancer Institute (DMR, NCI P50 CA69568). GB holds a National Health and Medical Research Council of Australia CJ Martin Biomedical Fellowship. EFN is a recipient of a Freemasons Foundation Postdoctoral Fellowship.
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Functional Motifs of the Androgen Receptor Elizabeth M. Wilson
Abstract Androgen receptor (AR) transcriptional activity stimulated by high affinity binding of testosterone or dihydrotestosterone involves a dynamic NH2and carboxyl-terminal (N/C) interaction between an AR NH2-terminal FXXLF motif (23FQNLF27) and a hydrophobic interaction surface in the ligand binding domain known as activation function 2 (AF2). The AR N/C interaction slows the dissociation rate of bound androgen, stabilizes AR and competitively inhibits activity from AF2. Functional significance of the androgen-dependent AR N/C interaction is supported by naturally occurring germline mutations in AF2 that decrease AR transcriptional activity and cause the androgen insensitivity syndrome by interfering with the N/C interaction without altering equilibrium androgen binding affinity. Gain-of-function AR somatic mutations in prostate cancer can enhance the AR N/C interaction in association with increased AR transcriptional activity. Melanoma antigen gene protein-A11 (MAGE-11) is an AR coactivator that competitively inhibits the AR N/C interaction by binding the AR FXXLF motif, but increases AR signaling through mechanisms that appear to contribute to the development and growth of prostate cancer. The androgen receptor (AR) is a ligand-activated nuclear receptor of the steroid receptor subfamily that mediates the developmental and behavioral effects of the biologically active, naturally occurring androgens, testosterone (T) and dihydrotestosterone (DHT). High-affinity androgen binding targets AR to the nucleus where it interacts with androgen response element DNA associated with androgen-regulated genes. The human syndrome of androgen insensitivity, in which 46XY genetic males have a mutation in the AR gene and are phenotypically female, demonstrates the functional requirement for AR to mediate the effects of androgen during normal male sex development. AR transactivation also contributes to prostate cancer development and progression. Structure and function relationships of AR interacting domains are the focus of this review.
E.M. Wilson Laboratories for Reproductive Biology and the Lineberger Comprehensive Cancer Center. Departments of Pediatrics, and the Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, NC 27599, USA, E-mail:
[email protected]
D. Tindall and J. Mohler (eds.), Androgen Action in Prostate Cancer, DOI: 10.1007/978-0-387-69179-4_11, # Springer Science + Business Media, LLC 2009
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AR has a multidomain structure characteristic of the steroid hormone family of nuclear receptors (Simental et al. 1991). The carboxyl-terminal ligand binding domain (LBD) binds T and DHT with similar high affinity (Wilson and French 1976). The central DNA binding domain binds androgen response element DNA associated with androgen-regulated genes. Naturally occurring mutations in the AR DNA binding domain result in the androgen insensitivity syndrome (AIS) and demonstrate that AR must bind DNA to mediate the biological effects of T and DHT (Quigley et al. 1995). A hinge region lies between the DNA binding domain and LBD and contains an AR nuclear-targeting signal (Zhou et al. 1994). The AR NH2-terminal region is the least conserved in AR between species and relative to other steroid receptors. The largely unstructured AR NH2-terminal region harbors a strong activation function 1 (AF1) that facilitates tissue-specific gene activation. AF1 is not influenced directly by ligand binding but depends on bound androgen for AR dimerization, nuclear transport, DNA binding, and transcriptional activation. AR transcriptional activity is also mediated by activation function 2 (AF2) in the LBD. AF2 bears no structural similarity to AF1, but both activation regions are involved in recruiting coactivator proteins that link to the transcriptional machinery of chromatin. Considerable research has focused on the structural and functional properties of AF2. Our understanding of AF2 derives in part from crystallographic studies made possible by the highly ordered arrangement of alpha helices in the LBD. AF2 is a hydrophobic surface of the LBD that lies contiguous to the ligand binding pocket and is structurally dependent on bound ligand (He et al. 2004). Functional studies on AR demonstrate an AF2 binding specificity unique among steroid receptors for a set of structurally related amphipathic alpha helical motifs.
1 AR N/C interaction Early studies on the structure and function of AR led to the identification and characterization of the androgen-dependent AR NH2-terminal and carboxyl-terminal (N/C) interaction (Langley et al. 1995, 1998; Wong et al. 1993; Zhou et al. 1995). The AR N/C interaction requires high-affinity androgen binding in the LBD and is mediated by an interaction between the AR NH2-terminal FXXLF motif sequence 23FQNLF27 in human AR and the AF2 region in the LBD (He et al. 1999, 2000, 2004) (Fig. 1). Original studies that led to the identification of the AR N/C interaction were based on determinants of ligand and DNA binding and AR stabilization.
1.1
Androgen Dissociation Kinetics
Of the two biologically active androgens, T dissociates about three times faster than DHT despite similar apparent equilibrium binding affinities determined from the ratios of association and dissociation rate constants (Wilson and French 1976).
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Fig. 1 Schematic of the AR FXXLF and WXXLF motifs. Full-length human AR (amino acid residues 1–919) contains the NH2-terminal FXXLF (23FQNLF27) and WXXLF (433WHTLF437) motifs, activation function 1 (AF1) and activation function 2 (AF2), DNA binding domain (DBD), and ligand binding domain (LBD) (He et al. 2001; Wilson 2007). (Copyright 2007, The Endocrine Society)
Consistent with the domain function of steroid receptors, the AR LBD functions relatively independently to bind T and DHT with high affinity. However, early studies demonstrated that deletion of the NH2-terminal region increases the dissociation rate of bound androgen in a manner reminiscent of the faster dissociation of T than DHT (Zhou et al. 1995). The basis for the different dissociation rates of T and DHT lies with the AR N/C interaction and is discussed further (see Sect. 3.2). When an AR fragment containing the DNA-and ligand binding domains is coexpressed with an AR fragment containing the NH2-terminal and DNA binding domains, the N/C interaction causes bound androgen to dissociate more slowly (Langley et al. 1998). These studies provided the first evidence that the AR NH2-terminal region directly influences the kinetics of androgen binding. Small deletions within the AR NH2-terminal region helped to pinpoint the NH2-terminal interacting region to the AR FXXLF motif (He et al. 2000; Zhou et al. 1995). Functional studies of naturally occurring mutations that cause AIS without altering apparent equilibrium androgen binding affinity provided evidence that the FXXLF motif in the AR NH2terminus binds AF2 in the AR LBD and is the basis of the AR N/C interaction (He et al. 1999; Langley et al. 1998). For AR, ligand dissociation rate is directly related to ligand potency as an AR agonist. DHT dissociates more slowly than T, and DHT is the more potent androgen than T. However, T is the predominant androgen produced by Leydig cells of the testis and by theca cells of the ovary and has important physiological effects in muscle and other tissues. Potency differences between T and DHT are related to the inability of T to fully stabilize the AF2 surface for AR FXXLF motif binding, with the result that T dissociates faster than DHT. AR antagonists with moderate to high binding affinity dissociate faster, with dissociation half-times from full-length AR of 5 min for AR antagonist RU56187 (Kemppainen et al. 1999) compared to 1 h for T and 3 h for DHT determined in whole cell cultures at 37 C (Askew et al. 2007; He et al. 2006; Zhou et al. 1995). Dissociation rates of the more classical AR antagonists, hydroxyflutamide and casodex, have not been determined due to their unavailability in radiolabeled form, but are expected to be rapid. AR antagonists bind with only moderate affinity and do not induce the AR N/C interaction in wild-type AR (Langley et al. 1995).
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Ligand and DNA Binding
The AR N/C interaction is one of the most androgen-selective properties of AR. While other steroids, including 17b-estradiol, progesterone and the synthetic progestin, medroxyprogesterone acetate, bind AR with moderate to high affinity, cause AR nuclear localization and activate androgen-responsive reporter genes (Kemppainen et al. 1992), none of these ligands induces the AR N/C interaction, and none is a potent androgen in vivo. Indeed, ligand specificity of the AR N/C interaction closely parallels ligand potency in vivo, providing support for its physiological importance. The AR N/C interaction is also induced in response to synthetic steroids that have anabolic activity in vivo (Kemppainen et al. 1999). Anabolic steroids, such as oxandrolone and fluoxymesterone, induce the AR N/C interaction despite lower equilibrium binding affinities. In the presence of androgen, AR binds androgen response element DNA as a homodimer (Wong et al. 1993). However, in the absence of a crystal structure for full-length AR bound to DNA, the precise structural arrangement is not known. Early studies using AR NH2- and carboxyl-terminal fragments provided some evidence that the N/C interaction influences AR binding to DNA. An AR fragment containing the DNA- and ligand binding domains but lacking the NH2-terminal region binds androgen response element DNA in the absence and presence of androgen. Dimerization of this carboxyl-terminal AR fragment presumably occurs through the DNA binding domain dimerization region. Ligand-independent DNA binding by the carboxyl-terminal AR fragment that lacks the NH2-terminal region raises the possibility that under certain physiological or pathological conditions, AR may bind DNA in the absence of androgen. In striking contrast, when the same AR fragment containing the DNA binding domain and LBD is coexpressed with an AR fragment containing the NH2-terminal and DNA binding domains without the LBD, DNA binding by this stable AR dimer complex requires androgen bound in the LBD (Kemppainen et al. 1999; Zhou et al. 1995). The results indicate that the AR N/C interaction influences AR DNA binding specificity and/or affinity.
1.3
AR Stabilization
AR differs from other steroid receptors because it is stabilized against degradation by high-affinity agonist binding, such as T, DHT, and synthetic androgen agonist derivatives such as methyltrienolone (R1881) (Kemppainen et al. 1992; Zhou et al. 1995). Other steroid receptors are typically down-regulated by agonist binding. Agonist-dependent stabilization of AR is another consequence of the androgendependent AR N/C interaction mediated by AR FXXLF motif binding to AF2 that appears to be critical for AR transcriptional activity (He et al. 2001; Langley et al. 1998). The extent to which a ligand stabilizes AR directly correlates with its ability
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to act as an agonist (Kemppainen and Wilson 1996). As noted earlier, ligands such as estradiol and progesterone, and the antagonist hydroxyflutamide, bind AR with moderate affinity, but do not stabilize AR against degradation due to their inability to induce the AR N/C interaction (Kemppainen et al. 1999). AR appears to be more stable in prostate cancer cell lines in the absence and presence of androgen, and in the CWR22 tumor after recurrence in the relative absence of circulating androgen (Gregory et al. 1998, 2001c). The mechanism for increased AR stabilization in castration-recurrent prostate cancer is not known, but occurs in association with increased AR nuclear localization and may be related to its interaction with coregulatory proteins. One mechanism that may contribute to increased AR stabilization in prostate cancer is the increased expression of the AR coregulator melanoma antigen gene protein-11 (MAGE-11 or MAGE-A11), which stabilizes AR in the absence or at low levels of androgen (see Sect. 5). Alternatively, there appears to be local androgen production in prostate cancer tissue that could stabilize AR (Mohler et al. 2004). On the other hand, local androgen production in prostate cancer cell lines cultured under serum-free conditions does not likely account for AR stabilization because of the lack of a biosynthetic substrate. The mechanisms that underlie increased AR stabilization in prostate cancer remain to be fully established and may be independent of the AR N/C interaction. Reports have suggested both direct and indirect mechanisms are the basis of the AR N/C interaction. Proposed indirect mechanisms involve coactivators such as CBP/p300, SRC1 and c-jun (Bubulya et al. 2001; Ikonen et al. 1997). A direct AR N/C interaction is supported by mammalian two-hybrid assays using GAL4 DNA binding domain and VP16 activation domain fusion proteins of the AR NH2-terminal region and LBD (Langley et al. 1995; Wilson et al. 2003). Since the original report, the interaction between the AR NH2-terminal domain and AF2 has been confirmed by several groups (Berrevoets et al. 1998; Hsu et al. 2005; Ikonen et al. 1997; Li et al. 2006; Schaufele et al. 2005). The biological significance of the AR N/C interaction is supported by its androgen specificity and inhibition by antagonists. Studies on AR AF2 mutations that cause AIS without altering apparent equilibrium androgen binding affinity (Langley et al. 1998), and more recently, experiments using FRET analysis of wild-type AR (Schaufele et al. 2005), support the possibility that the AR N/C interaction results in an antiparallel AR dimer. There is also evidence for intramolecular domain interactions within the AR monomer (Klokk et al. 2007; Schaufele et al. 2005). The likelihood for both models is indicative of domain swapping between the AR FXXLF motif and AF2 during transition from the ligand-bound inactive monomer to the ligand-bound active AR dimer (Bennett et al. 1995). In this self-association model of protein structure, the same interaction interface mediates self-complementation within a closed functional dimer unit arising from opening the monomer and rotation about a hinge loop (Bennett et al. 2006). Because the protein interaction interface is the same for monomer and dimer, only the hinge region conformation is expected to change. Altered structure of the hinge region could provide a new interaction surface that modulates AR transcriptional activity (Askew et al. 2007; Haelens et al. 2007; Wang et al. 2001) and contribute to higher-order aggregate formation (Liao et al.
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1999). Abnormal AR aggregation in Kennedy’s disease results from expansion of the AR NH2-terminal glutamine repeat at residues 58–78 positioned proximal to the AR FXXLF motif at residues 23–27 (La Spada et al. 1991; Taylor et al. 2003).
2 AR FXXLF and WXXLF Motifs The androgen-dependent AR N/C interaction is mediated by AR NH2-terminal FXXLF and WXXLF motif binding to the AF2 surface of the LBD in the carboxylterminal region of AR (He et al. 2000; He and Wilson 2002). However, it is primarily AR FXXLF motif binding to AF2 that is the basis of the androgendependent AR N/C interaction that slows the androgen dissociation rate and stabilizes AR.
2.1
Motif Binding Preferences
Small deletions within the AR NH2-terminal region that increase the dissociation rate of bound androgen (Zhou et al. 1995) led to the identification of the AR FXXLF motif sequence 23FQNLF27 (He et al. 2000) in the NH2-terminus of full-length AR amino acid residues 1–919 (Lubahn et al. 1988a, b). The AR FXXLF motif is the principal interaction site in the NH2-terminal domain for the AR N/C interaction (He et al. 2000). The AR FXXLF motif, where F is phenylalanine, X is any amino acid, and L is leucine, forms an amphipathic alpha helix when bound to AF2 in the AR LBD. The AR FXXLF motif binds the same AF2 hydrophobic cleft that binds the LXXLL motifs of SRC/p160 coactivators. Fluorescent binding measurements demonstrate binding of the AR-20–30 FXXLF motif peptide is five- to tenfold higher affinity than binding of a peptide containing the third and strongest interacting LXXLL motif of transcriptional intermediary factor 2 (TIF2, SRC2) (Askew et al. 2007; He et al. 2004), a member of the SRC/p160 family of coactivators. Higher binding affinity of the AR FXXLF motif compared to the coactivator LXXLL motifs results from amino acid sequence changes in the AR AF2 binding site during the evolution of AR (He et al. 2004). FXXLF and LXXLL motif binding to AF2 appears to be facilitated by flanking charged residues that are complementary to oppositely charged clusters on the LBD surface bordering AF2 (Darimont et al. 1998). The AF2 charged residues include the so-called charge clamp residues Lys-720 and Glu-897 (K720 and E897, Fig. 2), which correspond to the charge clamp residues first described for estrogen receptor binding of the coactivator signature LXXLL motif (Heery et al. 1997; Henttu et al. 1997; Le Douarin et al. 1996). There are, however, additional residues that make up two charge clusters surrounding AF2 that complement the charge distribution flanking the AR FXXLF motif and SRC/p160 coactivator LXXLL motifs (He and Wilson 2003). A greater percentage of positively charged residues lie NH2-terminal
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Fig. 2 Charge clusters flanking AF2 in the AR ligand binding domain. Space-filled model of the DHT-bound AR ligand binding domain (Sack et al. 2001) illustrates positively charged residues Arg-726 (R726), Lys-717 (K717), Lys-720 (K720) (in red ), and negatively charged residues Glu709, Glu-893, and Glu-897 (E709, E893 and E897) (in blue) surrounding the AF2 hydrophobic floor (in yellow). Charge clamp residues are K720 and E897. AF2 charge clusters are complementary to the charge distribution flanking the FXXLF and LXXLL motifs, suggesting electrostatic forces facilitate orientation of motif binding to AF2 (He and Wilson 2003) (See Color Insert)
to the FXXLF and LXXLL motifs, and a greater proportion of negative charges are positioned carboxyl-terminal to the binding motifs. The complementary nature of the charge distribution suggests that electrostatic interactions orient FXXLF and LXXLL motif binding to AF2. High-affinity motif binding is subsequently mediated by hydrogen bonding to backbone atoms of charge clamp residues and extensive hydrophobic interactions with the AF2 floor. The characteristic charge polarity of both the FXXLF and LXXLL motifs may be a useful property in predicting new potential interacting motifs. On the other hand, in mammalian two-hybrid assays and crystallization studies, the minimal AR-20–30 region that optimally binds AF2 contains positively charged Arg-20, important for the AR N/C interaction (Steketee et al. 2002), but lacks the carboxyl-flanking negative charged residues. Superimposition of crystal structures of the AR LBD bound to R1881 (He et al. 2004) and T (Askew et al. 2007) indicates an overlapping, parallel alignment of the AR FXXLF motif relative to the coactivator LXXLL motif within the same hydrophobic groove of AF2. Some studies suggest a deeper hydrophobic groove in
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association with FXXLF motif binding compared to the bound coactivator LXXLL motif (Dubbink et al. 2004; Hur et al. 2004). Crystal structures of the AR FXXLF motif bound in the presence of R1881 suggest an induced fit movement of AF2 residue side chains while the LBD core remains relatively static (He et al. 2004). The AR FXXLF motif peptide forms hydrogen bonds to AF2 charge clamp residues Lys-720 and Glu-897. The SRC/p160 coactivator TIF2 LXXLL motif is phase ˚ away from Glu-897 toward Lys-720, placing it too distant to shifted by 2 A hydrogen bond to Glu-897, but maintaining hydrogen bonds to Lys-720. The results are in agreement with the demonstrated requirement for Glu-897 in the AR N/C interaction but not in SRC/p160 coactivator binding (Slagsvold et al. 2000). Loss of hydrogen bonding to Glu-897 likely contributes to the lower AF2 binding affinity for the LXXLL than FXXLF motif. AR preference for FXXLF over the LXXLL motif is supported by several lines of evidence. Peptide display screens using the AR LBD as bait have detected a variety of FXXLF-related motifs, but few if any LXXLL motif-containing peptides (Chang et al. 2005; Hsu et al. 2003; van de Wijngaart et al. 2006). A consequence of higheraffinity AR FXXLF motif binding is that the AR N/C interaction competitively inhibits SRC/p160 coactivator LXXLL motif binding to AF2 (He et al. 2001). Inhibition of AF2 recruitment of SRC/p160 coactivators by the AR N/C interaction appears to be one mechanism that shifts the predominant activation function of AR away from AF2 in the LBD to AF1 in the NH2-terminal region (He et al. 2004). Identification of FXXLF motifs in several putative AR coactivators in yeast twohybrid screens using the AR LBD as bait suggests that these coactivators regulate AR transcriptional activity by binding AF2 (He et al. 2002b; Hsu et al. 2003). The biological significance of FXXLF motif-containing AR coactivators is currently under investigation. The AR NH2-terminal WXXLF motif, where W is tryptophan, also interacts with AR AF2 in an androgen-dependent manner, but with lower affinity than the FXXLF motif (He et al. 2000, 2002a). The functional significance of the WXXLF motif remains to be fully established. Some recent studies suggest that the AR WXXLF motif contributes to ligand-independent AR transcriptional activity in castration-recurrent prostate cancer cells (Dehm et al. 2007). When expressed in multiple copies, but not as a single sequence as a GAL-4 fusion protein, the AR WXXLF motif region was transcriptionally active. The competitive relationship between AR FXXLF and coactivator LXXLL motif binding to AF2 raises the possibility that alternative forms of AR that lack the NH2terminal FXXLF motif region may be transcriptionally more active than full-length AR by providing greater access by AF2 for coactivator recruitment. This would be reminiscent of progesterone receptors A and B, where A lacks an extended LXXLL motif-related sequence (Tung et al. 2006). While an intriguing hypothesis, it is not strongly supported because of the questionable physiological relevance of shorter forms of AR. Previous studies have shown that AR is especially susceptible to proteolytic cleavage during isolation and storage (Wilson and French 1979). Shorter forms of AR that lack part of the AR NH2-terminal region (Gao and McPhaul 1998) can result from proteolytic degradation during isolation (Gregory et al. 2001a).
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It therefore seems unlikely that endogenous alternative forms of AR lack the AR NH2-terminal FXXLF motif region. On the other hand, mutagenesis studies have demonstrated that removing or mutating the AR FXXLF motif in the AR NH2terminal region increases accessibility of AR AF2 for coactivator recruitment and can increase AR transactivation when SRC/p160 coactivators are overexpressed (He et al. 2001). As discussed further (see Sect. 5), the increase in AR transcriptional activity caused by a mutation of the AR FXXLF motif led to the prediction and subsequent identification of the AR coregulator, MAGE-11. MAGE-11 binds the AR FXXLF motif, allowing AR to escape competitive inhibition by FXXLF motif binding to AF2, with the result that AR transcriptional activity is increased by the SRC/p160 coactivators. MAGE-11 binding to AR appears to be an important mechanism to increase AR transcriptional activity in normal cells of the male and female reproductive tract and in prostate cancer.
2.2
Sequence Conservation Through Evolution
The AR amino acid sequence is relatively well conserved through evolution, with greatest sequence homology within the highly structured DNA and ligand binding domains (Choong et al. 1998). However, relative to other steroid receptors, a greater number of sequence changes occurred through evolution in the AR AF2 site that has resulted in increased binding specificity and affinity for the FXXLF motif (He et al. 2004). The AR NH2-terminal domain is the least conserved of the AR domains across species and relative to other steroid receptors. One notable region in the AR NH2-teminal domain is the polymorphic glutamine repeat at residues 58–78 (Lubahn et al. 1988a). The AR glutamine repeat varies in length in the normal human population from 7 to 34 residues, with a mean of 21 residues (Choong and Wilson 1998; Edwards et al. 1992). Slight expansions in AR glutamine repeat length have been linked to infertility (Casella et al. 2003; Mifsud et al. 2001) and genital abnormalities of otherwise unknown etiology (Lim et al. 2001). Glutamine repeat expansions greater than 40 residues cause spinal bulbar muscular atrophy, also known as Kennedy’s disease (La Spada et al. 1991). Contraction of the glutamine repeat has been correlated with prostate cancer (Linja and Visakorpi 2004; Schoenberg et al. 1994). The precise functional significance of the AR glutamine repeat remains unclear, but its length appears to influence AR levels. Longer CAG repeat lengths are associated with reduced AR levels, and shorter repeats with higher levels of AR (Choong et al. 1996). Glutamine repeat length increases during primate evolution, with longest repeats found in chimpanzees and humans (Choong et al. 1998). The polymorphic human AR glutamine repeat (residues 58–78) is located relatively close to the FXXLF motif (residues 23–27) (Lubahn et al. 1988a). However, glutamine repeat length does not appear to directly influence the AR N/C interaction (Langley et al. 1995). The human AR FXXLF motif sequence FQNLF is conserved among mammals but degenerates in fish (He et al. 2002a).
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Alternative FXXLF motif sequences in fish AR are FQNVF in rainbow trout (Takeo and Yamashita 1999), YQNVF in eel (Todo et al. 1999), and YQSVF in goldfish (AY090897), indicating conservation of key hydrophobic residues in the i + 1, 4 and 5 positions of the motif. This raises the probability that the AR N/C interaction contributes to AR function throughout the vertebrate lineage and that sequence differences may be complemented by changes in AF2. The WXXLF motif sequence WHTLF at human AR NH2-terminal residues 433–437 is also conserved among mammals through Xenopus but degenerates in fish to a greater extent than the FXXLF motif. The less well-conserved WXXLF motif may reflect its minor role in the AR N/C interaction (He et al. 2000). On the other hand, conservation of the WXXLF motif through mammals suggests it serves an additional function important in AR transcriptional activity that remains to be fully established.
3 Functional Effects of AR Motif Interactions A number of assays have provided evidence that the androgen-dependent AR N/C interaction is important for AR function in vivo. Modulation of AR transcriptional activity by the AR N/C interaction is complex and involves high-affinity androgen binding and multiple interacting motifs, activation functions, and coregulatory proteins.
3.1
Regulation of AR Transactivation
Promoter-specific effects of the AR N/C interaction are widely recognized, but the basis for these differences is not fully understood. The AR N/C interaction increases to different extents AR transcriptional activation of enhancer/promoter regions in reporter gene assays (Berrevoets et al. 1998). AR transactivation of the prostatespecific antigen (PSA) enhancer and most other natural promoter and enhancer regions is inhibited by AR NH2-terminal mutations that disrupt the N/C interaction (He et al. 2002a). In contrast, AR activation of the mouse mammary tumor virus (MMTV) long terminal repeat enhancer region is largely independent of the AR N/ C interaction. However, studies in which these androgen response regions were incorporated into chromatin indicate that both MMTV and PSA depend on the AR N/C interaction for activation by AR (Li et al. 2006). Furthermore, both the AR NH2- and carboxyl-terminal regions were required for an intranuclear compartmentalization pattern similar to transcriptionally active full-length AR (Saitoh et al. 2002). One possible explanation for reporter gene differences is that the MMTV enhancer comprises a series of stronger androgen response elements compared to PSA and most naturally occurring enhancer regions when assayed in transient
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transfection assays. This is supported by transfection studies in prostate cancer cell lines where endogenous AR transactivates MMTV but not the PSA reporter gene (Gregory et al. 2004). Like the PSA enhancer, most naturally occurring androgen response elements contain multiple half-site sequences. It has been suggested that the AR N/C interaction enhances cooperativity between weak androgen response elements to increase selective AR gene activation (Scheller et al. 1998). Arrangement in chromatin may dampen the transcriptional response of MMTV making it more dependent on the stabilizing effects of the AR N/C interaction. The in vivo potency differences between T and DHT parallel their abilities to induce the AR N/ C interaction (Askew et al. 2007), providing further support that the N/C interaction is required for AR transcriptional activity in vivo. One of the transcriptional effects of the AR N/C interaction is to limit SRC/p160 coactivator recruitment by AF2 in the LBD. This was demonstrated in studies using full-length AR except with an internal deletion of the AF1 region in the NH2terminal domain (He et al. 2001). In the presence of androgen, the AR FXXLF motif competitively inhibits AF2 recruitment of SRC/p160 coactivator LXXLL motifs, and inhibition is relieved by loss-of-function mutations in the FXXLF motif. SRC/ p160 coactivator activation at AF2 is further limited by sequence changes in AF2 that result in higher-affinity FXXLF motif binding compared to the SRC/p160 coactivator LXXLL motifs. Androgen-dependent AR FXXLF motif inhibition of AF2 coactivator recruitment may provide a dynamic mechanism for AF1 to be the predominant activation domain (He et al. 2004) and for coordinate androgendependent gene regulation by AF1 and AF2. Discrepancies exist in the literature regarding the role of the AR NH2-terminal region in the recruitment of the SRC/p160 coactivators. Most reports concur that the AR NH2-terminal region interacts with members of the SRC/p160 coactivator family (Bevan et al. 1999; He et al. 1999). SRC/p160 coactivators are reported to interact with AR in a manner that is synergistic (Bevan et al. 1999), independent (Berrevoets et al. 1998), and dependent (He et al. 2002a) on LXXLL motif binding to AF2. Some of these differences may be attributed to promoter-selective effects for activation by AR. Our findings continue to support a dependence on SRC/p160 coactivator LXXLL motif binding to AF2 to mediate transcriptional effects of AR. The AR N/C interaction nevertheless contributes to coactivator interactions possibly through the formation of new interaction surfaces to increase AR transcriptional activity (Alen et al. 1999). The evolutionary diversity of the NH2-terminal AF1 region provides a variety of potential interaction sites for tissueand species-selective gene activation. In contrast, AF2 is highly constrained by the structural requirements of high-affinity androgen binding. Other steroid receptors such as the estrogen receptor lack an extended NH2-terminal AF1 region and are more dependent on AF2 for transcriptional activity. In prostate cancer, the AR AF2 region appears to gain activity in part through increased expression of SRC/p160 coactivators (Gregory et al. 2001b). Higher levels of SRC/p160 coactivators increase the activity of wild-type AR in the absence and presence of the AR N/C interaction (He et al. 2002a). Overexpression of FXXLF motif peptides was reported to suppress the growth of the recurrent
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CWR22 tumor xenograft (Hsu et al. 2005) further implicating the AF2 site in ARmediated prostate cancer cell proliferation. The AR coregulator MAGE-11 increases AR transcriptional activity in normal reproductive tissues and in prostate cancer by increasing exposure of AF2 as discussed further (see Sect. 5).
3.2
Potency Differences Between T and DHT
Fundamental to understanding the molecular basis of AR activation of gene transcription is to establish the structural and functional consequences of the AR N/C interaction. Original studies to characterize AR FXXLF motif binding to AF2 were based on changes in androgen dissociation rate (He et al. 2000; Zhou et al. 1995). While the AR N/C interaction slows the dissociation rate of high-affinity agonists that stabilize AR, SRC/p160 coactivator LXXLL motif binding has only a minimal effect on androgen dissociation rate even when the LXXLL motif is inserted in the AR NH2-terminal region to replace the FXXLF motif (He et al. 2001). The ineffectiveness of LXXLL motif binding to slow androgen dissociation reflects the lower binding affinity of AF2 for LXXLL motifs compared to the FXXLF motif in AR and AR coactivators (Askew et al. 2007; He et al. 2004). T and DHT bind AR with similar apparent equilibrium binding affinities (Wilson and French 1976), yet T is a less potent androgen in vivo. The weaker inherent activity of T is demonstrated by the 5a-reductase syndrome, in which 46XY genetic males have a female phenotype at birth because of insufficient levels of 5a-reductase, the enzyme that converts T to DHT (Imperato-McGinley et al. 1974). Despite T levels within the normal range, virilization of the newborn male with 5a-reductase deficiency does not occur due to inadequate levels of DHT. Recently, pharmaceutical companies have identified tissue-selective synthetic nonsteroidal AR agonist analogs (Ostrowski et al. 2007; Wilson 2007). This raises the possibility for ligandselective regulation of AR target genes through mechanisms that include ligandspecific coactivator recruitment. However, there is as yet no evidence that T and DHT regulate different genes because higher levels of T elicit the same response as lower concentrations of DHT (Grino et al. 1990; Maes et al. 1979; Mahendroo et al. 2001). Recent biochemical and structure studies investigating the molecular basis for potency differences between T and DHT indicate that T-bound AR exhibits greater conformational heterogeneity at the AF2 surface (Askew et al. 2007). Even slight destabilization of the AF2 surface appears to compromise the AR N/C interaction to an extent that accounts for the faster dissociation rate and reduced in vivo potency of T. T is a 19-carbon steroid, which differs from DHT by a ~4,5 double bond that increases the polarity of T (Fig. 3). Polarizing effects of the A ring double bond in T appear to be transferred through Met-745, a ligand binding pocket residue whose side chain extends toward Leu-712 in the AF2 floor. Crystal structures of the T-bound AR LBD with FXXLF or LXXLL peptide indicate two conformations of Leu-712, whereas similar structures of DHT-bound (Estebanez-Perpina et al. 2005; Hur et al. 2004; Sack et al. 2001) and R1881-bound AR LBD (unpublished results)
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Fig. 3 Conversion of testosterone to dihydrotestosterone by 5a-reductase. Testosterone (T) and dihydrotestosterone are 19-carbon, biologically active androgens that differ by a ~4,5 double bond in ring A of T (Wilson 2007). (Copyright 2007, The Endocrine Society)
indicate a single conformation of Leu-712. The importance of Leu-712 in AR function and the AR N/C interaction is indicated by the naturally occurring AR-L712F mutation that causes complete AIS without altering high-affinity androgen binding (He et al. 1999, 2006). Functional differences between T and DHT are linked to their effects on AF2 binding of the FXXLF and LXXLL a-helical motif regions. The apparent slight destabilizing effect of bound T on the AF2 motif binding surface is supported by fluorescence polarization measurements. Fluorescein-labeled FXXLF and LXXLL motif peptides bind AF2 of the AR LBD with twofold lower affinity with bound T than DHT (Askew et al. 2007). Furthermore, in the absence of FXXLF motif binding, T and DHT dissociate with similar rates from an AR fragment containing the LBD, whereas T dissociates three times faster than DHT from full-length AR due to the FXXLF motif-mediated N/C interaction. These results indicate that the faster dissociation rate and weaker in vivo potency of T are a consequence of weaker AR FXXLF motif binding to AF2. Thus T is a weaker androgen than DHT in vivo because of the polarizing effects arising from ring A of T that cause local conformational heterogeneity at the AF2 surface. Weaker AR FXXLF motif binding causes faster dissociation of T from fulllength AR (Askew et al. 2007). Because AF2 is also the binding site for SRC/p160 coactivator LXXLL motifs, bound T similarly weakens LXXLL motif binding contributing to its weaker potency. The greater stabilizing effect of the less polar DHT increases AF2 binding affinity for the AR FXXLF and SRC/p160 coactivator LXXLL motifs making DHT a more potent androgen in vivo.
4 Natural AR Gene Mutations Alter Motif Binding Phenotypic expression of naturally occurring AR gene mutations occurs in part because AR function is not required for life (Goh et al. 2007; Quigley et al. 1995). Localization of the AR gene on the X chromosome (Brown et al. 1989) further confounds the potential detrimental effects of AR mutations on male reproductive function and health. In the 46XY genetic male, more than 300 different single amino acid AR mutations result in a range of phenotypes that reflect the degree to
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which AR function is disrupted (Quigley et al. 1995, 2004). The AIS phenotype spans normal male genital development with infertility, to partial and complete AIS characterized by different degrees of feminization of the external genitalia. Later in life, AR somatic mutations can arise in prostate cancer in association with the increasing genome instability of cancer. The frequency of AR mutations in early stage prostate cancer is low, but increases with progression to castration-recurrent disease after androgen-deprivation therapy (Gelmann 2002; Shi et al. 2002; Taplin et al. 1999). Unlike germline mutations that disrupt AR function and cause AIS, AR somatic mutations in prostate cancer can increase AR transcriptional activity through a gain-in-function. Many of the well-characterized AR mutations in prostate cancer are positioned near the AF2 site in the LBD. Like loss-of-function mutations that cause AIS, somatic AR LBD mutations in prostate cancer positioned near AF2 alter the AR N/C interaction through mechanisms that are independent of changes in equilibrium androgen binding affinity.
4.1
AR AF2 Germline Mutations in the Androgen Insensitivity Syndrome
The many different single amino acid AR mutations that cause AIS (http://ww2. mcgill.ca/androgendb/data.htm) localize predominantly in the highly structured regions of the AR DNA and ligand binding domains (Matias et al. 2000; Quigley et al. 1995). In contrast, the major AR NH2-terminal AF1 activation region is surprisingly free of missense mutations, and a naturally occurring mutation in the AR NH2-terminal FXXLF motif has not been reported. The detrimental effects of most AR LBD mutations result from disruption of equilibrium androgen binding affinity (Quigley et al. 1995). However, there are a few AR LBD mutations that cause partial or complete AIS without disrupting equilibrium androgen binding affinity. These have been of particular interest because they implicate a function for AR not directly related to ligand binding affinity. AR mutations that selectively inhibit AR FXXLF and coactivator LXXLL motif binding were helpful in early studies to characterize the region of the LBD involved in the AR interdomain N/C interaction (He et al. 1999; Langley et al. 1998). It soon became clear that the AF2 site for coactivator LXXLL motif binding was the same site required for the AR N/C interaction. Since then, a number of naturally occurring mutations have been identified that cause AIS by interfering with the AR N/C interaction (Ghali et al. 2003; He et al. 1999; Jaaskelainen et al. 2006; Langley et al. 1998; Quigley et al. 2004; Thompson et al. 2001). A universal finding regarding AF2 mutations that cause AIS is diminished AR FXXLF motif binding and increased dissociation rate of bound androgen. This is consistent with properties of the AR N/C interaction and the ability of the AR FXXLF motif to slow the androgen dissociation rate. The studies provide further evidence that the normally slow androgen dissociation rate from wild-type AR is a requirement for AR function in vivo. The near wild-type equilibrium androgen
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binding affinities of the AR AF2 mutants, yet faster androgen dissociation rates, are consistent with the properties and potency differences between T and DHT described earlier. Equilibrium binding affinity can be determined from the ratio of association and dissociation rate constants. Thus, fast and slow dissociating ligands can display high-affinity binding if there is a reciprocal change in association rate (Wilson and French 1976). This suggests that AF2 mutations cause more rapid association rates, although this has not been rigorously measured. The results parallel our findings with T and DHT since these biologically active androgens have similar equilibrium binding affinities, but the slower dissociation rate of DHT renders it a more active androgen. When T is the only predominant androgen during male sex development in individuals with the 5a-reductase syndrome, the phenotypic effects are similar to AIS. But unlike AIS, rising T levels at puberty in a 5areductase syndrome patient compensate for the lack of DHT and virilization occurs. Details of the AR AF2 interaction surface for FXXLF and LXXLL motif binding were revealed through biochemical studies of AR mutants that cause AIS and from the crystal structure of the wild-type AR LBD (He et al. 2006). AR AF2 residues Gln-733, Phe-725, and Ile-737 form hydrophobic Cluster A that interacts with Phe-27 of the AR 23FQNLF27 motif (Fig. 4). Cluster A is relatively distant from the ligand binding pocket so that the predominant effect of a Cluster A mutation is decreased FXXLF and LXXLL motif binding with an indirect effect on the bound ligand. Decreased FXXLF motif binding accounts for the faster androgen dissociation rate from the full-length AR mutants, whereas dissociation rates of mutant and wild-type AR fragments containing the LBD were similar. A second AF2 hydrophobic Cluster B lies closer to the ligand binding pocket and is formed by Ile-737, Ile-898, and Leu-712. Cluster B interacts with Phe-23 of the AR 23 FQNLF27 motif. Mutations in Cluster B have direct effects on FXXLF and LXXLL motif binding and on the bound ligand, and cause faster androgen dissociation rates from the full-length AR mutants and from the carboxyl-terminal fragments containing the LBD. AR mutation V889M, which causes complete AIS, results in a very rapid androgen dissociation rate from both full-length AR and the carboxyl-terminal fragment, suggesting that Val-889 functions in a ligand gateway at the base of helices 10, 11, and 12. AF2 mutations that cause partial and complete androgen insensitivity by disrupting FXXLF and LXXLL motif binding to AF2 without altering equilibrium androgen binding affinity indicate that the AF2 site is important for AR function in vivo. However, it is difficult to differentiate the in vivo significance of AF2 in terms of AR FXXLF and SRC/p160 coactivator LXXLL motif binding since the two motifs interact at essentially the same AF2 binding site and are similarly disrupted by AF2 mutations that cause AIS (He et al. 2004, 2006).
4.2
AR Somatic Mutations in Prostate Cancer
AR transcriptional activity increases in prostate cancer through a number of mechanisms that include mitogen signaling (Gregory et al. 2004) and somatic
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Fig. 4 Structure of the AR AF2 binding surface with bound AR FXXLF motif. Wild-type AR residues that undergo germline mutations and cause partial or complete androgen insensitivity syndrome (AIS) (cyan) are distributed within the AF2 binding surface for the AR FXXLF (23FQNLF27) motif, where the latter is represented by Phe-27 (F27), Phe-23 (F23), and Leu-26 (L26) (magenta). AF2 residues mutated in individual cases of AIS form hydrophobic Cluster A comprising Gln-733 (Q733), Phe-725 (F725), and Ile-737 (I737) (cyan). Cluster A interacts with Phe-27 of the AR FXXLF motif and lies more distal than Cluster B to the ligand binding pocket. Naturally occurring mutations in Cluster A cause AIS by disrupting AR FXXLF motif binding, which increases the dissociation rate of bound androgen. Cluster B comprises Ile-737 (I737), Leu712 (L712) and Ile-898 (I898) (cyan). Cluster B interacts with Phe-23 of the AR FXXLF motif and lies closer to the ligand binding pocket than Cluster A. AIS mutations in Cluster B alter AR FXXLF motif binding and have direct effects on bound ligand. Note that Leu-712, positioned at the AF2 surface near the ligand binding pocket, was identified in two conformations in T-bound AR LBD crystal structures, indicating that bound T imparts greater conformational heterogeneity in AF2. Bound DHT and R1881 result in a single conformation of Leu-712. Val-889 (V889) (cyan) has a gatekeeper function in ligand binding. Charge clamp residues Lys-720 (K720) and Glu-897 (E897) are in yellow, and the bound synthetic androgen, methyltrienolone (R1881) is in green. Sites in wild-type AR somatic AF2 mutations identified in prostate cancer, His-874 (H874), Val-715 (V715), and Thr-877 (T877), are in gold (He et al. 2006) (See Color Insert)
mutations in the AR gene (Culig et al. 1993; Shi et al. 2002; Tan et al. 1997; Veldscholte et al. 1992). The frequency of AR mutations in prostate cancer is low, but increases during tumor progression to castration-recurrent disease in association with increased genome instability (Gelmann 2002; Taplin et al. 1999). Wellcharacterized somatic mutations that increase ligand-dependent AR transcriptional
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activity include T877A in the LNCaP prostate cancer cell line (Veldscholte et al. 1992), H874Y in the CWR22 human prostate xenograft and CWR-R1 prostate cancer cell line derived from the castration-recurrent CWR22 xenograft (Gregory et al. 1998, 2001c; Tan et al. 1997), V715M (Culig et al. 1993), and V730M (Culig et al. 1993; He et al. 2004; Newmark et al. 1992; Peterziel et al. 1995) identified in prostate cancer specimens. Mechanisms underlying the increase in AR activity by these mutations differ even though each involves the AF2 motif binding site. Val-730 is in the hydrophobic core of AF2. The V730M somatic mutation in prostate cancer increases the AR AF2 interaction with the LXXLL motif of SRC1 (He et al. 2004). His-874, Val-715, and Thr-877 lie in a structural boundary ˚ from Trp-741, a ligand binding between AF2 and the ligand binding pocket, 5 A pocket residue whose side chain changes position depending on which ligand is bound (Askew et al. 2007; He et al. 2006). H874Y and V715M increase the AF2 interaction with the FXXLF and LXXLL motifs, which for H874Y, is independent of a change in motif binding affinity (Askew et al. 2007; Duff and McEwan 2005; He et al. 2006). T877A increases the intramolecular space around ring D of the bound steroid, allowing a broader spectrum of ligands to function as agonists, while maintaining an essentially wild-type response to T and DHT (He et al. 2006). AR-T877A enhances the AR N/C interaction in response to partial agonists and traditional antagonists primarily by changes in ligand binding specificity (Langley et al. 1995). A notable feature of the H874Y, V715M, and T877A somatic mutations that arose in prostate cancer is that each slows the dissociation rate of bound androgen from full-length AR and from an AR fragment that contains the DNA and ligand binding domains and lacks the NH2-terminal region (He et al. 2006). This result indicates that each of these mutations stabilizes androgen in the ligand binding pocket independent of the AR N/C interaction. The stabilizing effects of the H874Y, V715M, and T877A mutations on bound ligand may provide an explanation for previous reports suggesting transcriptional-growth-promoting effects of partial agonists in LNCaP cells that are not associated with the AR N/C interaction (Sathya et al. 2003). In addition, sufficiently high pharmacological concentrations of partial AR agonists and antagonists may contribute to the activity of wild-type and mutant AR (Kemppainen et al. 1992; Sathya et al. 2003; Song et al. 2004; Wong et al. 1993) as demonstrated by the flutamide withdrawal syndrome (Scher and Kelly 1993). Lack of a requirement for the AR N/C interaction for activation of the MMTV promoter enhancer region (He et al. 2002a) suggests further that some androgen-responsive genes may not require the AR N/C interaction. This may depend on the level of specific AR coregulatory proteins and the arrangement of specific transcription factor binding sites in the promoter and enhancer regions. Increased levels of SRC/p160 coactivators in prostate cancer tissue and cell lines provide a mechanism more broadly applicable to increases in AR transcriptional activity in response to androgens and other ligands (Gregory et al. 2001b, c). Crystal structures of the AR-H874Y LBD bound to T and AR FXXLF peptide indicate that the H874Y mutation in helix 10 results in the formation of new direct hydrogen bonds to residues in helices 4 and 5 that replace a water-mediated
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hydrogen bond network (Askew et al. 2007). In two-hybrid assays, AR FXXLF and LXXLL motif binding increases as a result of the H874Y mutation, but motif binding affinity is unchanged. The H874Y mutation appears to stabilize the LBD core and AF2 helix 12 through direct helix-to-helix hydrogen bonding that renders AF2 a more effective motif binding site. The predominant transcriptional effect of H874Y is to render DHT-like activity to T. Thus, several well-characterized AR somatic mutations in prostate cancer increase AR transcriptional activity through structural effects on AF2. These mutations are most likely selected during tumor progression by their ability to increase AR transcriptional activity and prostate cancer growth in response to a broader range of ligands. The results are consistent with a critical role for AR in prostate cancer development and progression and point to the AF2 binding surface as a mechanism for increased AR transcriptional activity in castration-recurrent prostate cancer that arises in an environment of low circulating androgen. Increased coactivator recruitment by AF2 in wild-type AR is also facilitated by increased levels of the SRC/p160 family members during prostate cancer progression (Gregory et al. 2001b).
5 AR Coregulator MAGE-11 Binds the AR FXXLF Motif AR FXXLF motif binding to AF2 is an important control mechanism regulating AR transcriptional activity. We discussed how the AR N/C interaction enhances the potency of endogenous androgens by stabilizing AR but inhibits SRC/p160 coactivator recruitment by AF2. These observations suggest that AR coregulators that alter the AR N/C interaction could either inhibit or enhance AR transcriptional activity. Inhibition of AR transcriptional activity by p53 and cyclin D1 has been linked to interference of the AR N/C interaction (Burd et al. 2005; Shenk et al. 2001). Two-hybrid interaction screens of a human testis library using the AR FXXLF motif region as bait identified melanoma antigen gene protein-11 (MAGE-11 or MAGE-A11) as an AR coregulator that binds the FXXLF motif and increases AR transcriptional activity by exposing AF2 for coactivator recruitment (Bai et al. 2005).
5.1
AR Coregulator MAGE-11
Identification of MAGE-11 as an AR coregulator that selectively binds the AR NH2-terminal FXXLF motif demonstrates that the AR FXXLF motif serves at least two functions in modulating AR transcriptional activity. In addition to binding AF2 in the androgen-dependent AR N/C interaction, the AR FXXLF motif serves as the principal interaction site for MAGE-11 (Bai et al. 2005). Mutagenesis studies demonstrated different flanking sequence requirements for AR FXXLF motif binding to AF2 and MAGE-11. In two-hybrid assays and crystal structures, the short
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AR-20–30 amino acid peptide RGAFQNLFQSV is necessary and sufficient to bind AF2 (Askew et al. 2007; He et al. 2004; He and Wilson 2003) and requires androgen bound in the AR ligand binding pocket. In contrast, the minimal sequence required for MAGE-11 to interact with the AR FXXLF motif region is AR-16–36. AR residues Val-30 and Val-33 are required for MAGE-11 to bind the AR FXXLF motif region, whereas mutations at these sites have no effect on the AR N/C interaction (Bai et al. 2005). Thus, there is an overlapping but distinct FXXLF motif binding region for AF2 and MAGE-11. Furthermore, MAGE-11 does not interact with FXXLF motifs present in several putative AR coregulators that interact with the AR AF2 site. Thus, MAGE-11 binding to AR requires a longer alpha helical FXXLF motif region and is more selective than AR AF2 binding of the AR FXXLF motif. Another difference is that MAGE-11 binds the AR FXXLF motif in an androgen-independent manner. MAGE-11 more readily coimmunoprecipitates with AR in the absence than in the presence of androgen, although recent studies indicate that the interaction between AR and MAGE-11 in the presence of androgen is dynamic and influenced by growth factor signaling. EGF-dependent phosphorylation at MAGE-11 Thr-360 serves to target the monoubiquitinylation of MAGE-11 at Lys-240 and 245, which is required for MAGE-11 to interact with the FXXLF motif region of AR (Bai and Wilson 2008). The mechanisms whereby MAGE-11 increases AR transcriptional activity are being actively investigated. Thus far we know that binding of MAGE-11 to the AR NH2-terminal FXXLF motif in the presence of androgen increases AR transcriptional activity by interfering with the androgen-dependent AR N/C interaction and exposing AF2 for coactivator recruitment (Bai et al. 2005). MAGE-11 also increases AR transcriptional activity through mechanisms independent of AF2. This is evident using the AR NH2-terminal and DNA binding fragment AR-1– 660 that lacks the LBD and AF2 binding site. AR-1–660 is strongly activated by MAGE-11 in an FXXLF motif-dependent manner through mechanisms that remain to be established. A model for the interrelationship between the AR N/C interaction, MAGE-11 binding, and AR transactivation involving SRC/p160 coactivators is shown in Fig. 5. MAGE-11 binding to the AR FXXLF motif increases the turnover of AR in association with growth-factor-dependent phosphorylation and ubiquitinylation of MAGE-11 (Bai and Wilson 2008). The MAGE-11-dependent increase in AR turnover and transcriptional activity is reminiscent of agonist-induced down-regulation that characterizes other steroid receptors and demonstrates a link between receptor degradation and transcriptional activity (Dennis et al. 2001; Verma et al. 2004; Wu et al. 2007). In the presence of androgen, AR and MAGE-11 colocalize in a disperse pattern throughout the nucleus. This differs from the colocalization pattern of MAGE-11 with an AR DNA binding mutant within prominent subnuclear particles. Thus, AR and MAGE-11 are intimately linked within the nucleus dependent on the transcriptional status of AR. In the absence of androgen, MAGE-11 colocalizes with AR in the cytoplasm but is also detected in the nucleus unassociated with AR (Bai et al. 2005). Under these conditions, MAGE-11 binding to the AR FXXLF motif is unimpeded by the
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Fig. 5 Schematic of AR modulation by the N/C interaction, MAGE-11 and SRC/p160 coactivators. A dynamic equilibrium that regulates AR transcriptional activity includes the androgendependent AR N/C interaction and AR binding of MAGE-11 and SRC/p160 coactivators (Bai et al. 2005). In the absence of androgen, MAGE-11 (hatched rectangle) binds the AR FXXLF motif (Fx) and stabilizes AR. In the presence of bound testosterone, dihydrotestosterone (DHT) or synthetic AR agonist, the AR FXXLF motif binds AF2 in the AR ligand binding domain (LBD). The N/C interaction may occur within an inactive AR monomer and through domain swapping in the active AR dimer. The AR N/C interaction has an inhibitory effect on AR transactivation by competing with SRC/p160 coactivator LXXLL (Lx) motif binding to AF2. In the presence of androgen, AR transcriptional activity is increased by AR FXXLF motif binding to MAGE-11 through mechanisms that include, but are not limited to, increased AR AF2 recruitment of SRC/ p160 coactivators (Bai et al. 2005)
androgen-dependent AR N/C interaction. MAGE-11 stabilizes AR in the absence of androgen and at less than saturating levels of androgen, indicating at second mechanism involving the AR FXXLF motif that stabilizes AR and contributes to increased AR transcriptional activity.
5.2
Expression Pattern of MAGE-11
The physiological significance of MAGE-11 in modulating AR transcriptional activity remains to be fully established. MAGE-11 is a member of the MAGE gene superfamily of so-called cancer-testis or cancer-germline antigens and part of the 12 member MAGE-A subfamily coded at the Xq28 region of the human X chromosome (Chomez et al. 2001; Rogner et al. 1995; Simpson et al. 2005). MAGE-11 shares extensive sequence homology within its largest carboxyl-terminal exon with
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other members of the MAGE family (Bai and Wilson 2008). However, the MAGE11 gene has three additional short 50 coding exons, one of which contains a nuclear localization signal (Bai et al. 2005; Irvine and Coetzee 1999). Although AR and MAGE-11 are X-linked genes, naturally occurring mutations in the MAGE-11 gene have yet to be identified. MAGE-11 is expressed in human and nonhuman primates but not in other mammalian species such as rats and mice. Since a functional homolog for MAGE-11 has not been identified in rodents, MAGE-11 may have evolved as part of a mechanism for higher-level control of AR function in primates. Recent studies demonstrate that MAGE-11 is expressed in reproductive tissues of the human male and female, with temporal expression in human endometrium during the menstrual cycle (Bai et al. 2008). MAGE-11 mRNA levels are up-regulated by cyclic AMP and down-regulated by estradiol in Ishikawa and ECC-1 human endometrial cell lines. Studies are ongoing in primate models to determine the functional effects of MAGE-11 in male reproductive function. In prostate cancer, the levels of MAGE-11 appear to increase based on studies in prostate cancer cell lines. MAGE-11 mRNA levels correlate with AR expression since both are detected in LNCaP, CWR-R1, and LAPC-4 prostate cancer cell lines but are essentially undetectable in DU145 and PC-3 cells. The functional role of MAGE-11 in prostate cancer remains to be fully established. However, MAGE-11 binding to the AR FXXLF motif contributes to increased SRC/p160 coactivator recruitment by AF2 and may contribute to ligand-independent AR activity through mechanisms that more directly involve the AR NH2-terminal AF1 transcriptional activation region.
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Berrevoets CA, Doesburg P, Steketee K, Trapman J, Brinkmann AO (1998) Functional interactions of the AF-2 activation domain core region of the human androgen receptor with the amino-terminal domain and with the transcriptional coactivator TIF2 (transcriptional intermediary factor 2). Mol Endocrinol 12:1172–1183 Bevan CL, Hoare S, Claessens F, Heery DM, Parker MG (1999) The AF1 and AF2 domains of the androgen receptor interact with distinct regions of SRC1. Mol Cell Biol 19:8383–8392 Brown CJ, Goss SJ, Lubahn DB, Joseph DR, Wilson EM, French FS, Willard HF (1989) Androgen receptor locus on the human X chromosome: regional localization to Xq11–12 and description of a DNA polymorphism. Am J Hum Genet 44:264–269 Bubulya A, Chen SY, Fisher CJ, Zheng Z, Shen XQ, Shemshedini L (2001) c-Jun potentiates the functional interaction between the amino and carboxyl termini of the androgen receptor. J Biol Chem 276:44704–44711 Burd CJ, Petre CE, Moghadam H, Wilson EM, Knudsen KE (2005) Cyclin D1 binding to the androgen receptor (AR) NH2-terminal domain inhibits activation function 2 association and reveals dual roles for AR corepression. Mol Endocrinol 19:607–620 Casella R, Maduro MR, Misfud A, Lipshultz LI, Yong EL, Lamb DJ (2003) Androgen receptor gene polyglutamine length is associated with testicular histology in infertile patients. J Urol 169:224–227 Chang CY, Abdo J, Hartney T, McDonnell DP (2005) Development of peptide antagonists for the androgen receptor using combinatorial peptide phage display. Mol Endocrinol 19:2478–2490 Chomez P, De Backer O, Bertrand M, De Plaen E, Boon T, Lucas S (2001) An overview of the MAGE gene family with the identification of all human members of the family. Cancer Res 61:5544–5551 Choong CS, Wilson EM (1998) Trinucleotide repeats in the human AR: a molecular basis for disease. J Mol Endocrinol 21:235–257 Choong CS, Kemppainen JA, Zhou ZX, Wilson EM (1996) Reduced androgen receptor gene expression with first exon CAG repeat expansion. Mol Endocrinol 10:1527–1535 Choong CS, Kemppainen JA, Wilson EM (1998) Evolution of the primate androgen receptor: a structural basis for disease. J Mol Evol 47:334–342 Culig Z, Hobisch A, Cronauer MV, Cato AC, Hittmair A, Radmayr C, Eberle J, Bartsch G, Klocker H (1993) Mutant androgen receptor detected in an advanced-stage prostatic carcinoma is activated by adrenal androgens and progesterone. Mol Endocrinol 7:1541–1550 Darimont BD, Wagner RL, Apriletti JW, Stallcup MR, Kushner PJ, Baxter JD, Fletterick RJ, Yamamoto KR (1998) Structure and specificity of nuclear receptor-coactivator interactions. Genes Dev 12:3343–3356 Dehm SM, Regan KM, Schmidt LJ, Tindall DJ (2007) Selective role of an NH2-terminal WxxLF motif for aberrant androgen receptor activation in androgen depletion independent prostate cancer cells. Cancer Res 67:10067–10077 Dennis AP, Haq RU, Nawaz Z (2001) Importance of the regulation of nuclear receptor degradation. Front Biosci 6:D954–D959 Dubbink HJ, Hersmus R, Verma CS, van der Korput HA, Berrevoets CA, van Tol J, Ziel-van der Made AC, Brinkmann AO, Pike AC, Trapman J (2004) Distinct recognition modes of FXXLF and LXXLL motifs by the androgen receptor. Mol Endocrinol 18:2132–2150 Duff J, McEwan IJ (2005) Mutation of histidine 874 in the androgen receptor ligand-binding domain leads to promiscuous ligand activation and altered p160 coactivator interactions. Mol Endocrinol 19:2943–2954 Edwards A, Hammond HA, Jin L, Caskey CT, Chakraborty R (1992) Genetic variation at five trimeric and tetrameric tandem repeat loci in four human population groups. Genomics 12:241–253 Estebanez-Perpina E, Moore JM, Mar E, Delgado-Rodrigues E, Nguyen P, Baxter JD, Buehrer BM, Webb P, Fletterick RJ, Guy RK (2005) The molecular mechanisms of coactivator utilization in ligand-dependent transactivation by the androgen receptor. J Biol Chem 280:8060–8068
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Gao T, McPhaul MJ (1998) Functional activities of the A and B forms of the human androgen receptor in response to androgen receptor agonists and antagonists. Mol Endocrinol 12: 654–663 Gelmann EP (2002) Molecular biology of the androgen receptor. J Clin Oncol 20:3001–3015 Ghali SA, Gottlieb B, Lumbroso R, Beitel LK, Elhaji Y, Wu J, Pinsky L, Trifiro MA (2003) The use of androgen receptor amino/carboxyl-terminal interaction assays to investigate androgen receptor gene mutations in subjects with varying degrees of androgen insensitivity. J Clin Endocrinol Metab 88:2185–2193 Goh KI, Cusick ME, Valle D, Childs B, Vidal M, Baraba´si AL (2007) The human disease network. Proc Natl Acad Sci USA 104:8685–8690 Gregory CW, Hamil KG, Kim D, Hall SH, Pretlow TG, Mohler JL, French FS (1998) Androgen receptor expression in androgen-independent prostate cancer is associated with increased expression of androgen-regulated genes. Cancer Res 58:5718–5724 Gregory CW, He B, Wilson EM (2001a) The putative androgen receptor-A form results from in vitro proteolysis. J Mol Endocrinol 27:309–319 Gregory CW, He B, Johnson RT, Ford OH, Mohler JL, French FS, Wilson EM (2001b) A mechanism for androgen receptor mediated prostate cancer recurrence after androgen deprivation therapy. Cancer Res 61:4315–4319 Gregory CW, Johnson RT, Mohler JL, French FS, Wilson EM (2001c) Androgen receptor stabilization in recurrent prostate cancer is associated with hypersensitivity to low androgen. Cancer Res 61:2892–2898 Gregory CW, Fei X, Ponguta LA, He B, Bill HM, French FS, Wilson EM (2004) Epidermal growth factor increases coactivation of the androgen receptor in recurrent prostate cancer. J Biol Chem 279:7119–7130 Grino PB, Griffin JE, Wilson JD (1990) Testosterone at high concentrations interacts with the human androgen receptor similarly to dihydrotestosterone. Endocrinology 126:1165–1172 Haelens A, Tanner T, Denayer S, Callewaert L, Claessens F (2007) The hinge region regulates DNA binding, nuclear translocation, and transactivation of the androgen receptor. Cancer Res 67:4514–4523 He B, Wilson EM (2002) The NH2-terminal and carboxyl-terminal interaction in the human androgen receptor. Mol Genet Metab 75:293–298 He B, Wilson EM (2003) Electrostatic modulation of steroid receptor recruitment of the LXXLL and FXXLF motifs. Mol Cell Biol 23:2135–2150 He B, Kemppainen JA, Voegel JJ, Gronemeyer H, Wilson EM (1999) Activation function 2 in the human androgen receptor ligand binding domain mediates interdomain communication with the NH2-terminal domain. J Biol Chem 274:37219–37225 He B, Kemppainen JA, Wilson EM (2000) FXXLF and WXXLF sequences mediate the NH2terminal interaction with the ligand binding domain of the androgen receptor. J Biol Chem 275:22986–22994 He B, Bowen NT, Minges JT, Wilson EM (2001) Androgen-induced NH2- and carboxyl-terminal interaction inhibits p160 coactivator recruitment by activation function 2. J Biol Chem 276:42293–42301 He B, Lee LW, Minges JT, Wilson EM (2002a) Dependence of selective gene activation on the androgen receptor NH2- and carboxyl-terminal interaction. J Biol Chem 277:25631–25639 He B, Minges JT, Lee LW, Wilson EM (2002b) The FXXLF motif mediates androgen receptorspecific interactions with coregulators. J Biol Chem 277:10226–10235 He B, Gampe RT, Kole AJ, Hnat AT, Stanley TB, An G, Stewart EL, Kalman RI, Minges JT, Wilson EM (2004) Structural basis for androgen receptor interdomain and coactivator interactions suggests a transition in nuclear receptor activation function dominance. Mol Cell 16:425–438 He B, Gampe RT, Hnat AT, Faggart JL, Minges JT, French FS, Wilson EM (2006) Probing the functional link between androgen receptor coactivator and ligand binding sites in prostate cancer and androgen insensitivity. J Biol Chem 281:6648–6663
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The Role of the Androgen Receptor Polyglutamine Tract in Prostate Cancer: In Mice and Men Diane M. Robins
Abstract The androgen receptor (AR) is critical in the initiation and progression of prostate cancer, and therefore may contribute to disease through its genetic variation. Particular scrutiny has focused on a polymorphic N-terminal glutamine (Q) tract (CAG repeat) that shows population heterogeneity. Abnormal expansion of this tract underlies late-onset neurodegeneration, and in vitro the length correlates inversely with transcriptional activity. Yet the question of whether length variation within the range of normal human alleles affects cancer has produced discordant epidemiological results, in part due to interacting genetic and environmental factors in human disease. To test Q tract length effects, the mouse AR gene was converted to the human sequence (h/mAr), creating alleles with 12, 21, or 48 CAG repeats. These mice were grossly normal, but molecular analysis revealed allele-dependent differences in target gene expression. Further, when crossed with mice transgenic for a prostate-directed oncogene (TRAMP), Q tract length-dependent differences in cancer initiation and progression were evident. TRAMP mice with short Q tract ARs exhibited earlier but more slowly progressing disease than mice with median or long Q tract ARs. Q tract length also affected disease progression after castration, but in directions opposite to those in intact mice – the AR12Q allele delayed tumor detection whereas mice with the AR48Q allele fared worse. These experiments provided evidence for a causal relationship between a human polymorphism and a cancer phenotype. In man, Q tract length effects may only be significant at extremes of variation within the normal range and may vary with stage of disease. The h/mAR mice provide an experimental paradigm in which to dissect mechanisms by which Q tract length affects development and progression of prostate cancer. Some of these mechanisms may lead to better predictors of response to therapy and new treatments targeted to the human AR.
D.M. Robins Department of Human Genetics, University of Michigan Medical School, Ann Arbor, MI, 481090618, USA, E-mail:
[email protected]
D. Tindall and J. Mohler (eds.), Androgen Action in Prostate Cancer, DOI: 10.1007/978-0-387-69179-4_12, # Springer Science + Business Media, LLC 2009
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1 Introduction Androgen signaling through its nuclear receptor (androgen receptor, AR) controls male differentiation, including the development and function of the prostate gland. Because of AR’s regulatory role in normal growth and homeostasis, it is also a pivotal component in diseases of the prostate. Prostate cancer in particular is a substantial health problem, with an etiology and course affected by many disparate factors. Risk of disease varies with population and with environment, and many cancers remain indolent while others progress rapidly to a lethal state. Underlying this heterogeneity in disease origin and trajectory is a nearly universal dependence on androgen in early stages of tumor growth and, despite therapeutic hormone depletion, on AR in later stages. Therefore, genetic variation in AR structure and expression may impact initiation and progression of disease. This chapter focuses on a highly polymorphic region of the AR, the polyglutamine tract (polyQ, Q tract, CAG repeat). Variable length of the CAG repeat first was noted in association with neurological disease and subsequently was suspected to be a factor in prostate cancer. Epidemiological studies, however, with different human populations and different patient criteria have produced discordant results. Dissection of glutamine tract effects in vitro has provided mechanisms underlying AR activity differences, but it is unclear whether these differences impact the function of AR during tumorigenesis. To address Q tract variation in vivo and to avoid the confounding genetic and environmental heterogeneity of man, a mouse model with variant human Ar alleles was created that represented extremes of Q tract length that were within the normal range. The mice demonstrated Q tract length effects in prostate cancer that varied with tumor stage and with strength of the androgen axis, which reflected well the complexity of human disease. Analysis of the Q tract in mice has lended support to a broader view of AR’s role in oncogenesis, which includes dictating context-dependent and opposing functions.
2 AR and the Polymorphic Polyglutamine Tract As a member of the nuclear receptor superfamily, AR consists of a highly conserved central DNA-binding domain (DBD), a moderately conserved C-terminal ligand-binding domain (LBD), and a variable N-terminal transactivation domain (NTD). The NTD of AR comprises over half of the encoded protein and bears little similarity to functionally homologous regions of other steroid receptors. Hormone binding to the LBD alters the stable association of AR with a cytoplasmic chaperone complex to a dynamic interaction that permits nuclear localization (Pratt et al. 2004). In the nucleus the DBD recognizes response elements in target genes. The NTD participates in recruiting diverse cofactors that orchestrate transcription, in part dependent on direct contact with the hormone-dependent activation function (AF-2) in the C-terminus (He and Wilson 2002). This N/C interaction is critical for
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optimal ligand binding and transactivation, and integrates differential effects of coactivators, chaperones, and post-translational modifications by ubiquitin ligases and kinases (Shen and Coetzee 2005). Promoter context-dependent interactions of the NTD with other DNA-binding proteins also enhance selectivity of AR for its targets (Robins 2004). Significant insight into nuclear receptor physiology is possible because the AR gene is located on the X-chromosome (Ar; Xp11-12). Since males are hemizygous for AR, mutations are phenotypically evident, and cases of partial to complete androgen insensitivity (AIS) have revealed structure/function correlations that are informative to the steroid receptor family in general. While mutations leading to androgen insensitivity mostly occur in the DBD and LBD (McPhaul 2002), abnormal expansion of an N-terminal CAG repeat encoding a polyglutamine tract underlies the androgen-dependent late-onset neurodegenerative disease, spinal and bulbar muscular atrophy (SBMA, or Kennedy disease) (La Spada et al. 1991). The Q tract is highly polymorphic, ranging from 9 to 37 repeats in the normal population, but occurrence of 40 or more glutamine residues leads to neurodegeneration. In contrast to the loss of AR function found in AIS, Kennedy disease, like other trinucleotide repeat expansion disorders, results from both a toxic gain of function due to misfolding and aggregation of the mutant protein, as well as a partial loss of normal protein activity (Lieberman and Robins 2008). Studies in vitro ascribe the loss of function to an inverse correlation between length of the Q tract and transcriptional strength of the AR (Mhatre et al. 1993; Chamberlain et al. 1994). Given the wide range in Q tract length in the population, whether its variation underlies other androgen-dependent pathologies, such as prostate cancer, should be investigated. In most individuals, the AR Q tract consists of between 15 and 30 contiguous glutamines from amino acid 58, with 21 CAG repeats in the reference human AR sequence (Zitzmann and Nieschlag 2003) (Fig. 1). In addition to the Q tract, there is a polymorphic polyglycine tract (G tract, GGN repeat) at amino acid 449, with 14–27 repeats. These trimeric repeats vary among ethnic populations in the United States, calling attention to them as possible genetic risk factors (Edwards et al. 1992). In an initial survey, the frequency of AR alleles with fewer than the median 22 CAGs was about 50% for Caucasians but much greater for African Americans and much less for Asian Americans, which parallels their differential risk of prostate cancer. These epidemiological observations suggest the hypothesis that the Q tract might be associated with prostate cancer risk, with higher risk deriving from greater activity of a shorter Q tract AR (Coetzee and Ross 1994). This was initially corroborated by finding a prevalence of short AR-CAG alleles in prostate cancer patients, especially those with advanced disease (Irvine et al. 1995), as well as somatic shortening of the CAG repeat in some tumors (Schoenberg et al. 1994). These findings are potentially important for risk assessment and prognosis, but subsequent research has produced conflicting results. Addressing the complexity requires first establishing that Q tract length differences in the normal range affect AR activity.
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Fig. 1 Polyamino acid tracts within human, mouse, and dog ARs. The diagram shows the receptor domains for transactivation, DNA binding (DBD), and ligand binding (LBD); the former shares 85% amino acid identity between man and mouse whereas the DBD and LBD are identical, except for the intervening hinge (h) domain. The region of coding sequence that was swapped to humanize the mouse AR was stippled, and includes the polyglutamine tracts (QI and QIII) and the polyglycine tract (G). Key amino acid positions are below the diagram for scale. Range in number of codon repeats within the polyamino acid tracts in normal populations is shown for the three species
3 Functional Analysis of the Glutamine Tract Glutamine-rich regions are functionally important in several transcription factors besides AR, including Sp1, TATA-binding protein, and the glucocorticoid receptor (Gerber et al. 1994). These domains form b-sheets that act as polar zippers to enhance affinity with other proteins in a nonspecific manner, or, for expanded Q tracts, to promote self-aggregation (Perutz et al. 1994). More recent biophysical studies of full-length ARs revealed that the relatively disordered NTD shows Q tract length-dependent differences in secondary structure, with longer tracts conferring greater flexibility (Duff et al. 2006). Functional subdomains within the NTD encompassing distinct transactivation surfaces (e.g., TAU1, TAU5) and the N/C interaction motifs (23FxxLF, 435WxxLF) may be differentially masked or exposed dependent on hormone-binding, post-translational modification, and cellspecific coregulators (Dehm and Tindall 2007). The Q tract may modulate these intra- and intermolecular interactions in a length-dependent manner by functioning as a spacer between domains. Initial mechanistic studies ascribed the reduced activity of long Q-tract ARs in transfection assays to decreased AR expression at both the mRNA and protein levels (Choong and Wilson 1998). Trinucleotide repeats are inherently unstable in DNA, and CAG repeats can form stem-loop structures in RNA that could be the target of RNA-binding proteins. Altered affinity of RNA-binding proteins due to CAG repeat length could impact both mRNA stability and translation efficiency. Precedent exists for this mechanism although such binding proteins have yet to be identified for AR. In the gene responsible for myotonic dystrophy (Yeap et al. 2004), an expanded CUG repeat in the 30 untranslated region of the mRNA was
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bound by a protein (CUGBP1) that caused aberrant RNA processing, which contributes to neurotoxicity (Mankodi et al. 2002). For AR, gene expression is also subject to complex cell-type dependent regulatory mechanisms that may be influenced by Q tract length. Furthermore, while decreased mRNA and protein levels may contribute to reduced activity of expanded Q tract ARs, significant differences in expression levels have not been noted for CAG repeat lengths within the normal range. The first AR-associated protein found to be sensitive to Q tract length differences was Ran/ARA24, which showed decreased interaction and activation of ARs with expanded Q tracts (Hsiao et al. 1999). While RAN/ARA24 behaved as an AR coactivator in transfection assays, it is better known for its roles in cell cycle progression, maintenance of nuclear structure, and nuclear transit of RNA and protein. Differential AR activity impacted by Q tract length thus may be indirectly linked via efficiency of nuclear localization or nuclear retention. However, RAN/ ARA24 has yet to show sensitivity to repeat length differences within the range of normal alleles. Such sensitivity, however, has been found for p160 coactivators. The effect is more likely due to Q tract length effects on AR’s structure than to direct interaction of the tract with other proteins (Irvine et al. 2000; Wang et al. 2004). In experiments where protein expression levels were controlled carefully, AR transactivation decreased linearly with increasing tract lengths by 20% over a range of 9Q to 42Q, and more precipitously by 80% for 50Q. The presence of p160 coactivators accentuated these modest differences. Furthermore, the short tract 9Q AR responded in transfection assays to lower concentrations of androgens than moderate or long tract ARs, primarily due to enhanced N/C interaction (Wang et al. 2004). Short tract ARs increased association with coactivators as well as components of the SWI/SNF chromatin remodeling complex, which increased transactivation and androgen-dependent cell proliferation in prostate cancer cell lines. Thus modest Q tract effects caused by several molecular mechanisms ultimately combine to impact transcriptional output. The effect of Q tract length on transactivation is not a simple linear relationship (Buchanan et al. 2004; Ding et al. 2004). AR N/C interaction seems to be optimally maintained over a critical size range of 16–29Qs. This range encompasses more than 90% of AR alleles in most populations, which suggests that pathologies may be associated with lengths at the extremes of the normal range rather than with subtle variations. This is supported by analysis of a somatic AR mutation where two nonconsecutive leucines disrupt the Q tract, presumably reducing flexibility of the NTD (Buchanan et al. 2004). For this AR-polyQ2L mutant, both protein levels and N/C interaction were reduced, but transactivation activity was increased. Thus very short or long Q tracts disrupt N/C interaction, which destabilizes AR and likely underlies reduced protein levels. This N/C interaction effect is distinct from the incremental effect of Q tract length on transactivation, for which each additional glutamine residue within the normal range may reduce structural order of the NTD, which results in reduced ability to recruit coactivators and other components of the transcriptional machinery. In contrast, interaction with corepressors appears less sensitive to differences in Q tract length (Buchanan et al. 2004). These studies
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support the notion that the Q tract does not possess an intrinsic function but instead enables contact between NTD motifs and AF-2 to position AF-1 for interaction with coregulators. How the multiple, alternative surfaces within AF-1 vary in their interactions dependent on Q tract length remains to be determined.
4 Epidemiology of AR Glutamine Tract Associations Prostate cancer has a significant genetic component, with twin studies suggesting that over 40% of cases are influenced by heredity, but whether this is due to common genetic variants or rare disease genes is unclear (Lichtenstein et al. 2000). Since AR is intimately involved in initiation and progression of prostate cancer, AR variation is a suspected risk factor. Although Q tract length differences within the range of normal alleles lead to demonstrable effects in vitro, studies to ascertain whether such variation impacts human physiology or cancer risk are difficult. Substantial evidence suggests that male fertility is impaired for longer Q tract lengths short of the neuropathological range (Casella et al. 2001; Davis-Dao et al. 2007). However, this association is less evident in European populations, which underscores a role for environmental factors (Yong et al. 2003). In hypogonadal men, such as those with Klinefelter’s syndrome, low androgen levels fail to saturate receptors, and therefore differences in AR activity may be more apparent (Zitzmann et al. 2004; Crabbe et al. 2007). In fact hypogonadal men with short CAG repeats were more responsive to testosterone replacement, which provides a physiological correlate to the hypersensitivity of short tract ARs in vitro (Wang et al. 2004). These syndromes provide support for the notion that Q tract length differences within the normal range affect AR activity in vivo. The hypothesis that short Q tract ARs increase prostate cancer risk was suggested by (1) the greater prevalence of these tracts in the high-incidence AfricanAmerican population, (2) identification of Q tract contraction in malignant but not benign prostate cells (Alvarado et al. 2005), and (3) in vitro evidence for greater transcriptional activity of short Q tract ARs, which might enhance oncogenic transformation. But association at the genetic level has been difficult to confirm for a variety of reasons. Out of nearly 100 studies examining Q tract length and prostate cancer, about half have found an association between short tract length and increased risk, earlier age of diagnosis or more advanced disease at diagnosis (e.g., Irvine et al. 1995; Giovannucci et al. 1997; Stanford et al. 1997). However, many studies, including some with large numbers of subjects, found no association (Zeegers et al. 2004; Freedman et al. 2005), and others found the opposite association of increased risk with long Q tract lengths (Edwards et al. 1999; Suzuki et al. 2002; Li et al. 2003; Lindstrom et al. 2006b). The G tract has not been examined as extensively, but there is a similar level of disagreement as to whether its variation is associated with risk of prostate cancer (e.g., Irvine et al. 1995; Stanford et al. 1997;
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Hsing et al. 2000; Chang et al. 2002; Zeegers et al. 2004). With regard to later stage disease and AR function, results also disagree on effects of CAG repeat length on response to androgen-deprivation treatment (Bratt et al. 1999; Suzuki et al. 2003). These conflicting conclusions are due to several factors. Smaller studies lack sufficient power to achieve statistical significance. The association with race is subject to differences in environment, socioeconomic status, admixture, and disparity in health care. Furthermore, while the median CAG repeat number is 19 in African Americans and 22 in Caucasian Americans (Edwards et al. 1992; Irvine et al. 1995), relatively few African Americans have tracts less than the 16 Qs that are the cut-off for transcriptional effects in vitro (Buchanan et al. 2004). Heterogeneity in both genetics and environment influences outcome, as evident in opposite associations found between Q tract length and prostate cancer risk in Swedish compared to Japanese populations (Li et al. 2003). Heterogeneity in prostate cancer progression complicates results, since some studies find associations with more advanced disease or earlier age at diagnosis but no association with overall risk (Hardy et al. 1996; Beilin et al. 2001; Cude et al. 2002; Santos et al. 2003; Shimbo et al. 2005; Sieh et al. 2006). Importantly, stronger associations are found in studies with populations either prior to or less reliant on PSA testing, since these patients present with more advanced tumors. Subsequent to PSA testing, malignancies that might never become symptomatic are diagnosed at greater frequency, increasing bias to the null hypothesis (Giovannucci 2002). Advances in genotyping technology have increased the number of genetic association studies for complex diseases such as prostate cancer, but have not resolved an association with Q tract length. A meta-analysis performed in 2004 detected a slightly increased risk associated with short CAG repeats, and this is probably an underestimation due to the inclusion of low-incidence populations (Zeegers et al. 2004). More recent studies focusing on the high-risk Swedish population found association of short Q tracts with advanced disease but disagreed with respect to overall risk. This may stem from differences in cut-off points – the study with a negative outcome defined short as 22 and long as >22 CAG repeats (Lindstrom et al. 2006b), whereas division of lengths by tertiles resolved a higher risk associated only with tracts 19 Qs (Andersson et al. 2006). Given the complexity of the epidemiological data, AR may be best viewed as a quantitative trait, the effect of which depends on variation at other loci that also vary in populations and are influenced by environment. Thus more compelling data include other genes in the androgen axis, including those encoding enzymes of testosterone synthesis, most notably cytochrome P450 (cyp17), and the enzyme that converts testosterone to the more active dihydrotestosterone, steroid-5-a-reductase type 2 (SRD5A2). Examining AR, cyp17, and SRD5A2 alleles together revealed haplotypes with a significant two-fold greater risk overall (Lindstrom et al. 2006a). This report underscores the multigenic nature of the androgen axis in the development of prostate cancer.
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5 Comparative Biology of Glutamine Tract Variation Insights into complex diseases are often gained from comparison to animal models. Dogs are the only species other than primates known to develop age- and hormonedependent prostate cancer (Cunha et al. 1987). Canine prostate physiology and prostate cancer progression are similar to man, and both have polymorphic AR Q tracts. There are two major CAG repeats in mammalian ARs, with the N-terminal one (CAG-I) more extensive in primates and the C-terminal one (CAG-III, at human amino acid 193) more extensive in rodents (Choong and Wilson 1998; Lu et al. 2001) (Fig. 1). A very short CAG-II just proximal to CAG-I is less variable between species. While most species have preferentially amplified either CAG-I or CAG-II, dogs are unusual in having long tracts at both positions, although CAG-I is shorter than in man. Only primates have an extensive G tract. Given the rich diversity of canine breeds, Q tract variation was assessed in multiple breeds and an association with prostate cancer incidence sought using data on dog prostate cancer incidence from the Veterinary Medical Data Base of Purdue University via collaboration with Dr. Vilma Yuzbasiyan-Gurkan of Michigan State Veterinary Medical Center. Of over 200,000 cases presenting for any medical problem, approximately 0.5% of male dogs had prostate cancer. In addition, incidence varied significantly among breeds. For example, 3.5% of intact male beagles presented with prostate cancer compared to only 0.5% of golden retrievers. Therefore, polymorphism in CAG-I and –III within and between breeds was analyzed for ten individuals of each of five breeds with low prostate cancer incidence (golden retriever, Siberian husky, cocker spaniel, dalmatian, and greyhound) and five breeds with high incidence (beagle, Scottish terrier, Rhodesian ridgeback, doberman pinscher, and Shetland sheepdog). Both canine Q tracts are polymorphic, with 9–12 Qs in CAG-I and 21–24 Qs in CAG-III (Shibuya et al. 1993). Two allele lengths were observed for CAG-I and 4 for CAG-III. While some variation in both tracts occurred within both groups, association of short tracts with tumorigenesis by breed did not reach statistical significance. More compelling data came from analysis of individual dogs with spontaneous disease. Of 13 histologically confirmed prostate cancer samples, ten had the short allele at CAG-I and either the shortest of the four alleles at CAG-III, or an even shorter allele absent from normal dogs. Moreover, two tumors were heterogeneous in CAG-III, displaying yet shorter alleles indicative of contraction during tumor progression. This pilot study supports the hypothesis that ARs with short Q tracts, in men or dogs, confer greater prostate cancer risk, and further, that such receptors arise and are selected for during tumorigenesis. Mice are preferable to dogs as models of human disease pathogenesis because of smaller size, faster generation time, and the homogeneous genetic background of numerous inbred lines. Although mice have a lobular prostate and develop spontaneous prostate cancer rarely, xenograft and germline modification techniques compensate for these deficiencies. All stages of disease can be sampled, in contrast to clinical specimens in which early events are more difficult to observe. However,
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the mouse Q tract differs significantly from the human (Fig. 1). Rodents have an abbreviated CAG-I and while rats have 20 Qs in CAG-III, the mouse tract is disrupted by histidines (Q8HQ3HQ2HQ4), likely influencing its flexibility as an interdomain spacer. Exclusive of the Q tract, the amino acid sequence of the mouse and human NTDs differ by 15%, in contrast to the DBD and LBD that are identical except for eight amino acids in the hinge region. In transfection experiments, the reduced transactivation strength of the rodent AR relative to human maps to the NTD (Chamberlain et al. 1994). Therefore, to create a mouse model to assess Q tract length effects on phenotypes and disease, genetic engineering was used to ‘‘humanize’’ the mouse AR, by swapping sequences encoding the human NTD into the mouse gene.
6 Development of ‘‘Humanized’’ AR Mice to Study Glutamine Tract Function To convert the mouse AR to the human sequence, targeting vectors for germline homologous recombination were constructed that used a backbone of a 129/Sv mouse genomic fragment encompassing mAR exon 1, which encodes almost the entire NTD. Amino acids 31–484 (including a median length Q tract of 21 residues) from human AR cDNA were substituted for the equivalent mouse sequences via conserved restriction sites, and a neomycin selectable marker was introduced into intron 1, near the 50 splice junction (Albertelli et al. 2006). Additional vectors were constructed to create AR alleles with 12 or 48 Qs, which represented extreme lengths within the normal variation range. These DNAs were electroporated into mouse embryonic stem (ES) cells where homologous recombination occurs via the substantial 50 flanking and intronic chromosomal sequences in the vector. The selectable marker was excised from ES clones by transfection of a plasmid expressing cre-recombinase, which produced a transcription unit with the humanized AR under the control of mouse regulatory sequences. This ‘‘h/mAR’’ sequence differs from human in retaining mouse codons for one N-terminal amino acid, 14 residues just before the DBD (ten of which are conservative changes) and eight amino acids (four conservative) in the hinge region. The targeted ES cells were introduced into mouse blastocysts, and chimeric progeny were used to establish lines of h/mAR mice that have been backcrossed onto the C57BL/6 background. Both sexes of the h/mAR mice were indistinguishable from wild-type mAR littermates in gross physiology including behavior, growth rate, body weight, and lifespan (Albertelli et al. 2006). All three strains were fertile with similar frequency of litters and number of pups per litter. In particular, while longer Q tracts were associated with reduced fertility in man, the 48Q male mice remained as fertile as the other genotypes even at older ages. A preliminary analysis of the androgen axis indicated no significant differences between genotypes in serum testosterone, LH or FSH levels, although individual values varied over a broad range. Whether the
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androgen axis is sensitive to Q tract length will be tested more stringently for mice that have been backcrossed additional generations and are individually housed to eliminate effects of social interaction. When seminal vesicle weight was used as a more direct indicator of androgen action, differences among genotypes became apparent; seminal vesicle weight was lower in h/mAR48Q mice at 6 months and greater by 2 years of age in h/mAR12Q mice. These data confirm an inverse correlation between AR activity and Q tract length, and suggest that minor effects may sum over time to detectable phenotypes. The h/mAR48Q mice were studied further since 48 CAGs may cause partial androgen insensitivity or Kennedy disease in man. However, gene defects often must be more severe in mice than man to model a human syndrome, perhaps due in part to differences in lifespan and, relevant to both trinucleotide repeats and cancer, differences in genomic instability. While transgenic ARs with 65Qs produced no phenotype in mice (Bingham et al. 1995), mice created by introducing 112Qs with our targeting vector replicated the pathology of Kennedy disease (Yu et al. 2006). The h/mAR48Q mice showed no evidence of neuromuscular deficiency, as determined by grip strength tests, even at advanced ages. Even though fertility remained normal, some variation in testis function was discernable at the molecular level. In particular, mRNA levels of Hsd17b3, an indicator of mature Leydig cell function, were significantly lower in 48Q than other mice, although levels were still within a normal expression range. Further, although AR protein levels were similar between genotypes, and no histological evidence of aggregation was observed in AR48Q cells as reported in Kennedy disease, this protein may be predisposed to aggregate since substantial amounts were found in the pellet fraction of whole cell lysates. Therefore, h/mAR48Q, while sufficient for male mouse differentiation and virility, may have suboptimal activity in some circumstances. This characterization confirmed that human and mouse ARs have sufficient conservation, as might their interactions with critical coregulators, to substitute functionally for one another at the organismal level. Furthermore, while minor phenotypic variations were detected for h/mARs with 12 or 48 Qs, these were mostly within the normal range of variation for these traits and did not produce pathology. These alleles in mice may model the extremes of Q tract length within the range of normal alleles similar to those found in man, which allows investigation of whether these variants influence prostate biology or disease. At a gross level, the three h/mAR strains appeared no different from wild-type mice in prostate morphology, with equivalent epithelial and nuclear AR at the immunohistochemical level. At a molecular level, however, differences in AR regulation were detected using quantitative RT-PCR. AR mRNA levels showed a trend to inverse correlation with Q tract length in 6-month-old mice. More significantly, the variation in expression of some AR target genes was consistent with greater transcriptional activity of the 12Q allele and reduced activity of the 48Q allele (Fig. 2). In particular, androgen-regulated probasin, a protein secreted from mature prostatic epithelium, was expressed at higher levels in 12Q than 21Q or 48Q h/mAR mice. Nkx3.1, a prostate-specific and AR-dependent homeobox gene, showed significantly lower levels in h/mAR48Q mice. In contrast, clusterin
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Fig. 2 Prostate gene expression was influenced by AR Q tract length. Prostate mRNAs from 3 to 8 mice at 6 months of age per h/mAR allele were quantified using real-time RT-PCR. Results are relative to levels in wild-type mAR mice. Probasin and Nkx3.1 were up-regulated by AR, clusterin was repressed. Asterisks indicate significant differences. Modified from Albertelli et al. (2006), Copyright 2006, The Endocrine Society
(an antiapoptotic protein also known as testosterone-repressed prostate message-2), which was down-regulated by AR (July et al. 2002), had higher expression in h/ mAR48Q mice. Q tract length appears to impact not just AR activated but also ARrepressed genes. Overall, differences in target gene expression due to h/mAR allelic variation were small, but consistently trended to greater activity of the 12Q allele and lesser activity of the 48Q allele, for both induction and repression. These results support the hypothesis that Q tract length impacts differential transcription of critical androgen-dependent genes, some of which may impact prostate cancer risk. A broader view of differential regulation by AR alleles was obtained from a preliminary comparison of prostate gene expression using Affymetrix oligonucleotide microarrays. Stringent statistical criteria did not reveal significant differences between AR alleles (including mAR), but when data were analyzed without adjusting for multiple comparisons, some intriguing patterns emerged (Albertelli 2007). For example, differentially expressed genes were frequently at highest levels in h/mAR48Q mice, which corroborated studies that reported more genes were repressed than activated by AR in the prostate (Desai et al. 2004). h/mAR12Q and h/mAR48Q expression often trended in the same direction relative to that of h/mAR21Q, which suggested that for many promoters the median Q tract length provides optimal activation (or repression), with deviation to either longer or shorter tracts having similar effects. Frequently only the 12Q or the 48Q differed from the other two alleles, which suggested that Q tract effects may be promotercontext dependent and encompass multiple mechanisms. Specific transcripts showing differential expression include some previously noted as either androgen-regulated or overexpressed in prostate cancer, such as several involved in inflammatory responses. Of particular interest are a few genes up-regulated in h/mAR48Q mice that are involved in Wnt signaling, especially Wnt5a, whose twofold greater expression was validated using Q-PCR. Crosstalk occurred between
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AR and Wnt signaling pathways and Wnt overexpression has been associated with androgen-stimulated prostate cancer (Yang et al. 2006). In this case, higher expression of Wnt5a in h/mAR48Q mice may be a factor in their development of aggressive castration-recurrent cancer (see later). More definitive analyses using laser capture microdissection will provide additional information on genes and pathways influenced by AR Q tract length.
7 Glutamine Tract Length Effects in Mouse Prostate Cancer h/mAR mice were crossed to the engineered model TRAMP (transgenic adenocarcinoma of the mouse prostate) to investigate the effect of Q tract length variation on oncogenesis. TRAMP mice carry the SV40 T antigen (Tag) oncogene driven by the prostate epithelium-specific probasin promoter and enhancer (Greenberg et al. 1995). TRAMP males develop prostatic intraepithelial neoplasia (PIN) and welldifferentiated prostate cancer that seems histologically similar to the human disease (Kaplan-Lefko et al. 2003). PIN generally develops by 12 weeks of age, and tumors are abdominally palpable somewhat later; metastasis occurs predominantly to lymph nodes, lungs, and liver, but not bone. Development of prostate cancer in TRAMP males is fully penetrant and can be delayed by castration, but ultimately progresses, indicating that disease becomes androgen independent, as in man. While T antigen is a more potent inducer of disease than in human prostate cancer, abrogation of Rb and p53 function occurs in both. TRAMP mice have been invaluable for studying early events in prostate cancer and for evaluating treatment and prevention strategies, although more recent models that incorporate inactivation of the tumor suppressor PTEN may more accurately mimic the origins of human disease (Wang et al. 2003). Regardless of how these models induce cancer and despite the genetic uniformity of mice, there is heterogeneity in time of initiation and rate of progression, which demonstrates the stochastic nature of tumorigenic events. Some of these events are genetic, as they vary with strain background, and some may be epigenetic and subject to diet, inflammation, and hormonal parameters. h/mAR-TRAMP mice and mAR-TRAMP littermates were compared to determine whether differences in Q tract length affected the development of PIN (Albertelli et al. 2008). PIN in mice is the precursor to carcinoma and is characterized by features including epithelial tufting, nuclear hyperchromasia, amphophilic cytoplasm, apoptotic debris, and cribriform architecture (Shappell et al. 2004). At 12 weeks of age, each prostate from ten mice per AR allele had varying levels of PIN in all prostatic lobes. The amount of dorsal epithelium associated with PIN per dorsal lobe was quantified by scoring into categories 1–10, which approximated the percentage of involvement (Fig. 3). A compelling trend was evident, although within each cohort interindividual differences precluded reaching statistical significance. More 12Q mice clustered at higher levels of PIN (70% in category 6 or above), while the majority of 48Q mice exhibited lower PIN
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Fig. 3 The amount of PIN was inversely proportional to Q tract length. The amount of epithelium involved in PIN at 12 weeks of age was assessed in sections of dorsal lobes, ten prostates per allele, and categorized by approximate % PIN involvement (categories 1–10, where 1 represents about 10% and 10 about 100% PIN involvement). Distribution per genotype showed a trend to inverse correlation with Q tract length (median categories of 7, 6, and 4 for 12Q, 21Q, and 48Q, respectively). Areas of PIN identified by H&E staining were examined in sequential sections for AR and Tag expression using IHC. Overall, AR and Tag expression was similar among Q tract variants, with AR fairly uniform in epithelium and stroma, while Tag expression was highest in areas of PIN. Modified from Albertelli et al. (2008), Copyright 2008, Oxford University Press
levels (60% in category 4). Furthermore, a higher grade of PIN, distinguished by greater expansion of the gland, cribriform proliferation, and more pronounced hyperchromasia, was detected in some of the 12Q and 21Q, but not 48Q, mice. Therefore, AR Q tract length had a notable affect on PIN by 12 weeks of age, with shorter tracts promoting more and higher-grade PIN than longer tracts. The higher levels of PIN may reflect increased proliferation rates or decreased apoptosis, which would also influence subsequent oncogenesis; whether these are affected by Q tract length is under study.
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The h/mAR mice expressed similar levels of prostatic AR mRNA in all genotypes, and immunohistochemistry (IHC) showed similar levels of AR protein in epithelial and stromal nuclei (Albertelli et al. 2006; Albertelli et al. 2008) (Fig. 3). AR in areas of PIN appeared slightly more intense than in normal epithelia, but this likely reflected the higher cell density in these regions. In contrast, although Tag did not show differential staining dependent on genotype, it was more prominent in areas involved in PIN than in normal epithelium. AR allele-dependent differences in Tag expression below the level of detection of IHC may exist, since expression of probasin, whose promoter drives the transgenic oncogene, varied with AR allele in the parental strains. Furthermore, functions of AR in stroma vs. epithelia may enter into higher Tag expression in regions of PIN compared to normal epithelia. Altogether, a cascade of events in neoplastic transformation may enhance Tag expression. In some regards, Tag is analogous to the translocation product TMPRSS2:ETS, in which an androgen-responsive promoter is fused to a transcriptional regulator of proliferation as a common and early event in human prostate oncogenesis (Tomlins et al. 2007). Fusions of this type may maximize the contribution of (or the sensitivity to) the androgen signaling axis in prostate cancer progression. Tumorigenesis was followed in h/mAR-TRAMP mice by abdominal palpation, which detects tumors only a few days later on average then their identification by more costly and time-consuming magnetic resonance imaging (Albertelli et al. 2008). Tumors become palpable at approximately 1 cm in diameter, at midstage disease. Kaplan-Meier analysis revealed a significant difference among genotypes in time to tumor detection (P value <0.0001; Fig. 4), with the median age of detection 10 weeks later for 48Q mice than for the other genotypes. Tumors were initially palpable in 12Q, 21Q, and mAR mice over a similar age range (median: 25 weeks for 12Q and 27 weeks for 21Q), but the kinetics of progression differed among genotypes. Comparing disease status at 29 weeks of age, by which time about half of the 21Q and mAR mice have a detectable tumor or have already died, overt disease was detected in 85% of the 12Q mice, and in less than one-third of the 48Q mice. Age of death further distinguished the genotypes by Kaplan-Meier analysis (P value <0.0001). mAR and 21Q mice survived to mean age of 30 weeks, compared to 42 weeks for 12Q and 55 weeks for 48Q mice. Taken together, the slightly greater PIN levels at 12 weeks, more tumors detectable by 29 weeks, and longer survival relative to 21Q mice suggest that in 12 Q mice tumors initiate earlier but progress more slowly than for the other AR alleles. This is corroborated by comparing ‘‘disease length,’’ noted as the time between initial tumor detection and death. Disease length is longer, and ranges more widely, in 12Q and 48Q mice than 21Q or mAR mice. Given the genetic homogeneity of these mice, stochastic events appear to increase heterogeneity in tumor progression as well as initiation. End-stage tumors were used to explore whether the different AR alleles played a role in heterogeneous tumor progression, using IHC and a tissue microarray. Shorter disease length corresponded to poorly differentiated or undifferentiated tumors with heterogeneous AR staining for all genotypes (Fig. 5). In contrast, the majority of 12Q and 48Q mice that had long disease lengths had well- or moderately
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Fig. 4 Q tract length affected age of tumor detection and survival in intact mice. The upper panel shows proportion of mice without a palpable tumor and the lower panel shows prostate cancer-free survival; P values are from logrank analysis. Number of mice per group (n) was: mAR = 18, 12Q = 13, 21Q = 19, 48Q = 19. Modified from Albertelli et al. (2008), Copyright 2008, Oxford University Press
differentiated tumors with high levels of AR expressed in most epithelial and stromal cells. IHC results were confirmed using Q-PCR and Western blot analysis of gross tumor samples. For all genotypes, greater differentiation and slower progression was associated with AR levels higher than benign prostate, and was influenced by Q tract length. In contrast, Tag levels were heterogeneous regardless of AR allele, level of expression, or disease length, which corroborated a general uncoupling from AR regulation that was evident in advanced tumors (KaplanLefko et al. 2003). These TRAMP tumors also differed in their extent of neuroendocrine phenotype, as indicated by synaptophysin expression. In man, neuroendocrine differentiation is associated with aggressive, castration-recurrent prostate cancer that mostly lacks AR expression (Shariff and Ather 2006). In TRAMP neuroendocrine cells are also associated with aggressive disease, but they can express AR (Kaplan-Lefko et al. 2003). Most 21Q tumors express moderate levels of both synaptophysin and AR. 48Q tumors showed the least synaptophysin expression, with only about half of the tumors positive for the marker and only half of those also showing AR expression. Tumors from 12Q mice were more similar to the human phenotype; synaptophysin and AR expression were mutually exclusive, with half of the tumors expressing synaptophysin and the other half expressing AR. Overall, synaptophysin expression in the TRAMP tumors, as in human disease, is most often associated with rapid
Fig. 5 Markers of prostate cancer progression in intact h/mAR-TRAMP mice. End-stage tumors were used to create a tissue microarray and sections were stained for AR (above) and synaptophysin (below) (with vertical rows from the same tumor). Two tumors are represented per genotype, the left one represents shorter disease length and the right one longer disease length; disease length in weeks is indicated in the black box in the upper right. 12Q and 48Q tumors of longer disease length had greater differentiation and AR expression, and less synaptophysin expression. Bar = 100 mM. Modified from Albertelli et al. (2008), Copyright 2008, Oxford University Press
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progression. A complementary pattern was shown for expression of clusterin, an antiapoptotic protein more often associated with more differentiated tumors (Caporali et al. 2004). Study of this mouse model of AR Q tract length polymorphism confirmed some expectations from epidemiological data. Disease initiation follows a linear trend from earlier occurrence in mice with shorter Q tracts to significant protection for longer Q tracts. Disease progression, however, follows a more complicated course. The effect on initiation likely stems from increased or earlier Tag activation by shorter Q tract ARs that hastens the time until some critical level for neoplasia is reached. Early androgen-dependent events in human prostate cancer may act similarly in the case of TMPRSS2 fusion genes (Tomlins et al. 2007). Tumors in mice with long Q tract ARs initiate later and progress slowly, perhaps modeling indolent disease more commonly found in aging men. In the 12Q mice, tumors progress along divergent paths – some show a more aggressive phenotype similar to that driven by the median Q tract AR, but most exhibit a slow-growing welldifferentiated disease. Some of this apparent complexity may reflect differences in the androgen axis in man and mouse and consequences of this in neoplasia. Since the AR Q tract length affects androgen-sensitive tumorigenesis in mice, h/mAR-TRAMP mice orchiectomized at 12 weeks of age were monitored to test whether this variation also influenced castration-recurrent cancer. Since adrenal androgen synthesis differs in mice and men, castration reduces serum testosterone to very low and perhaps undetectable levels. TRAMP tumors arise from preexisting PIN lesions and can be compared to those in men during androgendeprivation therapy. Remarkably, Q tract length elicited differences in tumor detection and progression in castrated mice, and in directions distinct from those in intact mice (Albertelli et al. 2008). The mice with short Q tract ARs trended to delayed tumor detection by Kaplan-Meier analysis (P value = 0.079) compared to those with median and long Q tracts, which followed a disease course very similar to each other in time to palpation and age of death (Fig. 6). This difference became apparent at 29 weeks of age when palpable tumors were present in only 15% of 12Q mice, whereas about half of the 21Q and 48Q mice had detectable tumors or had died. For all genotypes, the time from tumor palpation until death was shorter following castration than in intact animals, but was significantly longer for castrated 12Q mice than for the other alleles. In some animals, tumors either never became palpable or remained small, and these mice died of metastatic disease found at necropsy. Remarkably, half of the 12Q mice succumb in this manner, compared to only about 10% of the 21Q and 48Q mice. Furthermore, 2 of 14 castrated 12Q mice survived beyond the term of the experiment and were euthanized at the ages of 16 months and 2 years, with no gross evidence of disease upon necropsy. Thus the mice with short Q tracts followed distinct pathways of disease, one distinguished by delayed onset of large primary tumors and the other by late onset of aggressive metastatic disease. The prevalence of metastasis as cause of death in 12Q mice is reminiscent of epidemiological data indicating aggressive disease as a common feature of men with short Q tract ARs, such as found in the African-American population (Bennett et al. 2002).
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Fig. 6 Q tract length inversely affected tumor detection and survival in castrated mice. The upper panel shows proportion of mice without a palpable tumor and the lower panel shows prostate cancer-free survival; P values from the log-rank statistics are shown. Number of mice per group (n) was: 12Q = 13, 21Q = 16, 48Q = 14. Modified from Albertelli et al. (2008), Copyright 2008, Oxford University Press
Tumors from castrated TRAMP mice were mostly undifferentiated by histological analysis, in accord with the short survival time once tumors become palpable. AR IHC staining was modest and heterogeneous, but generally nuclear despite the presumed absence of testosterone. However, the small primary tumors of 12Q mice that died of metastatic disease were more differentiated with cytoplasmic as well as nuclear AR immunostaining that was distinctly higher overall than in the other genotypes (Fig. 7a). These small tumors also failed to express synaptophysin, in contrast to the larger palpable tumors from this group. Interestingly, the 48Q tumors also lacked synaptophysin expression, unlike 21Q tumors, even though these mice had similar ages of tumor detection and death, and similar gross histology. These phenotypic markers further illustrate that alternative courses of tumor progression are influenced by ARs with different Q tract lengths. The effect of AR Q tract length was accentuated by comparing intact and castrated mice of the same genotype. h/mAR21Q-TRAMP mice behaved similar to mAR-TRAMP mice, with a large proportion of those castrated exhibiting delayed tumorigenesis and death, but also with a substantial number developing tumors and succumbing earlier than intact counterparts. These alternative responses to castration were polarized in the 12Q and 48Q mice (Fig. 7b). In 12Q mice, tumor detection was significantly delayed in castrated compared to intact mice (P value
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Fig. 7 Prostate cancer progression in castrated h/mAR-TRAMP mice. (a) Representative endstage tumors from 12Q and 48Q castrated mice, stained for AR, at low (left) and high (right) magnification. The 12Q tumor (*) was a nonpalpable tumor from a mouse that died of metastatic disease. The 48Q tumor had a 7-week disease length. High magnification showed significant cytoplasmic AR in the 12Q tumor, and more nuclear AR in the 48Q tumor. (b) The response to castration varied with Q tract length. The 12Q AR shifted the balance to tumors developing later after castration, but the 48Q AR responded poorly to treatment. Modified from Albertelli et al. 2008, Copyright 2008, Oxford University Press
<0.0001), although an effect on survival was modest due to the long disease length of intact 12Q mice. In contrast, while 48Q mice showed little difference in time of tumor detection between intact or castrated mice, tumor progression was more rapid following castration and survival times were significantly shorter (P value = 0.011). Thus the short Q tract was associated with a positive response to earlier castration in mice. Androgen-independent cells are thought to exist in TRAMP mice prior to castration, and castration may synchronize these cells and drive selection for aggressive growth (Johnson et al. 2005; Wikstrom et al. 2005). This may be analogous to results of the finasteride prevention trial in man, in which reduced dihydrotestosterone levels decreased the number of prostate cancer cases but more of those occurring were of higher grade (Thompson et al. 2003). Since tumors arise later in 12Q mice following castration, and survival is decreased in 48Q mice by castration, AR strength may impact response to castration, with a stronger AR producing a more favorable outcome. Some studies have noted a similar effect in man, where low testosterone prior to treatment, which may suggest a weaker androgen axis, as in 48Q mice, correlated with poor prognosis (San Francisco
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et al. 2006). Overall, despite differences between the human and murine disease, the 12Q and 48Q mice provide a model in which response to hormonal treatment is genetically programmed. Further analysis may reveal tumor characteristics that could serve as early biomarkers in man for predicting response to androgendeprivation therapy and ultimately suggest distinct strategies for treatment.
8 Conclusions Humanized AR mice with alleles varying in Q tract length provide an in vivo model to test the role of this polymorphism in androgen-dependent traits and in the etiology of disease. In creating these mice, Q tract lengths were chosen at the extremes of those found in the normal human population to optimize detection of informative differences. The resulting strains are indistinguishable from wild-type mice at a gross level, but reveal AR allele-specific variation within the normal range in some physiological and molecular indicators of androgen action. Therefore modest phenotypic alterations, directly or indirectly due to differences in transcriptional activity of the variant Q tract ARs, are detectable and may be cumulative with age. These variations are accentuated in mice by their genetic and environmental homogeneity, but also may exist in man (Zitzmann and Nieschlag 2003). Our data and those of others suggest that they may be most penetrant for alleles with CAG repeat numbers at the extremes of the normal range. Polyglutamine-tract-dependent effects in the h/mAR mice are more pronounced in the context of cancer, with statistically significant differences in tumor detection and survival evident in the TRAMP model. The probasin promoter that drives the Tag oncogene is the likely initial oncogenic sensor of AR strength, but the critical differential gene activation may occur early or in a subset of cells since genotypic differences in Tag expression are not evident at a gross level. Moreover Tag expression is not the sole determinant of disease course, since significant differences occur in progression as well as tumor onset in these strains. Whether early hyperplasia becomes neoplasia depends not only on proliferative capacity of the epithelia cells but also on growth-promoting vs. inhibiting effects of stroma. These opposing forces are influenced overall by the androgen axis, and thus may be modulated directly and indirectly by AR Q tract effects and further exacerbated by neoplasia. Mechanisms that under homeostatic conditions limit the effects of genetic variation (e.g., AR alleles) and environmental change (e.g., hormone levels) may be overcome by the more global dysregulation accompanying oncogenesis. One such mechanism is sequestration of AR by chaperone molecules such as heatshock protein 90. Cryptic genetic variation and subclinical phenotypes may be kept in check by chaperones, but unmasked and amplified by stress or disease states. Cancer progression in TRAMP mice does not show a linear correlation with CAG repeat number, and short-term effects may differ from long term, complexities perhaps also underlying discordant results in man. Our studies suggest similar disease courses in intact 12Q and 48Q mice may occur through distinct mechanisms
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at the cellular level, due to disparate influences of these alleles on downstream targets involved in proliferation and differentiation. Early in disease, AR maintains its normal function as a tumor suppressor, so increased transcriptional activity associated with a short Q tract slows tumor progression by favoring differentiation. Later in disease, multiple events conspire to switch AR’s function to that of an oncogene and promote tumor growth (Litvinov et al. 2003). Thus reduced activity associated with the long Q tract delays tumor initiation and slows tumor progression in 48Q mice. Q tract variation is likely to affect AR activity differentially in stroma vs. epithelia due to the presence of differing amounts or types of cofactors and the array of active signaling pathways. Comparison of gene expression profiles in these two cell types and at different stages of disease may define multiple pathways and precise mechanisms by which these variant AR alleles influence androgen-dependent prostate cancer. In man, extremes within the normal range may favor divergent pathways of disease progression, although modest differences in CAG repeat number are likely insignificant. Comparison of disease progression in castrated TRAMP mice is complicated by the androgen dependence of the transgenic oncogene’s promoter. Nevertheless, striking differences highlight influences that may also operate in human disease, for instance via TMPRSS2 fusion genes. The divergent responses of the 12Q and 48Q alleles to castration suggest that an effect of Q tract length on ligand-independent AR activation may enter into castration-recurrent disease. Moreover, the slow growth and differentiated phenotype of 12Q tumors relative to the rapid progression of 48Q tumors after castration suggests some residual AR activity may be advantageous in treatment. This benefit pertains largely to the primary tumor in the TRAMP model since androgen-independent and probably AR-negative cells ultimately metastasize. Analysis of Q tract effects in clinical prostate cancer may be more difficult due to this dichotomy between slow tumor growth and aggressive metastasis. Detection of insignificant prostate cancer by PSA testing and disease heterogeneity may confound treatment effect further. These distinct pathways of progression following androgen-deprivation therapy suggest continued sensitivity to AR activity and the androgen axis. The role of AR during androgen-deprivation therapy will be clarified by examining the Q tract variant alleles in the context of an oncogenic model not directly driven by androgen, such as in mice with prostatespecific deletion of the Pten tumor suppressor. In summary, the Q tract variant h/mAR alleles provide a novel genetic paradigm that allows investigators to establish a causal relationship between a polymorphism and a phenotype. Extremes of the normal range of variation produce demonstrable differences in laboratory mice in a uniform genetic and environmental context. Some of these differences may be direct transcriptional effects and some may result indirectly from systemic modulation of the androgen axis. These differences are more apparent, and more complex, in cancer, which suggests that Q tract effects vary with cell type and disease stage. As a risk factor in man, extremes of the normal allelic range may have modest predictive value that take on more significance in combination with certain alleles for androgen metabolizing enzymes. Furthermore, extremes of Q tract length may affect cancer progression, and of more clinical
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relevance, response to androgen-deprivation therapy. Recent evidence from mouse models suggests ablation of AR function may not be the best strategy for prostate cancer, but rather in at least some situations may select for more aggressive ARnegative cells (Johnson et al. 2005; Wikstrom et al. 2005; Banach-Petrosky et al. 2007). Elucidation of mechanisms underlying different progression pathways may ultimately provide diagnostic tools to distinguish which patients will benefit from androgen-deprivation therapy and which may require additional or alternative treatments. Distinguishing downstream AR functions that promote differentiation from those that promote proliferation will lead to development of more targeted treatments. In these endeavors, the Q tract variant mice are a valuable preclinical model for testing therapies directed at the human AR.
Acknowledgments This chapter summarizes work by members of the laboratory over many years, but particularly Megan A. Albertelli, D.V.M., Ph.D., Arno Scheller, Ph.D., Orla A. O’Mahony, Ph.D., and Michele Brogley, M.S. Numerous colleagues providing essential discussions included, from the University of Michigan, Drs. Andrew Lieberman, Mara Steinkamp, and Christopher Krebs; and, from the Fred Hutchinson Cancer Research Center, Norman Greenberg. The Robins laboratory has been supported by the NIH (NIDDK-RO156356, NCI-P50-CA69568, NIH-T32-RR07008) and the DOD (DOD17-02-1-0099, W81XWH05-1-0105), as well as Core support from the University of Michigan Cancer Center (5 P30 CA46592) and the Michigan Diabetes Research and Training Center (5 P60 DK20572).
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The Androgen Receptor Coactivator-Binding Interface Eva Este´banez-Perpin˜a´ and Robert J. Fletterick
Abstract When hormone binds to the androgen receptor (AR), the ligand-binding domain (LBD) becomes ordered, displaying a new protein–protein interaction surface called AF2 (coactivator-binding pocket), which is a hydrophobic groove that fits AR coregulators. The association of coregulators with AR LBD is often a critical step for its transcriptional function. Existing pharmaceuticals block AR activity by disrupting AF2 surface’s ability to recruit coactivators. Such antagonists bind to the hormone-binding site inside the LBD core and perturb the structure of the most terminal helix of the LBD, distorting the AF2 surface. The AF2 pocket is also a potential candidate for pharmaceutical intervention by surface-directed small molecules that will directly block coactivator recruitment. Such molecules may be a novel generation of antiandrogens for treating prostate cancer.
1 Introduction The science addressed in this chapter is one snapshot in the cine´ma verite´ of the androgen receptor (AR) function: the nature of the interaction between its coregulatory partners and the coactivator-binding pocket present in its ligand-binding domain (LBD). The LBD is the heart of AR not only because it binds to steroid hormones, but because it also determines interactions with chaperones and partner transcription factors (Prescott and Coetzee 2006) (Fig. 1). It is believed that apo-AR (receptor in the absence of hormone), like other steroid receptors, is partly folded, and hormone binding is the pivotal folding key that triggers the formation of coregulator interaction surfaces (Feng et al. 1998). When hormone binds to the receptor, unknown conformational changes follow that drive disassembly of the AR-chaperone complex (Aranda and Pascual 2001). One key change in structure, hypothesized by R.J. Fletterick(*) Department of Biochemistry & Biophysics, University of California San Francisco, San Francisco, CA 94143-2240, USA, E-mail:
[email protected]
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Fig. 1 Hypothesized AR complexes. AR domains are represented as LBD (medium oval), DBD (small gray circle oval), and NTD (big gray oval). AR forms different macromolecular complexes depending on what ligand is present in its LBP. (a) In the apo-AR absence of ligand is complexed to chaperones (larger light gray sphere). (b) Upon DHT binding (small diamond), AR undergoes a series of conformational changes and the chaperones (HSP, smaller light gray sphere) are released; then AR is targeted to the nucleus and is (c) able to form productive complexes with coactivators (i.e., p160 family, larger oval). (d) After coactivator binding to AR AF2 pocket, recruitment of the transcriptional machinery (RNA POL II ) occurs with transcription or AR-dependent genes (DNA helix shown as schematic double helix). (e) When AR LBP binds antiandrogens (lightning), a series of conformational changes disrupt AF2, impeding coactivator recruitment. (f) AR-coactivator recruitment can be blocked by AF2- interacting compounds (star). (g) Allosteric modulators of AF2 may also block AR action
analogy with other NRs (Bourguet et al. 2000; Moras and Gronemeyer 1998; Wurtz et al. 1996), is a conformational change of the C-terminal helix 12 (H12) present in its LBD. Upon agonist binding, H12 and the C-terminal 20 amino acids (F domain) of AR fold against the body of the LBD (Nichols et al. 1998). In its new position, the F domain blocks the dimer assembly site known for other NRs, and H12 completes the hydrophobic coactivator-binding pocket (also known as AF2 site,
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Fig. 2 Structure of AR LBD. (a) AR LBD surface is represented in gray with the residues lining the AF-2 pocket highlighted in darker gray. (b) Secondary structure representation of AR LBD in gray with the buried DHT shown as a blue space-filling model. The position of helices 3, 5, and 12 is shown
Fig. 3 The AR AF2 pocket is a trifurcated groove formed by residues of helices H3 (Lys717, Val713), H5 (Asn733, Val730, Met734), and H12 (Glu897, Glu893, Met894). The floor of the pocket is formed by residues Phe725, Ile737, Asn738, Ile898, and Leu712
for trans-activation function 2), which is a charge-capped hydrophobic groove (Figs. 2–4) (Darimont 2003; Este´banez-Perpin˜a´ et al. 2005). This process is different from what happens when ligands bind to enzymes, which typically possess preformed and solvent accessible active sites. Binding of an agonist to AR completes the hydrophobic core of the receptor, reshaping its surface, which in turn
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Fig. 4 LXXLL (SRC2-3) and FXXLL (ARA70) peptides bound to AR LBD AF2 pocket. AR LBD surface is colored in pale gray. (a) AR LBD surface is shown in the absence of the LXXLL peptide. The three subsites S1, S2, and S3 are indicated. (b) Three hydrophobic residues of the hydrophobic motif of the SRC2-2 peptide, Leu 1 (L1), Leu 4 (L4), and Leu 5 (L5), are shown as sticks and spheres. The three leucine side chains of this biomotif interact with the correspondent subpockets of the AF2 groove. (c) SRC2-2 peptide is shown as sticks and spheres. (d) SRC22 peptide is shown as a ribbon running along the AR AF2 pocket surface. (e) AR LBD surface is shown in the absence of the FXXLF peptide. (f) The two phenylalanines and leucine side chains of the FXXLF biomotif (shown as sticks and spheres) interact with the correspondent subsites of the AF2 groove. (g) ARA70 peptide is shown as sticks and spheres. (h) ARA70 peptide is shown as a ribbon running along the AR AF2 pocket surface
allows assembly of coregulators on its surfaces (Este´banez-Perpin˜a´ et al. 2005; Hur et al. 2004). Both the N-terminal domain (NTD) and LBD recruit partnering proteins (Bevan et al. 1999; Zhou et al. 2002; McKenna et al. 1999; Hodgson et al. 2005; Lonard and O’Malley 2006). A curious confusion relates to competition for coactivator binding to the AR AF2 pocket. The AR NTD itself binds coactivators but also engages in a hormone-dependent interaction with its LBD, and this intramolecular interaction is known to regulate AR transcriptional activity by competing with coactivator recruitment to the LBD (He and Wilson 2002; Hsu et al. 2005). Apparently, both the NTD and coactivators bind to the AF2 surface (He et al. 2002). Coactivators may alter the self-assembly of AR and compete with AR NTD for binding to the AF2 groove (He et al. 2001). The NTD-LBD interaction also affects AR binding to chromatin (Li et al. 2006). Coregulators comprise coactivators and corepressors, which are structurally and functionally diverse proteins (Lonard and O’Malley 2006, 2007; McKenna and O’Malley 2002; Wang et al. 2005). Corepressors are believed to bind to the unliganded receptor (apo-AR) or to the antagonist-bound state, but are released from the receptor upon hormone binding (agonist-bound AR) (Lazar 2003; Burd et al. 2006). Several members of the p160 family (SRC, steroid receptor coactivators) and ARA family (androgen-receptor-associated proteins) of coactivators are
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involved in normal transcription and in the development and progression of prostate cancer (Chmelar et al. 2007; Yeh and Chang 1996; Agoulnik et al. 2005). Once bound to AR, coactivators are part of multiprotein complexes that include the transcriptional machinery (Shang et al. 2002; Choudhry et al. 2006) (Fig. 1). These multisubunit complexes contain many different enzymatic activities such as ubiquitylation, chromatin remodeling, or DNA methylation among others (Lonard and O’Malley 2006, 2007). Several lines of evidence support the idea that AR AF2 pocket can be a bona fide pharmaceutical target (Arnold et al. 2005, 2006, 2007; Este´banez-Perpin˜a´ and Fletterick 2007; Chang and McDonnell 2005). Small molecules have been reported to bind weakly to AR AF2 by Xray crystallography and fluorescent polarization assays (Este´banez-Perpin˜a´ et al. 2007a). Such compounds would serve as starting point chemical scaffolds to design novel antiandrogens. Such antiandrogens will be surface directed and would work as either steric blockers of the AF2 pocket or as allosteric modulators, reshaping the AF2 surface or adjacent putative regulatory surfaces (Este´banez-Perpin˜a´ and Fletterick 2007; Este´banez-Perpin˜a´ et al. 2007a).
2 AR Coactivator-Binding Pocket The LBD is the best understood of AR functional domains (Table 1, Fig. 2). The elucidation of the crystal structure of AR LBD revealed the structural determinants of AR agonist recognition (Matias et al. 2000; Sack et al. 2001; Askew et al. 2007), AR antagonist resistance development (Bohl et al. 2005a, b), coactivator recognition and recruitment to AR surface (Este´banez-Perpin˜a´ et al. 2005; Hur et al. 2004; He et al. 2004, 2006) (Fig. 2). There is no structure of AR LBD in either the apoform, in the antagonist-bound conformation, or as a part of the multidomain/fulllength AR. All AR LDB structures deposited in the Protein Data Bank (PDB) for either wild-type or mutant AR LBD are in the agonist-bound conformation (Fig. 2). A structure of AR DBD domain in complex with its hormone-response element has also been reported (Shaffer et al. 2004). Two ligand-binding sites on the AR LBD contribute to successful AR function. First is the ligand-binding pocket (LBP), which is formally defined as an allosteric site since it ‘‘builds’’ the active site located some distance away. The LBP contains embedded the hydrophobic hormone: testosterone or dihydrotestosterone (DHT). Second, the coactivator-binding pocket (AF2) located at the surface, which is the true active site (Fig. 1). Recruitment of coactivator proteins to the AF2 pocket is a crucial step in AR function. The AR AF2 groove is the most well-defined solvent-exposed protein–protein interaction site on the AR surface (Figs. 2–4). Peptides derived from specific coactivators represent good models for studying binding of coregulators to AF2. Several crystal structures are known for coactivator peptides bound to AR AF2 pocket at atomic resolution (Este´banez-Perpin˜a´ et al. 2005; Hur et al. 2004; Askew et al. 2007; He et al. 2004, 2006). These structures reveal the molecular
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Table 1 Structures of the androgen receptor (DBD and LBD) with atomic coordinates deposited in the Protein Data Bank Protein PDB code Comments Reference AR (P10275) 1I37, 1I38 Rat, wf/T877A mutant, DHT, Sack et al. (2001) ˚ 2.0 A ˚ 1XNN Rat, T877A mutant, HYQ, 2.2 A Salvati et al. (2005) ˚ 1E3G Rat, Metribolone (R1881), 2.4 A Matias et al. (2000) 1GS4 Human AR mutant Matias et al. (2002) ˚ (ARccr), 1.95 A 1R4I DBD bound to a direct HRE Shaffer et al. (2004) ˚ 1T73 DHT, FXXFF motif, 2.2 A Hur et al. (2004) ˚ 1T74 DHT, WXXLF motif, 2.0 A Hur et al. (2004) ˚ 1T76 DHT, WXXVW motif, 2.1 A Hur et al. (2004) ˚ 1T79 DHT, FXXLW motif, 1.8 A Hur et al. (2004) ˚ 1T7F DHT, LXXLL motif, 1.6 A Hur et al. (2004) ˚ 1T7M DHT, FXXYF motif, 1.6 A Hur et al. (2004) ˚ 1T7R DHT, FXXLF motif, 1.4 A Hur et al. (2004) ˚ 1T7T DHT, no peptide, 1.7 A Hur et al. (2004) ˚ 1XOW R1881, NTD peptide, 1.8 A He et al. (2004) ˚ 1XQ3 R1881, 2.25 A He et al. (2004) ˚ 2A06 R1881, Tif2(Iii) peptide, 1.89 A He et al. (2004) ˚ 1T5Z DHT, ARA70 peptide, 2.3 A Este´banez-Perpin˜a´ et al. (2005) ˚ 1T63 DHT, SRC2-3 peptide, 2.07 A Este´banez-Perpin˜a´ et al. (2005) ˚ 1T65 DHT, SRC2-2 peptide, 1.66 A Este´banez-Perpin˜a´ et al. (2005) ˚ 1XJ7 DHT, RAC3 peptide, 2.7 A Este´banez-Perpin˜a´ et al. (2005) ˚ 1Z95 R-bicalutamide, 1.8 A Bohl et al. (2005b) 2AX6 T877A mutant, Bohl et al. (2005a) ˚ Hydroxyflutamide, 1.5 A ˚ 2AX7 T877A mutant, S-1, 1.9 A Bohl et al. (2005a) ˚ 2AX8 W741L mutant, S-1, 1.7 A Bohl et al. (2005a) ˚ 2AX9 R-3 compound, 1.65 A Bohl et al. (2005a) ˚ 2AXA S-1 compound, 1.80 A Bohl et al. (2005a) ˚ 2AM9 Testosterone, 1.64 A Pereira de et al. (2006) ˚ 2AMA DHT, 1.90 A Pereira de et al. (2006) ˚ 2AMB Tetrahydrogestrinone, 1.75 A Pereira de et al. (2006) 2PIO DHT, 2-methylindole in AF2 Este´banez-Perpin˜a´ et al. (2007a) 2PIP DHT, 1H-INDOLE-3Este´banez-Perpin˜a´ et al. (2007a) CARBOXYLIC ACID (ICO) and 1-TERT-BUTYL-3-(2,5DIMETHYLBENZYL)-1HPYRAZOLO[3,4-D] PYRIMIDIN-4-AMINE (K10) in AF2, 1.80 A 2PIQ DHT, 3-[(4-AMINO-1-TERTEste´banez-Perpin˜a´ et al. (2007a) BUTYL-1H-PYRAZOLO [3,4-D]PYRIMIDIN-3-YL) METHYL]PHENOL in AF2, 2.40 A 2PIR DHT, Salicylaldehyde, 2.10 A Este´banez-Perpin˜a´ et al. (2007a) (continued)
The Androgen Receptor Coactivator-Binding Interface Table 1 (continued) Protein PDB code Comments 2PIT DHT, [4-(4-HYDROXY-3-IODOPHENOXY)-3,5-DIIODOPHENYL]-ACETIC ACID, 1.76 A 2PIU DHT, with two [4-(4-HYDROXY3-IODO-PHENOXY)-3,5DIIODO-PHENYL]-ACETIC ACID, 2.12 A 2PIV DHT, 3,5,3’TRIIODOTHYRONINE (T3 hormone), 1.95 A 2PIW DHT, 3,5,3’TRIIODOTHYRONINE (T3 hormone), 2.58 A 2PIX DHT, 2-[[3(TRIFLUOROMETHYL) PHENYL]AMINO] BENZOIC ACID (FLF, Flufenamic acid), 2.4 A 2PKL DHT, [4-(4-HYDROXY-3-IODOPHENOXY)-3,5-DIIODOPHENYL]-ACETIC ACID, 2.49 A 2QPY DHT, [4-(4-HYDROXY-3-IODOPHENOXY)-3,5-DIIODOPHENYL]-ACETIC ACID, 2.50 A
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determinants of the regulatory complex formation and specificity. A crystal structure of AR LBD has also been reported with a corepressor peptide (SHP) bound to its AF2 (Jouravel et al. 2007). The coactivator peptides studied by Xray crystallography are of two types: the first type comprised the actual domains or protein fragments from the family of physiological coactivators known to interact with the AR LBD (p160 and ARA family of coactivators) (Este´banez-Perpin˜a´ et al. 2005), and the second type is formed by libraries of peptides obtained by phage display that may mimic the natural binding domains (Hur et al. 2004). Natural regulatory peptides usually contain either the FxxLF motif (where F is phenylalanine, and X is any amino acid) present in the coactivator ARA70, as well as the LxxLL motifs (where L is leucine, and X is any amino acid) present in the p160 family member of coactivators. Peptides identified by phage display contain hydrophobic sequences with homologous motifs have also been reported: FxxLF, FxxFF, FxxYF, FxxLW, WxxLF, WxxVW, and LxxLL (Y and W are tyrosine and tryptophan, respectively) (Hur et al. 2004; Hsu et al. 2003). These hydrophobic amino acid sequence motifs are referred to as NR boxes (NR stands for Nuclear Receptor) (Fig. 4). AR LBD is an a-helical protein with 12 helices arranged in three layers (Matias et al. 2000; Sack et al. 2001). The AR AF2 surface is formed by residues of helices
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H3, H5, and H12 (Fig. 2). The AF2 hydrophobic pocket is a trifurcated groove formed by three moderately deep subpockets, each accommodating one of the three hydrophobic side chains of the NR motif (F1XXL4F5 and L1XXL4L5, respectively) (Figs. 2–4). The S1 subsite is the narrowest and deepest of the three, and it hosts the first hydrophobic residue: Leu1/Phe1 is embedded within this subsite (Fig. 4). S1 is lined by H5 residues M734, Q738, and H12 residues E897 and I898 (Figs. 3 and 4). The S2 subsite is the broadest and shallowest of the three and hosts the second hydrophobic residue: Leu4 is embedded within this subsite (Fig. 4). S2 is lined by H3 residues K720 and V713, and H12 residue M894 (Figs. 3 and 4). The S3 subsite is intermediate in size in comparison to the other two and quite deep. S3 is lined by H5 residues M734 and H3 residue K720 (Figs. 3 and 4). Both NR FXXLF and LXXLL motifs bind to the AR AF2 region as amphipathic a-helices with hydrophobic surfaces complementary to AF2 (Fig. 4). There are two clusters of oppositely charged residues on the AR LBD surface, at the opposite ends of the AF2 hydrophobic groove, that assist in orienting and registering the bound NR motifs in the AF2 groove. K720 and E897 are interesting because they function as ‘‘helix-capping’’ residues, forming the electrostatic ‘‘charge clamp’’ residues and stabilizing binding interactions of the FXXLF motif with the AF2 core via hydrogen bonding. At the start of the helix, Glu897 of AR forms hydrogen bonds to the backbone amide (NH) of Phe1 and preceding Lys. At the end of the helix, Lys720 of AR does not contact the carbonyl group of Leu4 or Phe5; instead, it forms hydrogen bonds to the carbonyl groups flanking residues at positions 6 and 7. The alternative NR-binding motif, LxxLL, interacts just like the FxxLF with Lys720, but its Leu1 residue does not contact Glu897 of AR. The structural basis of AR preference for peptides containing FxxLF motif over the canonical LxxLL sequence has been determined (Este´banez-Perpin˜a´ et al. 2005; Hur et al. 2004; He et al. 2004). Structural comparisons of several AR LBDs bound to different regulatory peptides revealed that side chains forming the AF2 pocket rearrange to bind multiple variants of the NR boxes. There are specific structural features in the AF2 site of AR that allow accommodation of the bulkier FxxLF motif. For example, residues M434 and K720 of the AR LBD assume different conformations to make room for FxxLF peptide, allowing its interaction with I737 of AR helix H4. In general, coactivator peptides cause the reformable AF2 pocket to open upon binding, resulting in a remarkable complementary fit between the AF2 surface and the bound FxxLF motifs. Interestingly, both LxxLL and FxxLF containing helical NR peptides interact with the same set of residues on the surface of the AR LBD; however, these residues are engaged in different binding interactions. Furthermore, ˚ the major axes of the helices docked into the AF2 groove shift by more than 1 A between the two classes of regulatory peptides. The described studies focused on the central region of the interface between the regulatory peptides and the AR LBD. However, the interactions may be more extensive in vivo as neighboring features of coactivators find footholds on the AR surface. The sequences beyond the conserved hydrophobic motif hot spot help to confer specificity to the receptor–coactivator coupling (Este´banez-Perpin˜a´ et al. 2005).
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Table 2 Mutations at the binding pocket (LBP), and AF2 pocket associated with prostate cancer and androgen-insensitivity syndromes (AIS) Residues Mutations in prostate cancer Mutations in AIS AF2 pocket V713 Not described Not described V716 Not described Not described K717 Not described Not described K720 K720E Not described R726 R726L R726L V730 V730M Not described Q733 Not described Q733H M734 Not described Not described I737 Not described I737I Q738 Not described Not described M894 Not described Not described E897 Not described Not described I898 Not described I898T
Structural analysis of the corepressor peptide SHP bound to the AR AF2 region reveals that SHP peptide adopts a conformation similar to that observed for the bound LxxLL coactivator peptides (Jouravel et al. 2007). Several mutations in the AF2 pocket that affect Lys720, Ile737, Val730, Gln733 are implicated in prostate cancer and androgen-insensitivity syndromes (http://androgendb.mcgill.ca/) (Table 2). These mutations affect coactivator recruitment to AR. Specifically, mutations affecting residues Ile737 and Phe725 have been shown to disrupt the AR NTD/AF2 interaction without affecting hormone binding (Quigley et al. 2004). Many residues in the AF2 pocket are conserved among several members of the nuclear receptor family (Table 3). There are nuclear receptor coactivators that bind to several nuclear receptors while others do not exhibit promiscuity and are selective to either one or another. The variety coactivator–nuclear receptor partnerships increase the complexity of nuclear receptors transcriptional regulation (Lonard and O’Malley 2007).
3 The AF2 Pocket may be a Drugable Interaction Surface Because the AF2 pocket plays a pivotal role in AR function, it may be a target for therapeutical intervention. In particular, blocking associations of AR with coregulatory partners might lead to alternative treatments for prostate cancer. Compounds targeting the regulatory AR LBD surfaces have been proposed as alternative antiandrogens (Este´banez-Perpin˜a´ et al. 2005, 2007a; Este´banez-Perpin˜a´ and Fletterick 2007; Chang and McDonnell 2005). Drugs that would bind to the ARcoregulator assembly site should be less susceptible to mutation of the AR protein by cancer cells because two proteins form the contact, and any mutation of the AF2 surface to render the drug ineffective might also affect coregulator binding.
AF2 residues
L712 V713 V716 K720 F725 R726 V730 M734 Q738 M894 E897 I898
V571 I572 V575 K579 F584 R585 L589 M593 Q597 M752 E755 I756
L354 V355 I358 K362 F367 V368 L372 V376 E380 D538 L541 E542
L726 L727 V730 K734 F739 R740 I744 I748 Q752 M908 E911 V912
M777 I778 V781 K785 F790 K791 L795 I799 Q803 M959 E962 I963
Table 3 Structurally conserved amino acids in AF2 among several nuclear receptors NR AR GR ERa PR MR PDB code 1T5Z 1P93 1ERE 1A28 2AA7 I226 T227 V230 K234 F239 S240 C244 I248 K252 L400 E403 V404
TRa 2H79 T281 R282 D285 K288 L290 P291 P297 Q301 K306 L454 E457 V458
TRb 1BSX L276 F277 V280 K284 F289 S290 L294 V298 R302 F450 E453 M454
RXRa 1FBY
V293 Q294 T297 K301 F306 V307 L309 V315 K319 L468 Q470 I472
PPARg 1PRG
I234 Q235 I238 K242 F247 R248 S252 I256 K260 L413 E416 V417
VDR 1RJK
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Surface targeted antiandrogens directly blocking the AR coregulator assembly may be therapeutically useful alone or paired with the current LBP-antiandrogens. Small molecules would have to block AR associations by preferentially populating the surface normally filled by the amphipathic a-helices provided to AR by coregulator proteins (Este´banez-Perpin˜a´ et al. 2005; Este´banez-Perpin˜a´ and Fletterick 2007) (Fig. 4). The first example of small molecules that block coactivator recruitment both in vitro and in vivo has been reported by Arnold et al. for the thyroid receptor b (TRb) (Arnold et al. 2005, 2006, 2007; Este´banez-Perpin˜a´ et al. 2007b). These compounds (aromatic b-amino ketones (BAK)), identified in large-scale chemical screens, are representing a novel class of potent TRb antagonists. As the crystal structure revealed, these compounds bind irreversibly to one of the exposed cysteine residues located in TRb AF2 pocket (Este´banez-Perpin˜a´ et al. 2007b). In addition, peptide antagonists for the AR-coactivator recruitment have been identified using phage display (Chang et al. 2005). The existence of peptides that target the AR AF2 pocket together with the discovery of TRb AF2-binding compounds set a precedent that could also be applied to AR for discovery of novel antiandrogens targeting its surface. The same research strategy that led to the discovery of TRb specific blockers also proved to be successful for AR, identifying its AF2 site as a pharmaceutical target. X ray crystallography combined with fluorescent polarization techniques have identified compounds that bind weakly to the AR AF2 surface. These compounds belong to different chemotypes including thyroid hormones (TRIAC and T3), purine analogs, and small heterocyclic molecules such as 2-methylindole. Most of these compounds exhibit weaker association at AF-2 site due to their poor fit. Subsite S1 of the AF2 that hosts F1 or L1 of the signature motif F1XXL4F5/L1XXL4L5 seems to be the hot spot for compounds that bind at AF2. Research strategies aiming at improving binding to the AF2 subsites S2 or S3 might yield higher affinity compounds.
4 AF2 is Allosterically Regulated by an Adjacent Surface-Exposed Hydrophobic Pocket It has been observed that the AF2 pocket is reshaped not only by ligand binding to the LBP but also by small molecules binding elsewhere on the LBD surface. Recent structural and mutational studies have provided evidence that a novel hydrophobic surface-exposed pocket termed BF-3 (Binding Function 3) is able to remodel the AF2 surface and affect coactivator recruitment (Fig. 2). This surface pocket is adjacent to the AF2 site and appears to modulate it allosterically (Este´banezPerpin˜a´ et al. 2007a). The BF-3 surface comprises H1 (Pro723, Phe673, and Ile672), H3–5 (Gly724 and Asn727) and H9 (Phe826, Glu829, Glu837, Arg840, and Asn833) (Fig. 2). The crystal structures of ternary complexes of DHT-bound AR LBD with coactivator peptides (containing either LXXLL or FXXLF motif) revealed that TRIAC (a thyroid hormone) can bind to BF-3, causing coregulator peptides bound to AF-
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2 to become disordered. Atomic resolution structural analysis revealed that binding of TRIAC to BF-3 remodels the adjacent AF-2 pocket weakening coactivator binding (Este´banez-Perpin˜a´ et al. 2007a). Mutating residues that form BF-3 inhibits AR function and its AF-2 activity in vitro and in vivo. Mutations at BF-3 can lead to prostate cancer and androgen-insensitivity syndrome. Specifically, mutations of Gln670, Ile672, and Leu830 are associated with prostate cancer (Buchanan et al. 2001a, b; Shi et al. 2002), and Leu830, Pro723, Gly724, and Arg840 are mutated in androgen-insensitivity syndrome (http://www.androgendb.mcgill.ca/) (McPhaul 2002). Targeted mutagenesis of Asn727 and Arg840 in the BF-3 site abolished the AR LBD activity, similar to inhibition observed with mutations in the AF2 region (Este´banez-Perpin˜a´ et al. 2005). Curiously, the same mutations resulted in a changed conformation of the bound TRIAC as revealed by Xray crystallography. Similar to mutating Asn727 and Arg840, substitutions of Phe673, Pro723, Glu724, Glu737 reduce AR activity. In contrast, mutations in the vicinity of the BF-3 site (affecting residues Gln670, Ile672, Glu829, and Asn833) increase the AR AF2 activity up to fivefold (Estebanez- 222 Perpina´ et al. 2007a). The BF-3 site could be present in other nuclear receptors. Consistent with this speculation, part of the site, the H3–H4 loop, contains a signature sequence (Brelivet et al. 2004). Superposition of available crystal structures reveals conservation of BF-3 residues in the subfamily of steroid nuclear receptors. Mutations in equivalent regions of estrogen and glucocorticoid receptor are shown to affect coactivator binding (Tanenbaum et al. 1998; Milhon et al. 1997). Acknowledgments We thank Elena Sablin, Debra Singer, and Leslie Cruz for their useful comments on the manuscript.
References Agoulnik I, Vaid A, Bingman WEIII, Erdeme H, Frolov A, Smith CL, Ayala G, Ittmann MM, Weigel NL: Role of SRC-1 in the promotion of prostate cancer cell growth and tumor progression. Cancer Res. 2005, 65:7976–7983. Aranda A, Pascual A: Nuclear hormone receptors and gene expression. Physiol Rev 2001, 81:1269–1304. Arnold LA, Estebanez-Perpina E, Togashi M, Jouravel N, Shelat A, McReynolds AC, Mar E, Nguyen P, Baxter JD, Fletterick RJ, Webb P, Guy RK: Discovery of small molecule inhibitors of the interaction of thyroid hormone receptor with transcriptional coregulators. J Biol Chem 2005, 280(52):43048–43055. Arnold LA, Estebanez-Perpina E, Togashi M, Shelat A, Ocasio CA, McReynolds AC, Nguyen P, Baxter JD, Fletterick RJ, Webb P, Guy RK: A high-throughput screening method to identify small molecule inhibitors of thyroid hormone receptor coactivator binding. Sci STKE 2006, 341:13. Arnold L, Kosinski A, Este´banez-Perpi–a E, Robert J, Fletterick Guy RK: Inhibitors of the interaction of a thyroid hormone receptor and coactivators: preliminary structure-activity relationships. J Med Chem 2007, 50:5269–5280.
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Coregulators and the Regulation of Androgen Receptor Action in Prostate Cancer Irina U. Agoulnik and Nancy L. Weigel
Abstract The importance of androgen receptors in primary prostate cancer is well established and some form of androgen blockade is the primary treatment for metastatic prostate cancer. There is increasing evidence that the androgen receptor (AR) continues to play a role in castration resistant disease despite the decrease in serum androgens. Thus, factors that modulate AR activity are potential therapeutic targets. AR is a transcription factor that regulates its target genes by recruiting a complex of coregulators with multiple enzymatic activities. These coregulators remodel chromatin, modify receptor, other coregulators, and general transcription factors, as well as affect splicing decisions. Here we summarize current evidence for changes in expression of coregulators in prostate cancer and their function in prostate cancer cell lines. Many of these coregulators have pleiotropic functions and modulate transcription factors other then AR. Thus, they may have both AR dependent and AR independent roles in prostate cancer.
1 Introduction Androgens, testosterone and dihydrotestosterone (DHT), acting through the androgen receptor (AR) are required for the development and maintenance of the prostate. Moreover, prostate cancer is an androgen-dependent disease (Arnold and Isaacs 2002). Prostate cancers that have escaped the prostate capsule are treated with androgen-deprivation therapy that is effective initially. However, most of the prostate cancers recur and become resistant to androgen-deprivation therapy. Despite this, there is strong evidence that castration-recurrent tumors continue to depend on the AR signaling pathway. AR is a ligand-activated transcription factor that assembles a series of coactivators and corepressors on the promoter of its target
N.L. Weigel(*) Baylor College of Medicine, Houston, TX, USA. E-mail:
[email protected]
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genes to regulate transcription of genes, which include prostate-specific antigen (PSA). AR activity is regulated by coactivators, corepressors, cell signaling, and the concentration of available AR ligands. Evidence that AR is reactivated and plays a role in prostate cancer growth and survival in androgen-depleted conditions is derived from both in vitro and in vivo studies. Although C4-2 prostate cancer cells grow in castrated nude mice and in androgen-depleted medium in vitro, reducing AR expression using AR-specific siRNA strongly inhibits cellular proliferation and basal PSA expression (Agoulnik et al. 2005). Although these cells do not require exogenous androgens, they remain AR sensitive for cell growth and expression of PSA. In vivo, castration-recurrent disease is detected typically by an increase in serum PSA levels, which suggests that AR is reactivated under these conditions. The level of AR in primary tumors is associated with markers of more aggressive disease (Li et al. 2004). Moreover, AR expression typically increases during progression to recurrence after castration (Chen et al. 2004a). These findings led to increased interest in identifying the coregulators and cell signaling pathways that regulate androgen-dependent and androgen-independent AR action in prostate as these factors are potential therapeutic targets to more completely block AR action.
1.1
Androgen Receptor
After translation, AR is folded and maintained in a form capable of binding hormone by a series of chaperone proteins that include hsp90. AR dissociates from this complex upon ligand binding, forms homodimers, and translocates into the nucleus. AR can regulate transcription in multiple ways (Fig. 1) (Agoulnik and Weigel 2006). In the best understood mechanism of action, upon agonist action, AR dimerizes, binds specific DNA sequences named AR response elements (ARE), and recruits coactivator complexes that modify chromatin to facilitate recruitment of general transcription factors and Pol II to induce transcription. A well-known gene regulated in this manner is PSA produced by both normal epithelium and prostate cancer cells; serum levels of PSA are used as a marker to evaluate tumor burden in prostate cancer patients. Another important AR target gene regulated in this manner is TMPRSS2, a gene whose promoter is frequently fused to ETS factor oncogenes in prostate cancer (Tomlins et al. 2005). AR also represses transcription, as in the case of maspin, a tumor suppressor from the serpin family (He et al. 2005). Although this is also accomplished through direct DNA binding, the associated factors that cause repression rather than induction have not been identified. AR also binds directly to other transcription factors and regulates their transcriptional activity presumably functioning as a coactivator for these factors (Lu et al. 2000). Finally, recent reports indicate that hormone-bound AR interacts with modulator of nongenomic action of estrogen receptor (MNAR) and src and thus activates cell signaling pathways such as MAPK-p42/p44 and PI3K
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Fig. 1 Modes of androgen receptor action AR is folded in the cytoplasm with the aid of heat-shock proteins (HSPs) and is maintained in a complex with HSPs in the absence of ligand. AR, upon binding androgen (triangles), sheds HSPs and modulates transcription in at least four different ways. (1) AR dimerizes in an antiparallel orientation, binds to AREs, recruits coactivator complexes that remodel chromatin, and facilitates binding of the general transcription machinery (GTF) and RNA Polymerase II (Pol) to initiate transcription. (2) Agonist-bound AR is recruited to repressive AREs, assembles coregulator complexes of as yet unknown content, and actively represses transcription. (3) Liganded AR binds heterologous transcription factors (TF) and presumably recruits coregulators to enhance transcriptional activity. (4) AR binds to src/MNAR complexes that activate kinase cascades, which results in altered transcriptional activity of transcription factors (TF) that do not necessarily physically interact with AR. AR, coregulators, GTFs, and polymerase II are all subjects to regulation by phosphorylation (phosphogroups are depicted as four pointed stars) that is initiated, in part, by receptor tyrosine kinases (YK) and/or G-protein-coupled receptors (GPCR)
(Castoria et al. 2003, 2004; Unni et al. 2004). In addition to the hormone-dependent pathways, there is increasing evidence that activation of some cell signaling pathways is sufficient to induce AR activity in the absence of hormone (Culig et al. 1994; Ueda et al. 2002). The exact mechanism of ligand-independent activation is not known but, as discussed later, it utilizes some of the same coactivators as liganddependent activation complexes. AR and many of its coactivators are phosphoproteins and their activity is regulated by phosphorylation.
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Coregulators
Regulation of transcription requires a series of steps mediated in part by the enzymatic activities of AR-interacting proteins. These steps include chromatin remodeling, initiation of transcription, elongation, and termination. During the activation process, AR is post-translationally modified to modulate activity and subsequently to target it for degradation through a proteasome-mediated pathway. Inhibition of the proteasome pathway hinders AR-dependent transcription (Kang et al. 2002; Lin et al. 2002). Many modifications of the active chromatin proteins are reversed and in some cases DNA is methylated during transcriptional repression. Although many coregulators have intrinsic enzymatic activity, others serve as scaffolds to promote interactions between multiple proteins. The first steroid receptor coactivator, SRC-1, was described in 1995 (Onate et al. 1995). The number of candidate coregulators capable of regulating the activity of steroid receptors has surpassed 300 (http://www.nursa.org/). Clearly, a single receptor molecule cannot interact with so many proteins simultaneously. A partial explanation for the large number of proteins is that coactivators typically function as large multiprotein complexes, conduits of multiple signaling pathways in the cell. Multiple complexes of distinct compositions must be recruited sequentially to chromatin in order to regulate transcription. While some factors may be required for transcription of all target genes, others may be receptor, promoter, or cell type specific. From the perspective of prostate cancer, the challenge is to identify the coregulators that are important for target gene-specific regulation of androgendependent and androgen-independent AR activity.
2 Coregulators in Prostate Cancer Because of the importance of AR in prostate cancer and its significance as a therapeutic target, the contributions of various coactivators to AR activity and their relative expression levels in prostate cancer have been active areas of research. Interpretation of some of these results is complex. Most, if not all, of the AR coactivators identified to date also regulate other nuclear receptors and many of these receptors are expressed in the benign and malignant prostate. Moreover, many of the proteins participate in the regulation of a wide variety of transcription factors. Thus, while changes in AR-mediated gene transcription in response to overexpression or reduction in coregulator expression are likely due to direct effects on AR, caution is needed in interpreting changes in cell proliferation or the consequences of altered expression in prostate cancer as AR-dependent activities. In addition, the overall activity of coregulators depends not only on the level of expression but also on a number of post-translational modifications, which include acetylation, sumoylation, phosphorylation, and ubiquitination. The exact effect of each modification on AR activity and levels of modifications in tissue is impossible to assess at present. The expression of coactivators in benign prostate and prostate cancer has
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been evaluated on both the transcriptional and the protein levels. In most cases, comparisons of the mRNA levels in samples of benign and cancer tissue do not consider the changed ratio between cells of stromal and epithelial origin; the stromal compartment is a major contributor in benign tissue, while cells of epithelial origin predominate in prostate cancer. Thus, it is difficult to determine accurately whether observed changes are prostate cancer specific. Because immunohistochemical studies can compare expression of the coregulators in specific cellular populations, they are more accurate in determining whether expression of a particular coregulator is changed in malignant compared to benign epithelial cells. Therefore, this chapter summarizes the available immunohistochemical data on coregulator expression in prostate cancer and benign prostate with emphasis on coregulators with enzymatic activity. In addition, current data are reported that describe a role for coregulators in AR action in prostate cancer cell lines.
2.1
Histone Acetyltransferases
The first nuclear receptor coactivator identified, SRC-1, is a histone acetyltransferase (HAT) (Spencer et al. 1997). Many of the subsequently identified AR coactivators are also HATs or function as scaffold proteins that recruit other HATs. Histones are major substrates for these enzymes. A change in global levels of histone acetylation is associated with cancer and is predictive of clinical outcome. The global histone modification pattern is an independent predictor of recurrence in patients with low-grade prostate cancer (Seligson et al. 2005). Although histones are important substrates, some of the HATs acetylate other coactivators and even AR itself.
2.1.1
The p160 Coactivators
This small family consists of three 160-kDa proteins that potentiate activities of nuclear receptors and some other transcription factors, although the range of transcription factors activated by these proteins is coactivator specific. The family includes SRC-1, SRC-2 (GRIP1, TIF2), and SRC-3 (AIB1, p/CIP, ACTR, RAC-3, TRAM-1) (Xu and O’Malley 2002). SRC-1 and SRC-3 coactivators are HATs, and all three recruit additional HATs to AR. The p160 coactivators have multiple LXXLL motifs, which interact with the ligand binding domains of AR and serve as platforms for recruitment of secondary coactivators such as methyltransferases and acetyltransferases. However, the most important p160 interaction site in AR is in the amino-terminal AF1 region, which interacts with a glutamine-rich region in the p160 coactivators (Bevan et al. 1999).
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SRC-1 The first demonstration that SRC-1 plays a physiologically significant role in prostate came from analysis of SRC-1 null mice (Xu et al. 1998). In castrated mice, androgen-induced prostate growth was less than in wild-type littermates. In SRC-1 null mice the level of TIF2 was increased twofold, possibly as a compensatory response. If SRC-1 and TIF2 have overlapping functions, as might be predicted from molecular biology studies, the role for SRC-1 in prostate growth may be underestimated. SRC-1 is expressed in benign prostate epithelium and in all the commonly studied human prostate cancer cell lines. SRC-1 expression was reduced using siRNA to determine whether it is required for androgen-dependent gene induction. SRC-1 proved necessary for maximal expression of the androgen-induced target gene, PSA, in LNCaP cells (Agoulnik et al. 2005). Coactivators are thought generally to play roles in activation, rather than repression, of transcription. However, depletion of SRC-1 resulted in derepression of the androgen-repressed tumor suppressor, maspin. SRC-1 expression was reduced in C4-2 cells grown in androgen-depleted medium and PSA expression measured to evaluate the role of SRC-1 in androgen-independent activities of AR; PSA expression was reduced, which indicated that SRC-1 also plays a role in androgen-independent AR activity. Although PSA expression is used as a measure of AR action, AR-dependent growth in LNCaP cells was induced by a selective AR modulator (SARM) without induction of PSA (Sathya et al. 2003). Thus, induction of PSA may be an imperfect surrogate for AR activity. However, depletion of SRC-1 from androgen-sensitive LNCaP cells or androgen-independent C4-2 cells reduced proliferation measured using [3H]-thymidine incorporation (Agoulnik et al. 2005). In contrast, depletion of SRC-1 had no effect on proliferation of the AR-negative prostate cancer cell lines PC-3 and DU145, which suggests that the effect of SRC-1 on prostate cell proliferation is AR dependent. Several reports describe SRC-1 expression in prostate cancer. In a small study, Gregory et al. (2001) found that SRC-1 expression was increased in castrationrecurrent prostate cancer compared to androgen-stimulated clinically localized prostate cancer and benign prostatic hyperplasia. When SRC-1 expression was examined in a tissue microarray containing androgen-stimulated prostate cancer and corresponding benign samples from more than 500 patients, SRC-1 expression was highly variable (Agoulnik et al. 2005). The average SRC-1 expression levels in benign prostate versus prostate cancer did not differ, but higher expression correlated with several markers characteristic of more aggressive prostate cancer. In contrast, expression in a set of metastatic tumors was higher than in the primary tumors. An unexpected finding was a highly significant correlation between SRC-1 expression in benign prostate and prostate cancer in the same patient. This implies that higher levels of SRC-1 expression in benign prostate may contribute to the development of more aggressive prostate cancer. Although the correlations between SRC-1 expression and prostate cancer are not striking, SRC-1 activity is regulated by phosphorylation. Rowan et al. (2000)
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identified multiple phosphorylation sites in SRC-1. At least two of these sites are phosphorylated by p42/p44 MAPK, and these sites play a role in the SRC-1dependent stimulation of AR activity induced by IL-6 (Ueda et al. 2002). IL-6 treatment induces p42/p44 MAPK activity and this activity is required for the induction of AR activity. Moreover, p42/p44 MAPK activity is increased in advanced prostate cancer (Gioeli et al. 1999); advanced prostate cancer may have elevated SRC-1 activity without significant changes in expression.
SRC-2 Accumulating evidence suggests that SRC-2 plays a role in prostate cancer and its relapse during androgen-deprivation therapy. Some of the concerns in inhibiting AR action in prostate cancer include factors that increase AR action at low androgen levels or facilitate activation by other ligands. SRC-2 is capable of both these activities. Overexpression of SRC-2 not only potentiates AR action, but the fold induction is higher at suboptimal levels of androgen (Agoulnik et al. 2006); depletion of SRC-2 using siRNA reduced androgen-dependent induction of several primary AR target genes, including PSA and TMPRSS2 (Agoulnik et al. 2006). Unlike SRC-1, SRC-2 does not play a role in androgen-dependent repression of maspin. Depletion of SRC-2 using siRNA reduced proliferation of not only the androgen-sensitive LNCaP cell line but also AR-negative PC-3 cells. Thus, SRC-2 modulates AR signaling, but it also has an AR-independent role in cellular proliferation. SRC-2 expression was repressed by androgens, which was a direct action of AR (Agoulnik et al. 2006). Analysis of the DNA regions to which AR is recruited in response to androgen treatment identified AR binding sites both in an SRC-2 intron and in the immediate upstream promoter. A small fragment of 50 flanking sequence linked to the coding sequence of luciferase was sufficient to recapitulate the repression caused by androgens. Repression required AR with an intact DNA and ligand binding domain and the presence of ligand (either agonist or antagonist). SRC-2 is also a target of the p42/ p44 MAPK pathway. In androgen-independent CWR-R1 cells, EGF treatment increased SRC-2 expression, phosphorylation, and interaction with AR, and increased transcriptional activity of AR (Gregory et al. 2004). SRC-2 expression also has been studied in prostate cancer. Gregory et al. examined 24 primary prostate specimens that included eight specimens of androgenstimulated benign prostatic hyperplasia, eight specimens of androgen-stimulated clinically localized prostate cancer, and eight specimens of castration-recurrent prostate cancer for SRC-2 expression. SRC-2 was elevated in castration-recurrent prostate cancer compared to either androgen-stimulated benign or malignant prostate (Gregory et al. 2001). In a large-scale tissue microarray study of primary prostate tumors and adjacent normal prostate from over 500 patients, Agoulnik et al. (2006) found that expression of SRC-2 increased with progression of prostate cancer. Increased expression correlated with a shorter time to biochemical recurrence (rising serum PSA levels). SRC-2 levels had no prognostic significance in patients with low levels of AR. Patients with higher levels of AR recurred sooner if they also expressed high levels of
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SRC-2 compared to those with lower levels of SRC-2 expression. Thus, in primary tumors, SRC-2 likely facilitates AR action despite the fact that it also has an AR-independent effect on cellular proliferation. Analysis of samples taken from patients during failed androgen-deprivation therapy revealed maximal expression in all nine samples, which supports the idea that androgen-deprivation therapy leads to increased expression of SRC-2; increases in SRC-2 expression may activate pathways that lead to stimulation of AR-dependent and AR-independent proliferation of prostate epithelium.
SRC-3 As inferred from one of its names, AIB1 (amplified in breast cancer 1)/SRC-3 is overexpressed in breast cancer (Anzick et al. 1997). Genetic disruption of SRC-3 in mice results in pleiotropic phenotypes that include dwarfism, delayed sexual maturation of females, and smaller prostate size in males (Xu et al. 2000; Yan et al. 2006). SRC-3 regulates expression of multiple IGF and Akt signaling pathway proteins, at least in part, through coactivation of AP-1 transcription factors (Yan et al. 2006; Zhou et al. 2003, 2005). SRC-3 also is an AR coactivator and is recruited to the PSA promoter and enhancer (Wang et al. 2005). Depletion of SRC-3 using siRNA reduced proliferation of prostate cancer cells, including ARnegative prostate cancer cells, which confirms that SRC-3 has diverse functions in prostate cancer cells (Zhou et al. 2003). Moreover, prostate cancer cells artificially overexpressing SRC-3 increased in size and grew faster in nude mouse xenografts. Similar to other members of the p160 family, SRC-3 is a phosphoprotein. Six phosphorylation sites were identified that are important for AR coactivation and at least one of them is an ERK1/2 target (Wu et al. 2004). Immunohistochemical analysis of tissue microarrays containing tissues from more than 500 patients showed nuclear SRC-3 staining in basal and luminal epithelium, as well as in malignant cells. SRC-3 expression was higher in primary prostate cancer than benign prostate; higher SRC-3 expression in prostate cancer correlated with increased proliferation and reduced apoptosis, which are characteristics of aggressive prostate cancer, and shorter time to biochemical recurrence (Zhou et al. 2005). Consistent with cell-based studies, SRC-3 expression in clinical prostate cancer positively correlated with phospho-Akt staining. In a mouse prostate reconstitution model, artificial overexpression of AR and Akt, but not of either alone, was sufficient to induce prostate cancer (Xin et al. 2006). Thus, SRC-3 may potentiate AR activity not only through its role as a classical AR coactivator, but also by increasing Akt activity.
2.1.2
Acetyltransferases
The acetyltransferases discussed are sometimes called cointegrators or secondary coactivators. They are broad in their action, can interact with numerous transcription
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factors and coregulators, and acetylate both histones and other nuclear proteins, which include AR and AR coregulators. Acetylation of AR on lysines in the hinge region selectively augments AR transcriptional activity on some target genes, enhances p300 binding, and reduces NCoR/HDAC/Smad3 corepressor binding (Fu et al. 2003). Transfection of AR mutants that mimic hyperacetylation in its hinge region into prostate cancer cells increases prostate cancer cell survival and proliferation in soft agar and nude mouse xenografts (Fu et al. 2003).
p300 The p300 coactivator acetylates histones, receptors, and coactivators (Fu et al. 2004; Kalkhoven 2004). p300, but not its homolog CBP (CREB binding protein), may have a role in nonsteroidal as well as steroid-mediated AR activation. Activation of AR by IL-6 requires p300 (Debes et al. 2002). Depletion of p300 from cells reduces AR activity and the level of AR acetylation. Treatment with bombesin, a neuropeptide, also activates AR in the absence of ligand by increasing p300 HAT activity and the AR acetylation level (Desai et al. 2006; Gong et al. 2006). Analysis of p300 expression in prostate cancer shows that expression of p300 correlates with increased proliferation, pathological characteristics of more aggressive disease, and decreased time to biochemical recurrence after treatment (Debes et al. 2002). The expression of p300 is subject to androgen regulation; androgen treatments reduces p300 protein levels without affecting its mRNA levels (Heemers et al. 2007). P/CAF (p300/CBP-Associated Factor) P/CAF is an acetyltransferase that interacts with AR (Fu et al. 2004), and AR coactivators such as CBP and the p160 coactivators, and thus can be recruited to AR through these coactivators. AR is acetylated by P/CAF potentiating the activity of AR through direct post-translational modification as well as through its actions on histones and other proteins/(Fu et al. 2004). Tip60 (Tat-Interacting Protein, 60 kDa) Tip60 is a HAT that has multiple substrates which include histones, AR, and p53 (Fu et al. 2004; Sykes et al. 2006). Acetylation of the AR’s hinge region is required for Tip60 coactivation of AR. While acetylation of AR and histones increases AR activity, cell proliferation, and survival (Fu et al. 2004; Popov et al. 2007), acetylation of p53 induced apoptosis in response to DNA damage (Sykes et al. 2006). Analysis of Tip60 expression in prostate cancer revealed a shift in its subcellular localization, from diffuse distribution in androgen-stimulated benign prostate and primary prostate cancer to nuclear accumulation in castrationrecurrent prostate cancer (Halkidou et al. 2003). Studies in prostate cancer cell lines showed recruitment of Tip60 to the PSA promoter in both androgen-sensitive and
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androgen-independent cell lines, which suggests a role for Tip60 in both androgendependent and nonsteroidal activation of AR (Halkidou et al. 2003). Furthermore, androgen depletion increased Tip60 expression and nuclear accumulation, while androgen treatment decreased Tip60 levels.
2.1.3
Deacetylases
Acetylation of histones is an important reversible process in the regulation of transcriptional activation and silencing. The relative acetylation levels of histones and other proteins involved in transcription is maintained by the balancing action of HATs and histone deacetylases (HDAC). The three classes of HDACs are based on their homology to yeast transcriptional repressors. Classes I and II are the classical HDACs (Gregoretti et al. 2004), and Class III contains the newly discovered family of sirtuins (Michan and Sinclair 2007).
Classical HDACs Classes I and II are phylogenetically related to the yeast deacetylases, Rpd3 and Hda1, respectively. HDAC1, 2, 3, and 8 belong to class I and HDAC4, 5, 6, 7, 9, 10, and 11 belong to class II (Waltregny et al. 2004). HDACs from both classes are expressed in the prostate and can be inhibited by HDAC inhibitors, such as trichostatin A. HDAC1 is expressed predominantly in benign and malignant epithelium rather than stroma (Waltregny et al. 2004). HDAC8 is expressed uniquely in stromal cells of the prostate (Waltregny et al. 2004). Most HDACs are recruited to AR in multicomponent repressor complexes such as NCoR (nuclear receptor corepressor), SMRT (silencing mediator of retinoid and thyroid receptors), or DAX1 (dosage-sensitive sex-reversal, AHC, on the X-chromosome, gene 1) complexes. Unlike other deacetylases, HDAC7 binds to AR directly and inhibits receptor independent of its acetylation sites (Karvonen et al. 2006). Deacetylase activity is partially dispensable for HDAC7 repression of AR (Karvonen et al. 2006).
Sirtuins Class III HDACs are the Sir2 (silent mating type information regulation 2) family of NAD + dependent enzymes, SIRT1–7, and they are insensitive to class I and II HDAC inhibitors. SIRT1 deacetylates AR in a NAD-dependent fashion and inhibits its association with p300 and its transcriptional activity (Fu et al. 2006). As a result, SIRT1 inhibits NAD- and androgen-sensitive cellular proliferation (Fu et al. 2006). SIRT1 is thought to be a key protein in calorie-restriction-mediated life span extension and delay in carcinogenesis (Bordone and Guarente 2005; Cohen et al. 2004). However, expression of SIRT1 does not increase in the prostates of calorie-restricted mice (Huffman et al. 2007). Moreover, prostate cancer cells
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express higher levels of SIRT1 than benign epithelial cells as determined using immunohistochemical analysis of human benign prostate and prostate cancer (Huffman et al. 2007). Similarly, immunohistochemical analysis revealed that TRAMP mice expressed higher levels of SIRT1 in poorly differentiated adenocarcinomas compared to those with less advanced disease (Huffman et al. 2007). The intriguing observation that expression of some HATs and HDACs increases with the progression of prostate cancer underscores the complexity of AR regulation by coactivators. The preinitiation complexes may be promoter specific; consequently, an increase in any one coregulator does not change all AR-regulated genes to the same extent.
Corepressors Some of the best characterized corepressors of AR are NCoR, SMRT, and DAX-1 (Agoulnik et al. 2003). Both NCoR and SMRT are recruited to the PSA promoter in an androgen-dependent manner (Shang et al. 2002). Though they have no enzymatic activities of their own, they act as scaffolding proteins that bring HDACs to the AR complexes and thus mediate repression. In addition, all three corepressors interfere with the carboxy- and amino terminal interaction of the AR dimer (Agoulnik et al. 2003). Limited information is available regarding expression of these proteins in prostate cancer. DAX-1 is a member of the nuclear receptor family, but lacks the classical DNA binding domain. DAX-1 is expressed abundantly in the luminal epithelium of the prostate and is localized to the nuclei. Expression is reduced drastically in benign prostatic hyperplasia, that may increase AR signaling (Agoulnik et al. 2003). These corepressors have diverse functions independent of AR signaling. SMRT facilitates DNA repair (Yu et al. 2006), NCoR sequesters p85-a, a regulatory subunit of PI3 kinase, which reduces its activity (Furuya et al. 2007), and DAX-1 directly binds to RNA and is found in polyribosomal complexes (Lalli et al. 2000).
2.2
Methyltransferases as AR Coactivators
Methyltransferases are enzymes that methylate specific arginine or lysine residues in histones. Either amino acid can be monomethylated or polymethylated and the biological consequences depend upon the specific methylation (Daniel et al. 2005). The pattern of methylation is indicative of transcriptional status of the chromatin (Seligson et al. 2005). While histone acetylation generally is associated with transcriptional activation, histone methylation plays roles both in stimulation and inhibition of transcription. Methylation of histones was considered an irreversible epigenetic signature until recently. A number of demethylases are now known to actively demethylate the repressed chromatin and balance methylase activity.
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CARM1
CARM1 (Coactivator-associated arginine methyltransferase) methylates arginines 2, 17, and 26 of histone H3 (Chen et al. 1999). CARM1 is a secondary coactivator because it does not bind directly to AR but is recruited to receptor by the p160 coactivators (Chen et al. 1999). Thus, CARM1 activation of AR depends on the presence of p160 coactivators and their action is synergistic. In addition to modifying histones, CARM1 also methylates other proteins in the AR preinitiation complex, including p300 (Chen et al. 1999; Majumder et al. 2006). Depleting CARM1 in prostate cancer cells reduces AR transcriptional activity, diminishes proliferation, and induces apoptosis (Chen et al. 1999). CARM1 expression is higher in prostate cancer than benign prostate and is highest in castration-recurrent prostate cancer (Majumder et al. 2006).
2.2.2
G9a
The target for G9a methyltransferase activity is lysine 9 of histone H3, which is commonly associated with transcriptional repression. However, in the case of AR, G9a potentiates AR transcriptional activity synergistically with SRC-2, p300, and CARM1. G9a methyltransferase activity is dispensable for its role as an AR coactivator, which suggests that G9a functions as a scaffold rather than a methyltransferase (Lee et al. 2006).
2.2.3
PRMT1
Arginine 3 of histone H4 is the target for this arginine methyltransferase. PRMT1 binds to p160 coactivators and potentiates AR action like CARM1 (Wang et al. 2001).
2.3
Demethylases
Histone methylation originally was considered irreversible since the half-life of methyl lysine residues and histones are similar (Byvoet et al. 1972). Recent discoveries of demethylases show that histone methylation is dynamic in some regions of the genome (Metzger et al. 2005; Wissmann et al. 2007). Prostate cancer patients with a Gleason score of less than 7 have a lower 10-year recurrence rate if the percentage of cells with histone H3 Lysine 4 dimethylation staining is above the 60th percentile (Seligson et al. 2005). Increased methylation or decreased demethylation activity results in a more favorable prognosis for prostate cancer patients.
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LSD1
Lysine-specific demethylase 1 (LSD1) interacts with AR in a ligand-dependent manner to demethylate the repressive histone modifications, mono- and dimethyl histone H3-lysine 9 (Metzger et al. 2005). Depletion of LSD1 in LNCaP cells reduces androgen-sensitive PSA induction and cellular proliferation. Immunohistochemical analysis of LSD1 expression in prostate cancer tissue microarrays revealed higher levels of LSD1 staining in the nuclei of patients who relapsed after radical prostatectomy and Kaplan–Meier analysis revealed shorter time to biochemical recurrence in patients with an LSD1 staining score of 8–12 compared to less than 8 (Kahl et al. 2006). 2.3.2
JARID1B
JARID1B (also called Plu-1) is a demethylase whose overexpression results in removal of mono-, di-, and trimethyl groups from histone H3 lysine 4 without changing H3K36me3 and H3K9me3 methylation (Xiang et al. 2007). JARID1B directly interacts with AR and its overexpression increases AR transcriptional activity on a PSA luciferase reporter (Xiang et al. 2007). JARID1B is undetectable in benign prostate tissue but expression is high in prostate cancer (Xiang et al. 2007). The role of endogenous JARID1B on various types of AR-regulated promoters requires further investigation since JARID1B is a strong transcriptional repressor in some promoter contexts; it interacts directly with the class I and II HDACs and indirectly with NCoR (Barrett et al. 2007). Alternatively, JARID1B’s primary activity in prostate cancer may be AR independent. Microarray analysis of the global knockdown and overexpression of JARID1B in breast cancer cells show that it represses the majority of its target genes (Scibetta et al. 2007).
2.3.3
JHDM2A
Unlike LSD1, which is constitutively associated with the PSA promoter (Yamane et al. 2006), JHDM2A interaction with AR and recruitment to AR responsive promoters is androgen dependent. JHDM2A can demethylate mono- and dimethyl H3 lysine 9. Knockdown of JHDM2A results in increased levels of dimethyl histone H3 lysine 9 at the promoters and decreased expression of the AR target genes PSA, TMPRSS2, and Nkx3.1 (Yamane et al. 2006).
2.3.4
JMJD2C
This demethylase interacts with LSD1 and is constitutively associated with ARregulated promoters (Wissmann et al. 2007). JMJD2C is a histone H3 lysine 9 tridemethylase that stimulates activity of AR cooperatively with LSD1 (Wissmann
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et al. 2007). Depletion of JMJD2C in LNCaP cells reduced both androgen-sensitive PSA induction and cellular proliferation (Wissmann et al. 2007). JMJD2C is expressed ubiquitously and is also found in benign prostate and prostate cancer as determined using immunohistochemistry (Wissmann et al. 2007).
2.4
Ubiquitin Ligases
AR activity and turnover is regulated, in part, by ubiquitin ligases and ubiquitination of AR and histones at AR responsive genes. Ubiquitin ligases conjugate the small protein, ubiquitin, to the epsilon amino group of lysines in target proteins (Haas 2007). Ubiquitination plays a prominent role in the regulation of protein stability through targeting proteins for proteasome-dependent degradation. Degradation typically involves the formation of polyubiquitin chains linked to one or more lysines in target proteins. However, ubiquitination and particularly monoubiquitination play roles in a variety of other protein functions (Sigismund et al. 2004). E6AP ubiquitin ligase interacts directly with AR and activates AR transcriptional activity in a ligand-dependent manner (Khan et al. 2006). Consistent with this hypothesis, prostates are smaller in E6AP null mice compared to wildtype littermates. AR activity on the probasin promoter is decreased despite increased levels of AR protein in E6AP knockout mice (Khan et al. 2006). These data suggest that efficient turnover is necessary for optimal AR activity. Inhibition of the proteasome inhibits AR activity (Kang et al. 2002; Lin et al. 2002). However, E6AP expression is decreased in prostate cancer relative to benign tissue (Gao et al. 2005).
2.5
Deubiquitinases
Histone H2A was the first protein shown ubiquitinated on a highly conserved residue, lysine 119 (Goldknopf and Busch 1977). An H2A deubiquitinase JAMM/ MPN+ (H2A-DUB) is required for optimal activation of the AR in prostate cancer cells (Zhu et al. 2007). H2A-DUB interacts directly with p/CAF and is recruited to the PSA promoter in response to androgen treatment causing a coordinate increase in acetylation of histone H3 and a decrease in monoubiquitination of histone H2A. H2A-DUB overexpression increases AR transcriptional activity and its depletion from LNCaP cells reduces expression of AR target genes including PSA and Nkx3.1 (Zhu et al. 2007). Immunohistochemical analysis of tissue microarrays reveals a remarkable reduction in ubiquitinated H2A in prostate cancer compared to benign prostate (Zhu et al. 2007).
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Small Ubiquitin-Like Modifier Proteins
A number of proteins in AR signaling are targets for sumoylation; these include AR, SRC-1, SRC-2, p300, and HDAC1. In contrast to ubiquitination, sumoylation does not target proteins for degradation. Mutations in SRC-2 sumoylation sites reduce its ability to activate AR (Kotaja et al. 2002). In contrast, mutation of the sumoylation site in the p300 carboxyl terminus increases p300-mediated transcriptional activity (Girdwood et al. 2003). AR is sumoylated in vivo on lysine residues 386 and 520 (Poukka et al. 2000). Mutation of these residues does not affect DNA binding but increases transcriptional activation by AR, which suggests a repressive role for small ubiquitin-like modifier (SUMO) conjugation in transcriptional activity. Multiple proteins are involved in AR sumoylation pathways. SUMO is an evolutionarily conserved protein; this suggests an important function in cells. There are four SUMO proteins in humans that vary slightly in length; only SUMO-1, -2, and -3 form covalent isopeptide bonds with the substrate proteins. SUMO-2 and -3 are most closely related (90% identity vs. about 50% identity with SUMO-1). SUMO-1 represses, while SUMO-2 and -3 increase AR activity (Callewaert et al. 2004; Poukka et al. 2000; Zheng et al. 2006). SUMO E2 ligase UBC-9 is an AR coactivator (Poukka et al. 1999), while other SUMO E3 ligases can either activate or repress AR activity (Kotaja et al. 2000). Very little is known about cell and endogenous promoter-specific actions of these proteins in benign prostate and prostate cancer. Sumoylation of proteins is dynamic and is reversed by SUMO proteases called SENPs. Humans express six SENPS that deconjugate SUMO from the large number of modified proteins. SENP1 is a SUMO protease that increases AR transcriptional activity (Cheng et al. 2004). The target protein for SENP1 is HDAC1, not AR. By desumoylating HDAC1, SENP1 reduces its histone deacetylase activity and therefore its ability to repress transcription (Cheng et al. 2004). SENP1 expression is upregulated in turn by androgens, which suggests a regulatory feedback mechanism (Bawa-Khalfe et al. 2007). SENP1 also is induced upon IL-6 treatment and androgens and IL-6 act synergistically to induce its expression (Cheng et al. 2004). Depletion of SENP1 causes a decrease in androgen-mediated cellular proliferation of LNCaP cells (Cheng et al. 2006). Cheng et al. (2004) investigated expression of SENP1 in normal prostate, high-grade prostate intraepithelial neoplasia (PIN), and prostate cancer using in situ hybridization and prostatectomy samples from 43 patients. Expression of SENP1 was increased in high-grade PIN and cancer.
2.7
Splicing Associated Factors
AR-induced transcription is a sequential multistep process involving receptor recruitment to promoters, chromatin remodeling, initiation, elongation, RNA splicing,
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and termination. The coregulators described play a critical role in the initial steps of transcription. Recent data suggest an important role for AR in determining the splicing signature of the transcriptome in benign prostate and prostate cancer. Many AR target genes have numerous splicing variants. For example, the primary target genes KLK2 and KLK3 (PSA) have numerous splicing variants and protein products generated from the alternatively spliced forms, which are present in benign prostate and prostate cancer (David et al. 2002). Another example is a tumor suppressor and the earliest marker of prostate formation, Nkx3.1. Although initial expression of Nkx3.1 in prostate epithelium precedes that of AR, later expression of Nkx3.1 depends completely on androgen signaling. At least five alternatively spliced variants of Nkx3.1 exist in humans (Shen and Abate-Shen 2003). The effect of receptor-mediated transcription on splicing can be described as a combination of two synergistic models. The first model is based on the kinetics of transcriptional elongation and suggests that more rapid transcription favors exon skipping. The second model is based on the fact that AR can recruit various splicing factors to the RNA polymerase II (Pol II) complex. AR coactivators increase the rate of transcription via different mechanisms and thus could induce a higher rate of skipping. A number of splicing factors that interact with AR are recruited to AR responsive promoters to affect splicing according to the second model. Splicing factors can both activate and repress AR activity. For example, U5snRNP/ANT-1 increases AR transcriptional activity (Zhao et al. 2002) while COBRA1, PSF, and p54nrb repress it.
2.7.1
Cofactor of BRCA1 (COBRA1)
COBRA1 is a BRCA1-interacting protein and is a subunit in the negative elongation factor complex, NELF-B. COBRA1 interacts with AR directly and represses its transcriptional activity. Overexpression of COBRA1 induces exon skipping in the CD44 minigene driven by the androgen responsive MMTV promoter (Sun et al. 2007).
2.7.2
PTB-Associated Splicing Factor (PSF) and p54nrb
These two RNA splicing factors interact with the Pol II holoenzyme, bind to AR complexes, and inhibit AR transcriptional activity by recruiting HDAC complexes and destabilizing AR interaction with the ARE (Dong et al. 2007). A liganddependent recruitment of PSF to the PSA promoter occurs simultaneously with release from the PSA enhancer (Dong et al. 2007). Because PSF and p54nrb directly interact with the C terminal domain (CTD) of Pol II and AR, they can serve as a molecular link among the transcriptional machinery, splice site recognition, and splicing.
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Signaling Molecules Kinases and Phosphatases
AR and its numerous coregulators are phosphoproteins and therefore their activity is subject to regulation by kinases and phosphatases. Tyrosine phosphorylation of AR is increased during progression to androgen independence; AR phosphorylation on tyrosine 534 is induced by activation of the Src kinase (Guo et al. 2006). AR phosphorylated on tyrosine 534 becomes more active at low androgen concentrations and mutation of this site abolished EGF-dependent AR activity (Guo et al. 2006). Phosphorylation of AR on tyrosine 534 is increased almost tenfold in castration-recurrent prostate cancer compared to benign prostate as determined using immunohistochemistry (Guo et al. 2006). Although some kinases and phosphatases influence receptor function through regulation of AR phosphorylation, others form more stable interactions with AR and function as coactivators.
2.8.2
Male Germ Cell-Associated Kinase
Male germ cell-associated kinase (MAK) is an AR coactivator. MAK is a direct target of AR and its expression is induced by androgens. Once expressed, MAK interacts directly with the AR, is recruited to AR-dependent promoters, and increases ligand-dependent transcriptional activity of AR and androgen-sensitive cellular proliferation (Ma et al. 2006).
2.8.3
Activated cdc42-Associated Tyrosine Kinase (Ack1)
Ack1 is a Her2-activated nonreceptor tyrosine kinase that phosphorylates AR on tyrosines 267 and 363 (Mahajan et al. 2007). Ack1 interacts directly with AR and is recruited to the PSA enhancer in an androgen-dependent manner (Mahajan et al. 2007). LNCaP cells expressing a constitutively active mutant of Ack1 formed tumors in nude mice faster than cells transfected with either vector or a kinase dead mutant of Ack1, and these tumors became resistant to castration after 2 weeks in animals (Mahajan et al. 2007). The Ack1 gene was amplified in primary prostate cancers and its transcript levels significantly increased in castration-recurrent prostate cancer (van der Horst et al. 2005). While of little importance for proliferation, overexpression of Ack1 significantly increases metastatic potential of prostate cancer cells (van der Horst et al. 2005). 2.8.4
Cdc25B
Along with multiple kinases, AR activity also is enhanced by the dual-specificity phosphatase, Cdc25B. Cdc25B interacts directly with AR and synergizes with
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P/CAF and p300 in potentiating AR activity (Ma et al. 2001). Ngan et al. (2003) showed that Cdc25B expression assessed using immunohistochemistry increased in prostate cancers and correlated with Gleason score. 2.8.5
Protein-Kinase-C-Related-Kinase-1 (PRK1)
PRK1 interacts directly with the amino terminus of AR and increases its liganddependent transcriptional activation (Metzger et al. 2003). Inhibition of PRK1 using a chemical inhibitor or PRK1 depletion with siRNA represses AR transcriptional activity (Metzger et al. 2008). Androgen treatment causes its association with AR and concomitant recruitment to both the PSA promoter and enhancer. As PRK1 is recruited to the promoter, it phosphorylates H3T11 and causes increased demethylation of H3K9, which is associated with inactive chromatin. When PRK1 is inhibited or depleted, androgen treatment does not cause H3T11 phosphorylation and the androgendependent induction of AR target genes, PSA and KLK2, is reduced. Immunohistochemical analysis of PRK1 expression in tissue microarrays containing samples of 20 benign prostates and 111 prostate cancers revealed that higher expression of PRK1 and higher phosphorylation levels of H3T11 positively correlated with Gleason score and other aggressive characteristics of prostate cancer (Metzger et al. 2008). 2.8.6
Cyclin D1
Cyclin D1 interacts with AR and represses AR activity. Cyclin D1 is expressed as two splice variants, a and b; the D1a isoform strongly represses AR activity whereas the D1b is less effective (Burd et al. 2006). Cyclin D1a overexpression reduces proliferation of AR-dependent prostate cancer cell lines, while cyclin D1b mildly stimulates proliferation. Growth of prostate cancer cell lines that do not express AR is unaffected by overexpression of either cyclin D1a or cyclin D1b (Burd et al. 2006). A frequent polymorphism in the Cyclin D1 gene, G/A 870, is associated with increased risk of prostate cancer (Comstock et al. 2007). Cells with this polymorphic change in the gene have an increased tendency to express the cyclin D1b variant (Burd et al. 2006). Immunohistochemical analysis of cyclin D1 expression revealed that cytoplasmic localization of Cyclin D1 correlates with lower Ki-67 index (indicator of cellular proliferation), while nuclear staining positively correlated with higher Gleason score, increased proliferation, and nuclear localization of p21Cip1 (Comstock et al. 2007).
2.8.7
Cyclin D3/CDK11
Cyclin D3 forms a complex with the 58-kDa isoform of cyclin-dependent kinase 11 (CDK11p58) and AR, phosphorylates AR on serine 308, and represses AR induction of ARE, MMTV, and PSA-driven luciferase reporters, and endogenous PSA expression in LNCaP cells (Zong et al. 2007). Ectopic expression of cyclin D3 in
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LNCaP cells increased accumulation of cells in the G1 phase of the cell cycle, but not in PC-3 cells, which suggests cyclin D3 may interfere with the mitogenic cellular response to androgens (Olshavsky et al. 2008); depletion of CDK11p58 resulted in a decline in BrdU incorporation in androgen-treated LNCaP cells and expression of cyclin D3 was necessary for this response (Zong et al. 2007). Total expression of cyclin D3 declined with prostate cancer progression and showed a negative correlation with cellular proliferation. (Olshavsky et al. 2008). 2.8.8
Isomerase and Wnt/b-Catenin Signaling
Pin1 is a peptidyl-prolyl isomerase that catalyzes cis/trans conversion of proteins upon binding to their phosphorylated Ser/Thr-Pro motifs. Multiple protein targets are regulated by Pin1 isomerase. Pin1 interferes with the ability of AR to inhibit the b-catenin/Tcf4 pathway that is in turn regulated by wnt signaling (Chen et al. 2006). b-catenin itself modestly increases AR activity and broadens ligand specificity (Truica et al. 2000). Wnt-1 and b-catenin are expressed at higher levels in lymph node and bone metastases than in primary cancers, and their expression positively correlates with Gleason score and serum PSA levels (Chen et al. 2004b). Expression analysis of Pin1 showed increased expression in prostate cancer compared to benign prostate tissue and predicted progression-free survival. Patients with high Pin1 expression have over eight times higher risk for recurrence than patients with low Pin1 expression (Ayala et al. 2003). A further increase in Pin1 expression was observed in metastatic prostate cancers compared to primary tumors using immunohistochemical evaluation of tissue microarrays (Chen et al. 2006). 2.8.9
PELP-1 (Proline-, Glutamic Acid-, and Leucine-Rich Protein-1)/ MNAR (Modulator of Nongenomic Activity of Estrogen Receptor)
MNAR is a scaffolding protein that brings together AR, general coactivators, such as p300 and CBP, and other coactivators, such as FHL2. In addition, MNAR complexes in the cytoplasm contain AR and src kinase and can activate downstream kinases, such as p42/p44 MAPK (Unni et al. 2004). In a limited MNAR expression analysis in prostate cancer, MNAR expression was higher in advanced prostate cancer specimens than in benign prostate tissue or low-grade prostate cancer (Nair et al. 2007).
3 Conclusion AR recruits a series of coactivator complexes to modulate transcription of target genes. Some of the coactivator/coregulators may be required regardless of the target, whereas others are needed to regulate subsets of target genes. Coregulators possess a wide range of enzymatic activities or serve as scaffold proteins to recruit other enzymes. Once recruited they covalently modify chromatin, other coregulators,
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transcription factors, and Pol II thus modulating transcription initiation, elongation, and splicing. Therefore, there are a large number of coactivators that are required for AR-mediated transcription. Cell-based studies using siRNA that transiently deplete individual coactivators typically show reductions in AR transcriptional activity of greater than 50%. These findings suggest that cellular levels of coactivators are limiting and that in an acute situation, other closely related coactivators do not compensate for the loss of one coactivator. However, in vivo, the situation may be somewhat different. For example, the SRC-1 null mouse expresses twice the level of the closely related p160 coactivator, SRC-2/TIF2 (Xu et al. 1998). Receptors function as scaffolds to recruit a variety of coregulators and act to integrate the input from a variety of signaling pathways. The initial studies of the role of post-translational modifications in regulating coactivator function and transcription factor selectivity suggest that one explanation for the apparent redundancy in coactivators is that the individual coactivators respond to unique sets of signaling pathways (Wu et al. 2004). Thus, they may aid in coordinating cellular response to hormone with the level of activity of specific cell signaling pathways. The analyses of coactivator expression comparing benign tissue and prostate cancer or correlating expression with Gleason score often show a correlation between increased coactivator expression and prostate cancer, Gleason score, or time to biochemical recurrence. Global chromatin modifications are also somewhat predictive of patient outcome; however, the exact coregulators that cause these changes in chromatin are not known. The coregulators modulate activity of multiple transcription factors and, in some cases, also have activities distinct from their roles as coregulators, but the correlations between many of these, their ability to facilitate AR action, and altered levels in prostate cancer suggest that they are major factors in potentiating AR activity in prostate cancer. Some coactivators interact with AR through regions in the hormone binding domain and antagonists that alter the conformation of the hormone binding domain reduce these interactions limiting AR activity. However, the p160 coactivators interact with the amino terminal AF-1. Developing means to block interaction of coactivators with this important surface is a potential new target to block AR activity and prostate cancer progression. However, to our knowledge there has been no report showing that expression of one or even a combination of coactivators is an independent predictor of outcome. It is possible that expression of the enzymatic classes of coregulators will be more predictive.
References Agoulnik, I. U., and N. L. Weigel. 2006. Androgen receptor action in hormone-dependent and recurrent prostate cancer. J Cell Biochem 99: 362–72. Agoulnik, I. U., W. C. Krause, W. E. Bingman, III, H. T. Rahman, M. Amrikachi, G. E. Ayala, and N. L. Weigel. 2003. Repressors of androgen and progesterone receptor action. J Biol Chem 278: 31136–48.
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Androgen Receptor Coregulators and Their Role in Prostate Cancer Latif A. Wafa, Robert Snoek, and Paul S. Rennie
Abstract The limiting factor in the survival of a patient with prostate cancer is the rate of progression to the noncurable androgen-independent (AI) stage of disease. The androgen receptor (AR) is a critical regulator of prostate cancer development and is involved in AI progression. Coregulators are proteins that interact directly with AR to enhance (coactivators) or reduce (corepressors) its transcriptional activity. Currently, over 165 AR coregulators have been discovered. In this chapter, we focus on a subset of the most well-characterized AR coregulators that are associated with prostate cancer. The first part of our review discusses the mechanisms by which classical type I and nonclassical type II AR coactivators/corepressors regulate AR transcriptional activity. The second section focuses on the role of coregulators in prostate cancer, including their expression profile in prostate cancer patients, tumor cell growth effects, and potential as therapeutic targets. In view of their involvement in prostate cancer progression, it is anticipated that further study of AR coregulators will provide more treatment options for increasing survival of patients with AI prostate cancer. Prostate cancer is the most commonly diagnosed nonskin cancer in men and the second leading cause of cancer death in North America (Jemal et al. 2004). While frequently curable in its early stages, many patients will present with locally advanced or metastatic disease for which there are currently no curative treatment options (Albertsen et al. 2005). Although androgen deprivation therapies, which block the growth promoting effects of androgens, are often used to treat advanced disease, progression to a lethal state variably referred to as castration-resistant, hormone refractory, castration-recurrent, or AI is the usual outcome, giving rise to a median survival of approximately 19 months (Albertsen et al. 2005; Kent and Hussain 2003; Martel et al. 2003). Some brief survival extensions can sometimes be achieved using current docetaxel-based chemotherapy (Petrylak 2005; Tannock et al. 2004). However, to have any major impact on current survival rates, more
P.S. Rennie(*) Department of Urologic Sciences, Faculty of Medicine, University of British Columbia, Prostate Centre at Vancouver General Hospital, Vancouver, BC, Canada, V6H 3Z6, E-mail:
[email protected]
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effective treatment strategies need to be developed. Because androgens mediate their effect on target cells through their receptor, it is thought that the AR is important in the development of AI disease. While the molecular mechanisms responsible for the development of AI prostate cancer are largely unknown, typically they do not appear to involve loss of AR expression (Taplin and Balk 2004). In over 80% of locally advanced AI prostate cancers, high levels of nuclear AR have been observed (Tilley et al. 1994), and in bone metastases, the amount of AR present is often higher than in primary tumors (Hobisch et al. 1995). There is evidence that in most cases, some form of inappropriate activation of AR is linked to recurrent, AI growth of prostate cancers (Taplin and Balk 2004; Rennie and Nelson 1998). In fact, in vivo knockdown of AR can delay AI progression (Cheng et al. 2006). One can envision a variety of molecular alterations that could lead to continued or amplified AR signaling following surgical or medical castration, such as gain of function AR mutations (Culig et al. 2003; Gaddipati et al. 1994; Marcelli et al. 2000; Newmark et al. 1992; Shi et al. 2002; Taplin et al. 2003) and amplification/overexpression of wild-type AR (Ford et al. 2003; Linja et al. 2001; Visakorpi et al. 1995). However, the available evidence suggests that the most commonly occurring mechanisms for progression to the AI stage are likely to be epigenetic, involving ligand-independent (or ligand-reduced) activation of AR either through convergence of cell signaling pathways and/or altered activity and expression of AR coregulators (Taplin and Balk 2004; Rennie and Nelson 1998; Sadar et al. 1999; Ueda et al. 2002). The latter is the focus of this chapter. Section 1 will describe the various types of AR coregulators that have been identified and Sect. 2 will discuss their role in prostate cancer and how they may provide therapeutic targets.
1 Coregulators of Androgen Receptor Activity 1.1
Overview of Androgen Receptor Coregulator Proteins
Transcriptional activity of androgen receptor (AR), as well as other members of the nuclear receptor superfamily, is modulated by coregulatory proteins. Generally, coregulators can be defined as proteins that interact directly with nuclear receptors to enhance (coactivators) or reduce (corepressors) transactivation of target genes, without significantly altering the basal transcriptional rate (McKenna et al. 1999). Coregulator proteins are not considered to possess specific DNA-binding ability (Xu et al. 1999). A list of more than 270 coregulators is available on the Nuclear Receptor Signaling Atlas (NURSA) website (http:www.nursa.org). Overall, a subset of 165-plus coregulators have been identified to interact with AR (Heemers and Tindall 2007). Many reviews have been written on coregulators (Heemers and Tindall 2007; Chmelar et al. 2007; Burd et al. 2006a; Kumar et al. 2005; Lonard et al. 2007; Dehm and Tindall 2007; Culig et al. 2005; Wang et al. 2005; Rosenfeld et al. 2006; Smith and O’Malley 2004) and this chapter will focus on a subset of the most well-characterized coregulators that are associated with prostate cancer.
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Since coactivators use multiple mechanisms to influence AR transcription, they can be categorized, based on their functional characteristics, into two major types: classical/type I and nonclassical/type II coactivators (Heinlein and Chang 2002). Type I coactivators exert their function on AR while the receptor is at the target gene promoter and facilitate chromatin remodeling, DNA occupancy, or recruitment of basal transcription factors associated with the RNA polymerase (pol) II holocomplex (Sect. 1.2). Type II coactivators function primarily by modulating the appropriate folding of AR, ligand binding to AR, or facilitating the N-/ C-terminal interaction of AR. These events can contribute to AR protein stability, alter ligand-binding capability, or influence the subcellular distribution of AR, which ultimately leads to an increase in AR transcriptional activity. As discussed in Sect. 1.3, a wide array of type II coactivators has also been found for AR, which can act at many stages of receptor activation. In contrast to coactivators, corepressors suppress nuclear receptor transcriptional activity. Similar to coactivators, corepressors can be classified into two major types: classical/type I and nonclassical/type II corepressors (Sect. 1.4). Type I corepressors are characterized by their ability to bind nuclear receptors to repress transcription through the formation of nonproductive interactions with basal transcriptional machinery or through chromatin remodeling (Heinlein and Chang 2002). Type II corepressors function through other mechanisms such as inhibition of AR nuclear translocation/DNA-binding and disruption of both intermolecular interactions of AR and AR binding to coactivators. A partial list of the more established type I/II AR coactivators and corepressors is provided in Tables 1 and 3, respectively. A subset of reported AR coactivators that function through unique or unknown mechanisms is included in Table 2. More recently, involvement of coregulator proteins in the nongenomic actions of AR and signal transduction cascades has also been demonstrated (Sect. 1.5).
1.2 1.2.1
Type I Classical Coactivators of AR SRC/p160 Coactivators
The first identified and most extensively characterized of AR coregulators is the steroid receptor coactivator (SRC) family consisting of three 160-kDa proteins. Members of this family include SRC1, TIF2 (transcriptional intermediary factor 2) or SRC2, and AIB1 (amplified in breast cancer 1) or SRC3 (Onate et al. 1995; Ding et al. 1998; Alen et al. 1999). The SRC/p160 coactivators have been shown to interact with the AR-N-terminal domain (AR-NTD) and activation function 2 (AF2) surface of the AR-ligand-binding domain (AR-LBD) to enhance liganddependent transactivation of the receptor (Ikonen et al. 1997; Needham et al. 2000; Wang et al. 2001a; Callewaert et al. 2006). SRC1, SRC2, and SRC3 share similar structural organization that includes an N-terminal tandem basic helix-loophelix domain, a C-terminal glutamine rich region, and three LXXLL motifs in the
344 Table 1 Type I/II AR coactivators Name Interacting domain of AR Type I classical coactivators SRC/p160 family SRC1/NCoA-1 NTD, DBD, LBD SRC2/TIF2/GRIP1 NTD, DBD SRC3/AIB1/ACTR LBD and other domains Chromatin remodeling CBP/p300 NTD, DBD p/CAF NTD, DBD SRC1 and SRC3 NTD, DBD, LBD SWI/SNF Precise domain unknown Link to basal transcriptional machinerya RNA Pol II subunit LBD (RBP-2) TFIIB Interacts with SRC1 TFIIF NTD TFIIH NTD Type II nonclassical coactivators Molecular chaperonesa hop (p60) Precise domain unknown hsp40 (Ydj1) LBD hsp70 LBD hsp90 p23
LBD Precise domain unknown Modulation of ligand binding and AR stabilization ANPK (PKY) DBD ARA70 DBD, LBD
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Selected references
Heinlein and Chang 2002; Onate et al. 1995 Heinlein and Chang 2002; Ding et al. 1998 Heinlein and Chang 2002; Ozers et al. 2007
Fronsdal et al. 1998 Reutens et al. 2001 Spencer et al. 1997; Chen et al. 1997 Marshall et al. 2003
Lee et al. 2003 Takeshita et al. 1996 McEwan and Gustafsson 1997 Lee et al. 2000
Chmelar et al. 2007; Freeman et al. 2000 Caplan et al. 1995; Fan et al. 2005 Veldscholte et al. 1992; Smith and Toft 1993; Shatkina et al. 2003 Marivoet et al. 1992; Fang et al. 1996 Freeman et al. 2000; Thomas et al. 2006 Moilanen et al. 1998 Heinlein and Chang 2002; Yeh and Chang 1996 Shatkina et al. 2003; Froesch et al. 1998
BAG-1L (hsp70 NTD, LBD cochaperone) Cellular trafficking of AR Caveolin-1 NTD, LBD Lu et al. 2001 Filamin Hinge domain Ozanne et al. 2000 Gelsolin DBD, LBD Nishimura et al. 2003 Ran/ARA24 NTD Hsiao et al. 1999 Supervillin NTD, DBD Ting et al. 2002 The organization of coactivators is based on their known functions and the mechanism by which they can enhance AR activity NTD N-terminal domain, DBD DNA-binding domain, LBD ligand-binding domain a These subcategories are not typically classified as coactivators but have been included here due to the close nature of their effects on AR with type I and II coactivators
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Table 2 Other AR coactivators Name Mode of action Selected references b-Catenin Binds to AR-DBD and LBD to enhance AR Truica et al. 2000; transactivation, possibly by increasing receptor nuclear Mulholland et al. 2002 translocation ARA160 Binds to AR-NTD and can enhance AR transactivation Hsiao and Chang 1999 cooperatively with ARA70 coactivator in prostate cancer cells ARA267 Interacts with both N- and C-termini of AR to increase Wang et al. 2001b AR activity and can synergize with other coactivators, such as Ran/ARA24 and p/CAF ART-27 Binds to the NTD of AR and enhances transcription in Taneja et al. 2004 prostate cancer cells BRCA1 Tumor suppressor that binds to AR N-/C-termini and Yeh et al. 2000 synergistically enhances AR transcription with ARA70 CAK An AR-NTD interacting coactivator of AR and Lee et al. 2000 kinase moiety of the general transcription factor complex TFIIH HNF-3a DNA-binding protein that interacts with AR-DBD/hinge Gao et al. 2003 domain to promote assembly of AR-dependent transcription complexes, resulting in increased receptor activity hsp27 Binds to AR-NTD and LBD and enhances AR Zoubeidi et al. 2007 transcriptional activity Ku70 and Interact directly with AR-LBD, enhance AR transcription, Mayeur et al. 2005 Ku80 and can bind to prostate-specific antigen promoter in an androgen-dependent manner DDC Binds to AR-NTD and LBD to enhance AR transcriptional Wafa et al. 2003 activity through a ligand-dependent mechanism MAK Enhances AR transactivation potential in an androgenMa et al. 2006 and kinase-dependent manner in prostate cancer cells and can synergize with SRC3 RanBPM Binds to AR-NTD and DBD to enhance AR Rao et al. 2002b transcriptional activity Rb Tumor suppressor binds the NTD and LBD of AR Yeh et al. 1998 and enhances receptor transactivation in prostate cancer cells Tip60 Interacts with the LBD and hinge domain of AR and Brady et al. 1999 can increase ligand-dependent receptor transcription This list includes AR coactivators that have been shown to enhance receptor transactivation through unique or unknown mechanisms AR androgen receptor, DBD DNA-binding domain, LBD ligand-binding domain, NTD N-terminal domain, ART-27 AR-trapped clone-27, BRCA1 breast cancer-susceptibility gene 1, CAK Cdkactivating kinase, HNF-3a hepatocyte nuclear factor-3a, DDCL-Dopa decarboxylase, MAK male germ cell-associated kinase, Rb retinoblastoma protein, Tip60 TAT-interactive protein
central portion of the protein that are necessary for interaction with nuclear receptors (McKenna et al. 1999). Notably, binding of the LXXLL motifs of SRCs to the AR-LBD is weaker than with the LBD AF2 of other steroid receptors (e.g. glucocorticoid and estrogen receptors), possibly due to competition with the AR-NTD for
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Table 3 Type I/II AR corepressors Name Interacting domain of AR Type I classical corepressors Histone deacetylase recruitment HDAC1 DBD, LBD NCoR LBD SMRT NTD, LBD
Selected references
Gaughan et al. 2002 Cheng et al. 2002 Dotzlaw et al. 2002; Liao et al. 2003
Type II nonclassical corepressors Inhibition of AR nuclear translocation and DNA binding Calreticulin DAX-1
DBD LBD
Dedhar et al. 1994 Holter et al. 2002
Disruption of AR N-/C-terminal interaction and coactivator binding Akt Precise domain unknown Lin et al. 2001 Cyclin D1 NTD, hinge domain Burd et al. 2006a; Reutens et al. 2001; Knudsen et al. 1999a GSK3b NTD, LBD Wang et al. 2004 p53 Precise domain unknown Shenk et al. 2001 SHP NTD, LBD Gobinet et al. 2001, 2005 Other corepressors of AR Alien NTD HBO1 DBD, LBD PSF Unknown PTEN DBD, LBD
Moehren et al. 2007 Sharma et al. 2000 Dong et al. 2007 Lin et al. 2004
The organization of corepressors is based on their known functions and the mechanism by which they can repress AR activity NTD N-terminal domain, DBD DNA-binding domain, LBD ligand-binding domain
binding with the C-terminal AF2 of AR (He et al. 1999, 2001). However, SRC coactivator LXXLL motif binding does play an important role in the regulation of AR activity and has been suggested to induce conformational changes in the AF2 of AR to modulate ligand-binding kinetics (He et al. 2006). The AR-LBD can also bind to FXXLF motifs contained in some AR coregulators, such as the type II AR-associated protein 70 (ARA70) coactivator, but this sequence is also present in the NTD of AR, which further adds to the competition between coactivators and the NTD for binding to the LBD of the receptor (He et al. 2002; Dubbink et al. 2004). SRCs can increase AR transactivation via their intrinsic histone acetyltransferase (HAT) activity, which allows maintenance of a transcriptionally open chromatin structure at the promoter of target genes (Spencer et al. 1997). Recently, SRC1 was shown to actually enhance the direct binding of AR to chromatin, presumably due to its HAT activity (Li et al. 2007). In addition, SRCs can act as platforms for the recruitment of secondary coactivators (Fig. 1), such as CREB-binding protein (CBP)/p300 and p300/CBP-associated factor (p/CAF) (see Sect. 1.2.2), which possess chromatin remodeling capabilities (Chakravarti et al. 1996; Blanco et al. 1998) and which can also bridge nuclear receptors to basal transcriptional machinery (see Sect. 1.2.3) (Takeshita et al. 1996).
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Fig. 1 Type I AR coactivators. Type I/classical coactivators exert their function on androgen receptor (AR), while the receptor is at a target gene promoter. This is typified by the steroid receptor coactivator (SRC) family which binds to AR, contains intrinsic histone acetyltransferase activity, recruits secondary coactivators that possess chromatin remodeling capabilities, and can also bridge to general transcription factors (GTFs) associated with the RNA polymerase II holocomplex. ARR androgen responsive region, l dihydrotestosterone
1.2.2
Chromatin Remodeling Coactivators
Chromatin remodeling protein complexes act upon the nucleosome (the subunit of chromatin composed of a short length of DNA wrapped around a core of histone proteins) by either disrupting the histone–DNA interaction or controlling the acetylation status of histones. This disruption of chromatin structure allows transcription factors to bind more readily to DNA and thus facilitate transcriptional activation (Rosenfeld and Glass 2001; Rhodes 1997). AR coactivators that contain intrinsic HAT activity include CBP or its homologue p300 (CBP/p300), p/CAF, and members of the SRC/p160 family, SRC1 and SRC3 (Spencer et al. 1997; Chen et al. 1997; Blanco et al. 1998; Ogryzko et al. 1996; Heery et al. 1997). CBP/p300 has been shown to increase AR-mediated transcription by three- to fivefold (Aarnisalo et al. 1998) and p/CAF can reduce cyclin D1-mediated repression of AR transactivation (Reutens et al. 2001). AR itself has also been shown to be acetylated by the coactivators CBP/p300, p/CAF, and TAT-interactive protein (Tip60) (Fu et al. 2000; Gong et al. 2006; Gaughan et al. 2002). Acetylation of AR at the KXKK motif (amino acids 630–633) increases coactivator protein interactions and enhances AR transactivation (Fu et al. 2003). Also, members of the SRC family, such as SRC2, can be recruited simultaneously with CBP/p300 by AR to the prostate-specific antigen (PSA) promoter/enhancer for activation of transcription (Black et al. 2004; Kim et al. 2005). The switch/sucrose nonfermentable (SWI/SNF) multiprotein complex, which is able to perturb the conformation of the nucleosome by diminishing the interaction between DNA and histones, can also enhance transcription of AR at the PSA promoter (Marshall et al. 2003). The normal N/C-terminal interaction of AR is required to recruit the SWI/SNF complex, which in turn remodels chromatin to allow AR to bind to androgen response elements (Li et al. 2006). Both SWI/SNF
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and CBP/p300 HAT activity may be required for androgen-dependent activation of AR. SWI/SNF can be targeted to chromatin by CBP/p300, which itself is recruited through interaction with SRC coactivators (Huang et al. 2003). Thus, multiple cofactors required for activation are not always recruited through direct interactions with steroid receptors but may involve a coordination of cofactor–cofactor interactions (Marshall et al. 2003; Chiba et al. 1994; Ratajczak 2001; Lambert and Nordeen 2003).
1.2.3
Role of Basal Transcriptional Machinery in Coactivation of AR
The transcriptional activation of steroid receptors ultimately requires the recruitment of RNA pol II to the promoter of target genes (Roeder 1996). RNA pol II is recruited through the assembly of the transcriptional preinitiation complex, formed by general transcription factors (GTFs). A large preinitiation complex consisting of three major components (RNA pol II, GTFs, and mediator), composed of 58 subunits, assembles before initiation of transcription occurs (Kornberg 2007). Coactivators of steroid receptors, such as SRC1, CBP/p300, and p/CAF, can also exert their activities by facilitating communication between the receptor and basal transcriptional machinery (Heinlein and Chang 2002). SRC1 interacts with TATAbinding protein and TFIIB and also recruits CBP/p300 to the transcriptional start site (Takeshita et al. 1996). In turn, CBP/p300, along with p/CAF, can bind directly with subunits of RNA pol II. AR can also interact directly with GTFs, such as TFIIF and TFIIH, through its NTD (McEwan and Gustafsson 1997; Lee et al. 2000). GTFs are not considered coregulator proteins since they can alter the basal transcription rate (McKenna et al. 1999). Analyses of AR-mediated transcription suggest that the orchestrated interaction of AR with TFIIF and TFIIH may increase efficiency of transcriptional elongation from androgen target genes, such as PSA (Lee and Chang 2003). AR may regulate transcription by enhanced assembly of the transcriptional initiation complex, by regulating promoter clearance, and during the elongation step of transcription (Lee and Chang 2003; Choudhry et al. 2006). Enhanced communication of AR with the basal transcriptional machinery, through other coactivators or direct GTF interactions, allows for more efficient transcription and an additional level of control in regulating AR activity.
1.3 1.3.1
Type II Nonclassical Coactivators of AR Role of Molecular Chaperones in Coactivation of AR
Chaperones prevent the irreversible aggregation of unfolded or partially folded proteins through recognition of and binding to their hydrophobic regions (Bukau and Horwich 1998). Although, chaperone proteins are not usually designated as coactivators, they interact with steroid receptors and modulate their activity. In the
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absence of ligand in the cytoplasm, the structural conformation of the AR-LBD necessary for receptor activation is preserved by multiple cycles of binding and release with components of a multichaperone complex, which consist of heat-shock proteins (hsp) and cochaperone molecules (Prescott and Coetzee 2006). The minimal complex believed crucial for ligand-responsive signaling of steroid receptors consists of hsp70 (hsc70), hsp40 (ydj1), hop (p60; hsp organizer protein), hsp90, and p23 (Prescott and Coetzee 2006; Pratt and Toft 1997; Dittmar et al. 1998; Kosano et al. 1998). The interaction of AR with these proteins can maintain the receptor in a stable, partially unfolded conformation that is primed for high-affinity binding with androgens (Bohen et al. 1995). Upon binding of AR to ligand, the chaperone heterocomplex mediates trafficking of the receptor into the nucleus, possibly by facilitating interaction with dynein, a cytoplasmic protein that can drive active nuclear transport of the receptor along the cytoskeleton (Owens-Grillo et al. 1996; Davies et al. 2002). Hsp70, hsp40 (Rassow et al. 1995; Caplan and Douglas 1991), and hsp27 (Zoubeidi et al. 2007) have also been implicated in receptor translocation across intranuclear membranes. Within the nucleus, the chaperone complex dissociates from AR, but can still modulate receptor activity by promoting the dissociation of androgen from the receptor that returns AR to a primed state, which can be reactivated if ligand becomes available (Chmelar et al. 2007). However, in the absence of nuclear androgen, AR action is rapidly terminated and the receptor is degraded through the ubiquitin–proteosome pathway (Prescott and Coetzee 2006).
1.3.2
Coactivators of AR that Modulate Ligand Binding and Receptor Stability
The stability of AR protein depends on the ability of the AR-LBD to bind ligand (Krongrad et al. 1991). Binding of androgen to AR requires the proper folding of the receptor, which is regulated by the chaperone heterocomplex (see Sect. 1.3.1). Ligand binding induces the formation of an AF-2 coregulator-binding site (Dehm and Tindall 2007). Upon ligand binding, AR dimerizes, allowing the N- and C-termini of the receptor to interact. The occurrence of the N/C-terminal interaction of AR can result in decreased ligand dissociation and increased AR protein stability (Heinlein and Chang 2002). Hence, coactivators that can alter the folding of AR protein, receptor ligand binding, or the ability of the N- and C-termini to interact can potentially regulate AR transcriptional activity. Several proteins have been shown to coactivate AR transactivation through these mechanisms. A component of the hsp70 chaperone complex, Bcl-2-associated athagene-1 (BAG-1L), can increase AR transcription by interacting with the NTD and LBD of the receptor to facilitate proper folding (Shatkina et al. 2003; Froesch et al. 1998). Hsp90 can participate in the activation of AR by maintaining the receptor in a high-affinity ligand-binding conformation (Fang et al. 1996). The serine/threonine kinase AR-interacting nuclear protein kinase (ANPK) interacts with the AR-DBD and enhances AR-dependent transcription through stabilization
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of AR protein levels (Moilanen et al. 1998). The most well-established coactivator of AR that modulates receptor function through stabilization of the ligand-bound receptor is ARA70 (Yeh and Chang 1996; Miyamoto et al. 1998). ARA70 has been implicated in changing the conformation of cytosolic AR so that it binds and/or retains androgen more easily, which leads to a faster rate of nuclear translocation (Hu et al. 2004). Furthermore, estrogen has been shown to activate AR and ARA70 retards the dissociation of estrogen from AR (Hu et al. 2004). ARA70 is a key example of how coactivators can regulate AR activity in the cytoplasm, prior to or during nuclear translocation. L-dopa decarboxylase (DDC) is another cytosolic AR-interacting protein that enhances receptor transactivation, possibly through modulating receptor androgen-binding ability (Wafa et al. 2003).
1.3.3
AR Coactivators Involved in Cellular Trafficking of the Receptor
The transactivation of steroid receptors can also be enhanced by coactivators that modulate cellular transport of the ligand-bound receptor. Both an increase in the rate of nuclear translocation and retention of AR in the nucleus can result in higher levels of transcription. Major structural components of cells such as filamentous actin (f-actin) play an important role in the cellular trafficking of proteins along the cytoskeleton (Stossel et al. 1985). Proteins that bind to f-actin mediate the process of actin-bundling, which influences cellular morphology and regulates actin polymerization and depolymerization. Several actin-binding proteins have been shown to alter AR function, including filamin, supervillin, and gelsolin (Ozanne et al. 2000; Nishimura et al. 2003; Ting et al. 2002). The f-actin cross-linking protein filamin interacts with the hinge domain of AR and facilitates receptor nuclear translocation (Ozanne et al. 2000). However, paradoxically, this protein has also been implicated in repression of AR transactivation (Loy et al. 2003). Supervillin binds directly to the NTD and DBD of AR, while gelsolin interacts with the AR-DBD and LBD (Nishimura et al. 2003; Ting et al. 2002); both increase AR transactivation. The association of AR with these actin-binding proteins is thought to serve as a mechanism by which the receptor can migrate along the cytoskeleton. The scaffolding protein, caveolin-1, is a principal component of caveolae membranes involved in many signal transduction pathways and is also another coactivator of AR that modulates transport of AR into the nucleus (Lu et al. 2001). High levels of caveolin-1 expression result in increased AR transactivation and nuclear localization of phosphorylated AR (Li et al. 2003). In contrast to the mechanism of AR coactivation by caveolin-1, Ras-related nuclear G-protein (Ran)/ARA24 has been suggested to enhance AR transcription by maintaining higher receptor levels in the nucleus (Heinlein and Chang 2002; Hsiao et al. 1999). In addition, a Ran-binding protein, RanBPM (ran-binding protein in the microtubule organizing center), has also been shown to enhance AR transcription (Rao et al. 2002b). Ran/ ARA24 is responsible for the nuclear export of importin proteins, which interact with nuclear localization signal-containing cargo proteins to dock protein complexes onto nuclear pores and allow nuclear import (Dasso 2001). Hence, an
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Fig. 2 Type II androgen receptor (AR) coactivators. Type II/nonclassical coactivators can act at many stages of AR activation. These include modulation of AR folding by hsp chaperones, alteration of receptor ligand-binding capacity and AR protein stability in the cytosol, and influencing the trafficking of AR to the nucleus along the cytoskeleton. l dihydrotestosterone
increase in levels of Ran/ARA24 may result in rapid return of importins to the cytoplasm that in turn may result in more efficient translocation of proteins into the nucleus (Heinlein and Chang 2002). Figure 2 illustrates the various stages of AR activation at which type II coactivators can modulate receptor function.
1.4
Type I Classical and Type II Nonclassical Corepressors of AR
While the majority of AR coregulators identified have been coactivators, in recent years several corepressor proteins have been reported. The mechanisms by which corepressors suppress AR transcription seem to be as variable as that of coactivators. These include classical/type I histone deacetylase (HDAC) recruitment and nonclassical/type II mechanisms such as inhibition of the formation of active nuclear AR, inhibition of AR intermolecular interactions, or AR binding to coactivators and other unique processes (Wang et al. 2005). In addition to coactivators, corepressors may also be critical for regulation of AR transcription in a precise and efficient manner. A partial list of AR corepressors discussed here is provided in Table 3.
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HDAC Recruitment (Type I)
The acetylation status of histones plays a critical role in the regulation of transcription as discussed previously for coactivators (Sect. 1.2). In contrast to coactivators that utilize HAT activity to disrupt chromatin structure and activate transcription, histone deacetylation of core histones can restabilize chromatin to suppress transcription by nuclear receptors (Xu et al. 1999). Silencing mediator for retinoid and thyroid hormone receptors (SMRT) corepressor and nuclear receptor corepressor (NcoR) are the best characterized nuclear receptor-binding proteins that suppress AR transcription, presumably through recruitment of HDACs and by competing with coactivators for interaction with the receptor (Wang et al. 2005). SMRT has been shown to interact with both the NTD and the LBD of AR (Dotzlaw et al. 2002; Liao et al. 2003), while NCoR only binds to the AR-LBD (Cheng et al. 2002). Moreover, HDAC1 has been shown to bind the AR-LBD/DBD and downregulate AR transcription (Gaughan et al. 2002). Recently, small interference RNA used to knock down SMRT and NCoR in prostate cancer cells enhanced the recruitment of the coactivators SRC1 and CBP/p300 by ligand-bound AR, which suggests that these corepressors can compete with coactivators for binding to the active receptor (Yoon and Wong 2006).
1.4.2
Inhibition of AR Nuclear Translocation and DNA-Binding (Type II)
AR can be targeted by corepressor proteins at multiple stages during receptor activation. DAX-1 (dosage-sensitive sex reversal adrenal hypoplasia congenita critical region on the X chromosome gene 1) corepressor interacts directly with the AR-LBD and sequesters the receptor in the cytoplasm (Holter et al. 2002). Calreticulin, a calcium-binding protein, has been shown to bind directly to the AR-DBD and thereby inhibit AR binding to androgen response elements and reduce receptor transactivation (Dedhar et al. 1994).
1.4.3
Disruption of AR N-/C-Terminal Interaction and Coactivator Binding (Type II)
The N-/C-terminal interaction of AR plays an important role in stabilizing AR protein and facilitating androgen binding for enhanced transactivation (Heinlein and Chang 2002). Glycogen synthase kinase 3b (GSK3b) is a serine/threonine kinase that phosphorylates a wide range of substrates, including the NTD of AR (Wang et al. 2004). Through direct binding to the AR-NTD, GSK3b is thought to suppress AR transcription by inhibiting the N-/C-terminal interaction of the receptor. The tumor suppressor protein p53 also can function as a corepressor of AR through disruption of the N-/C-terminal interaction, which may inhibit receptor homodimerization (Shenk et al. 2001).
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Several corepressors have been found to alter AR transactivation through inhibiting AR association with coactivators. Cyclin D1, known to phosphorylate the cellular proliferation promoting Rb (retinoblastoma) protein, binds AR via its hinge domain and suppresses transactivation activity, possibly by disrupting association of AR with the p/CAF coactivator (Reutens et al. 2001; Knudsen et al. 1999a, 1999b). More recently, cyclin D1 has been shown to bind HDAC3, which suggests a role for this corepressor in chromatin remodeling (Petre-Draviam et al. 2005). Short heterodimer partner (SHP) is an orphan nuclear receptor that can repress AR transcriptional activity through competition with the SRC2 coactivator for binding to the receptor (Gobinet et al. 2001). More recently, SHP has also been shown to inhibit AR transactivation via recruitment of HDAC1 (Gobinet et al. 2005). The role of some AR corepressor proteins, such as Akt, is complex and controversial. Akt, an oncoprotein, is a serine/threonine kinase that plays a critical role in the phosphatidylinositol 3-kinase (PI3K)-mediated pathways. While some studies have shown Akt to be a positive modulator of AR transcriptional activity (Wen et al. 2000; Lin et al. 2003; Gregory et al. 2005), others have reported that Akt suppresses AR transactivation by binding and phosphorylating the receptor as well as inhibiting the interaction between AR and the coactivator ARA70 (Lin et al. 2001; Taneja et al. 2005).
1.4.4
Other Corepressors of AR (Type II)
Several other corepressors of AR have been reported, although the mechanism by which these corepressors suppress AR transactivation is not clear. PTEN (phosphatase and tensin homologue deleted on chromosome 10) tumor suppressor protein can interact with AR, inhibit nuclear translocation and promote AR protein degradation, which results in inhibition of receptor transactivation (Lin et al. 2004). HBO1 (histone acetyltransferase binding to the origin recognition complex-1 subunit protein) also binds to the DBD and LBD of AR to repress receptor transcriptional activity (Sharma et al. 2000). Recently, the NcoR Alien (Dressel et al. 1999) was shown to bind to the N-terminus of AR and inhibit endogenous PSA expression in LNCaP human prostate cancer cells (Moehren et al. 2007). RNA splicing factors, such as PSF (polypyrimidine tract-binding (PTB)-associated splicing factor), p54 nuclear RNA-binding protein (Dong et al. 2007), and COBRA1 (cofactor of BRCA1) (Sun et al. 2007), have also been shown to bind to AR and inhibit its transactivation.
1.5
Coregulator Involvement in Nongenomic and Rapid Signaling Events of AR
Addition of androgens to prostate cancer cells can result in activation of signaling pathways in a time frame too rapid to be accounted for by transcriptional activation of genes and thus has been termed nongenomic AR activity (Freeman et al. 2005).
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The criteria for nongenomic actions are rapid onset (within minutes), resistant to inhibitors of RNA and protein synthesis, and activated at physiological concentrations of androgens (Boonyaratanakornkit and Edwards 2007). For AR to be involved in nongenomic events, it must be located outside the nucleus. There appear to be two types of receptors involved in nongenomic signaling. One is a putative membrane steroid receptor, which can bind to nonpermeable analogues such as bovine serum albumin-bound testosterone, to increase intracellular calcium, activate PI3K, phosphorylate focal adhesion kinase, and rearrange actin (Papakonstanti et al. 2003; Lyng et al. 2000; Kampa et al. 2002). However, the identity of this androgen-binding membrane protein remains unknown (Boonyaratanakornkit and Edwards 2007). The other type of receptor involved in nongenomic signaling is the bona fide AR. Cinar et al. (2007) have reported a subpopulation of AR found in lipid rafts in LNCaP cell membranes. The most likely mechanism for AR membrane localization is palmitoylation of the highly conserved nine-amino acid motif in the ligand-binding domain of AR (Pedram et al. 2007). Whether cytosolic AR physically associates with the cell membrane or is retained by a complex of proteins, addition of androgens leads to the AR associating with signaling proteins and rapid Src tyrosine kinase, mitogen-activated protein kinase (MAPK), and PI3K nongenomic steroid signaling (Boonyaratanakornkit and Edwards 2007). The AR coregulator PELP-1 (proline-, glutamic acid-, and leucine-rich protein-1), also known as MNAR (modulator of nongenomic activity of the estrogen receptor), facilitates AR interactions with cytoplasmic signaling components by forming a complex with Src-AR (Unni et al. 2004). PELP-1 also interacts with coregulators CBP/p300, cyclin D1, Rb (Vadlamudi and Kumar 2007), and FHL2 (four and a half LIMdomain protein 2) (Nair et al. 2007). Rapid signal transduction pathways can also activate AR in the absence of androgens or under androgen-deprived conditions (Edwards and Bartlett 2005a). Growth factors, cytokines, and neuropeptides can activate AR through the MAPK, janus kinase/signal transducers and activators of transcription (JAK/STAT), and PKA/PKC (protein kinase A and C) pathways, respectively [see recent reviews by Edwards and Bartlett (2005a, 2005b); Lange et al. (2007); and Weigel and Moore (2007)]. In addition, some of these pathways can also activate AR coregulators (Edwards and Bartlett 2005b; Lange et al. 2007; Weigel and Moore 2007; Gioeli 2005) (Fig. 3). Epidermal growth factor (EGF) activation of MAPK results in the phosphorylation of the coactivators SRC1 (Edwards and Bartlett 2005b) and SRC2 (Gregory et al. 2004). EGF also increases AR coactivator SRC2 (Gregory et al. 2004) and activated CREB (cAMP response element-binding protein), which increases ART-27 (Nwachukwu et al. 2007) expression. Levels of the AR corepressor cyclin D1 are increased by MAPK (Lange et al. 2007), and the growth factor, heregulin (Casimiro et al. 2007). Interleukin-6 cytokine activation of AR requires coactivators p300 (Debes et al. 2002) and SRC1 (Ueda et al. 2002). Src is a coactivator of AR and transiently phosphorylates the receptor at Tyr534 in response to EGF (Guo et al. 2006; Kraus et al. 2006). Interleukin-6 (Guo et al. 2006) and the neuropeptide bombesin (Desai et al. 2006) can also activate AR via Src. Moreover,
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Fig. 3 Coregulator involvement in androgen receptor (AR) rapid signaling events. AR can be activated through signal transduction pathways that involve cytokines, growth factors, and neuropeptides. Only those pathways involving AR coregulators are depicted. Neuropeptide activation of PKA/PKC signaling can act upon AR itself or on coregulators through cross talk with the MAPK pathway. Growth factors activate MAPK, which results in activation of coactivators SRC1 and SRC2. Cytokine activation of AR requires the coactivators SRC1 and p300. Addition of androgens also leads to AR associating with Src tyrosine kinase and a rapid activation of both AR and Src, which can also activate the MAPK pathway. Boxes represent signal transduction proteins and circles depict AR coregulators. Arrows indicate connections but not necessarily direct interactions. l dihydrotestosterone
the Erk-2 (extracellular signal-regulated kinase) MAPK can phosphorylate AR at Ser514, which results in increased interaction with coactivator proteins (Yeh et al. 1999; Bakin et al. 2003; Rochette-Egly 2003; LaFevre-Bernt and Ellerby 2003).
1.6
Summary
AR activity is regulated by an ever enlarging number of coactivators and corepressors. However, the relative in vivo importance of modifying AR function via these coregulators and the relevance to pathological conditions remains to be demonstrated. The expression profile, tumor cell growth effects, and therapeutic potential of a number of AR coregulators in prostate cancer have been reported and are discussed in Sect. 2.
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2 AR Coregulators in Prostate Cancer 2.1
Association of AR Coactivators with Prostate Cancer
Since coactivators enhance AR function in vitro, their expression may be deregulated in prostate cancer to provide a growth advantage. Recently, increased SRC1 expression was associated with increased tumor aggressiveness in clinically localized prostate cancer (Agoulnik et al. 2005). In the same study, reduction of SRC1 expression significantly reduced growth of androgen-sensitive LNCaP prostate cancer cells, whereas this had no effect on growth of AR-negative PC-3 and DU145 human prostate cancer cell lines. SRC3 has also been shown to be required for proliferation of androgen-sensitive prostate cancer cells and for tumor growth through controlling the expression of key cell-cycle genes (Zou et al. 2006). Moreover, SRC3 is overexpressed in prostate cancer patients and its expression correlates inversely with apoptosis of tumor cells (Zhou et al. 2005). The expression profile and tumor cell growth effects of several other AR coactivators have been reported. For example, CBP/p300 expression has been found to be upregulated during androgen deprivation therapy in patients with prostate cancer (Comuzzi et al. 2004). Knockdown of CBP/p300 transcripts using small interference RNA inhibits prostate cancer cell proliferation (Debes et al. 2003). The microRNA miR-26a for which CBP is a predicted target (Volinia et al. 2006) is also downregulated in prostate cancer (Mattie et al. 2006; Porkka et al. 2007), which could lead to increased expression of this coactivator. The core subunit of the SWI/SNF coactivator that mediates direct interactions with AR has been shown to be involved in the proliferation of AR-sensitive prostate cancer cells (Link et al. 2005). Expression of the ARA70 type II coactivator has been reported to increase in high-grade prostate cancer and in androgen-deprived prostate cancer cells (Hu et al. 2004; Chang et al. 2005). The AR cytosolic coactivator and neuroendocrine marker, DDC, has been shown to increase in expression with neo-adjuvant androgen deprivation therapy of prostate cancers (Wafa et al. 2007). Immunostaining of the AR chaperone hsp27 increases in androgen-independent (AI) tumors compared to untreated prostate cancer (Rocchi et al. 2004). In addition, expression of another AR coactivator, gelsolin, has been found to increase in LNCaP xenografts and human prostate cancers after androgen deprivation therapy (Nishimura et al. 2003). Recently, Tip60 AR coactivator was shown to increase in nuclear accumulation in 87% of AI prostate cancer specimens compared to benign prostatic hyperplasia (BPH) samples (Halkidou et al. 2003). Immunohistochemically, PELP-1 has been shown to increase in high-grade tumors (Nair et al. 2007) and HIP-1 (huntingtin interacting protein 1), a novel AR coactivator that prevents receptor protein degradation in prostate cancer cells (Mills et al. 2005), also increases in prostate cancers (Rao et al. 2002a). The above studies suggest that coactivators of AR not only facilitate receptor transactivation in vitro but also promote cell growth and play a crucial role in activation of AR in clinical prostate cancer.
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Association of AR Corepressors with Prostate Cancer
Although effects of expression of the original AR corepressors SMRT and NCoR in prostate cancer are not clear, their ability to inhibit activated AR in prostate cancer cells suggests that loss of expression of these corepressors could facilitate tumor growth (Chmelar et al. 2007; Miyamoto et al. 2004). On the other hand, expression of cyclin D1 has been found to inhibit cell-cycle progression specifically in AR-dependent prostate cancer cells, which suggests that loss of this corepressor may promote proliferation of these cells (Burd et al. 2006b). However, the microRNA miR-20, whose predicted targets include cyclin D1 (Volinia et al. 2006), has been found to be downregulated in prostate cancer (Mattie et al. 2006). Indeed, Kaltz-Wittmer et al. (2000) found that the cyclin D1 gene was amplified in 25–37% of prostate cancer patients and Drobnjak et al. (2000) found cyclin D1 protein overexpression in metastatic relative to primary prostate cancer. While Akt has been shown to directly suppress AR activity, activation of AR by Akt through the PI3K-mediated pathway has been implicated in prostate cancer progression. Increased Akt activity can synergize with AR signaling to promote initiation and progression of prostate cancer to androgen independence in xenograft prostate cancer tumor models (Graff et al. 2000; Xin et al. 2006). Analysis of human prostate cancer tissues has demonstrated that, although there is neither Akt gene amplification nor enhanced protein expression in prostate cancer compared to normal prostate tissue, poorly differentiated tumors exhibit increased expression of a phosphorylated (activated) form of Akt compared to normal prostate tissue. This activated Akt may promote AI survival of prostate cancer cells through the activation of AR (Ghosh et al. 2003). The tumor suppressor proteins p53 and PTEN, both of which corepress AR activity, have been well documented to be mutated or lost in prostate cancer. PTEN is a critical regulator of prostate cancer growth and progression (Burd et al. 2006a). In fact, loss of PTEN, which occurs at a frequency of approximately 20–27% in prostate cancer, is associated with a decrease in survival rates (McMenamin et al. 1999; Halvorsen et al. 2003). It has also been shown that prostate-specific deletion of PTEN induces metastatic prostate cancer (Wang et al. 2003). Since the ability of PTEN to induce apoptosis in prostate cancer cells is reversible with androgen treatment, PTEN loss may result in enhancement of AR transactivation and resistance to cell death in prostate cancer cells (Li et al. 2001). Nevertheless, because PTEN plays an important role in regulation of many other cell survival pathways, such as PI3K/Akt, one cannot definitively conclude that its effects on prostate cancer growth are entirely due to loss of the PTEN corepressor function for AR (Paez and Sellers 2003). Mutations and loss of the p53 tumor suppressor protein are also crucial in cancer development and progression (Burd et al. 2006a). In prostate cancer, approximately 45% of tumors have been found to contain p53 mutations (Shi et al. 2004). It has been reported that there is a balance of AR and p53 expression during the androgenstimulated growth of prostate cancer, but this is lost during further progression
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(Cronauer et al. 2004). Hence, similar to PTEN, it is tempting to speculate that loss of p53 function results in increased tumor growth, predominantly through an elevation in AR activity due to loss of corepressor function. However, it is difficult to separate this effect from the normal tumor suppressor role of p53. Nevertheless, it is possible that loss of AR corepressors, such as PTEN and p53, is at least in part responsible for increased activation of AR and subsequent enhancement of prostate cancer growth.
2.3
Expression Profile of AR Coregulators Associated with Progression of Clinical Prostate Cancer
Increasing data suggest that conventional androgen deprivation therapy does not completely eliminate intracellular androgens from prostate cancer cells (Mohler et al. 2004; Mostaghel et al. 2007a). After 9 months of androgen deprivation therapy, approximately 25% of the precastration androgen levels remain in prostatic tissues (Mostaghel et al. 2007b). Since coactivators increase AR transcriptional activity, their overexpression can potentially promote progression of prostate cancer by sensitizing the receptor to lower concentrations of androgens (Heinlein and Chang 2004). Moreover, the simultaneous loss of corepressor expression could further add to the unopposed AR activation effect of coactivators. Lonard et al. (2007) examined the ONCOMINE expression profiling metaanalysis database (www.oncomine.org) and found 117 overexpressed and 86 underexpressed coregulators in prostate cancer samples. In addition, they reviewed the literature and cite ten coregulators that were upregulated (ARA55, CARM1, Daxx, PDEF, PIAS1, RAF1, SENP1, SRC1, TSG101, and VAV3). Heemers and Tindall (2005) reviewed publications of protein expression in prostate cancer samples and found nine upregulated coactivators (SRC1, SRC2, SRC3, CBP, p300, Tip60, CARM-1, b-catenin, and ARA70). However, Chmelar et al. (2007) analyzed a number of studies that compared steady state levels of coregulator transcripts and found a lack of concordance due to variability in methods of analysis and heterogeneity of the samples. To assess the role of coregulators in prostate cancer progression to androgen independence, the literature was reviewed. A total of 18 studies analyzed clinical prostate cancer sample coregulator protein levels immunohistochemically and included samples from patients treated by androgen deprivation therapy. Heterogeneity of disease even within the same patient (Shah et al. 2004), different treatments (Chmelar et al. 2007), quality of tissue and availability, and quality of antibodies all combined to make evaluation of these studies difficult. As shown in Table 4, most were coactivators and most were overexpressed in AI prostate cancer. The only corepressor that also increased in AI prostate cancer was cyclin D1, which may be a consequence of its role in cell-cycle progression as opposed to that of an AR corepressor. Moreover, other immunohistochemical studies of prostate cancer
Androgen Receptor Coregulators and Their Role in Prostate Cancer Table 4 AR coregulators altered in clinical prostate cancer Name Number of patients Alteration ARA55 6 paired PCa and AI 2 patients had increase in AI disease BAG-1 32 untreated, 18 Increased nuclear preoperative staining for AI hormone treatment disease BAG-1L 190 untreated, 73 AI Increased nuclear staining for AI disease CARM1 66 untreated, 12 AI Increased staining (Coactivatorfor AI disease associated arginine methyltransferase 1) CBP 0 untreated, 26 AI High nuclear expression Cyclin D1 86 PCa (primary), Increased staining 22 AI bone for AI disease metastases DDC 21 PCa, 21 < 3 months Increased with >6 ADT, 28 3–6 months ADT and months ADT, in AI disease 28 > 6 months ADT, 13 AI DJ-1 21 PCa, 21 < 3 months Increased in patients ADT, 28 3–6 with >6 months months ADT, ADT 28 > 6 months ADT GAK 21 PCa, 49 6 months Increased with ADT ADT, 28 > 6 and in AI disease months ADT, 13 AI Gelsolin 11 PCa, 16 androgen Higher expression deprivation with ADT HIP-1 114 PCa (localized), Higher expression 14 AI metastatic with ADT hsp27 35 PCa, 58 < 3 months Increased with ADT ADT, 52 3–6 and in AI disease months ADT, 57 > 6 months ADT, 30 AI SRC1 205 PCa, 49 AI Increased staining in AI disease but not significant SRC1, SRC2 8 BPH, 8 PCa, 8 AI More intense in AI disease SRC2 518 total PCa samples More intense in AI disease including 9 AI Tip60 10 BPH, 43 PCa, 15 AI
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References Fujimoto et al. 2007
Krajewska et al. 2006 Maki et al. 2007
Hong et al. 2004
Comuzzi et al. 2004 Drobnjak et al. 2000
Wafa et al. 2007
Tillman et al. 2007
Ray et al. 2006
Nishimura et al. 2003 Rao et al. 2002a Rocchi et al. 2004
Maki et al. 2006
Gregory et al. 2001 Agoulnik et al. 2006 Halkidou et al. 2003 (continued)
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Table 4 (Continued) Name Number of patients
Alteration References Increased nuclear staining in AI disease (87%) vs untreated tumors (37%) aSGT (small 30 PCa, 54 metastatic AR/aSGT ratio Buchanan et al. 2007 glutamine-rich increased in tetratricopeptide metastatic vs PCa repeat and in AI versus containing nontreated protein) metastatic cochaperone disease b-Catenin 21 AI metastatic 24% (5/21) positive Chesire et al. 2002 for nuclear staining This list of coregulators is based on studies of immunohistochemically stained clinical samples including patients treated with androgen deprivation therapy PCa untreated prostate cancer, AI androgen-independent, ADT androgen deprivation therapy and BPH benign prostatic hyperplasia, GAK cyclin G-associated kinase
have shown that the coactivators SRC3 (Gnanapragasam et al. 2001), b-catenin (Aaltomaa et al. 2005; Assikis et al. 2004), caveolin-1 (Karam et al. 2007), lysinespecific histone demethylase-1, and FHL2 (Kahl et al. 2006) all predict aggressive prostate cancer. These studies indicate that dysfunctional expression of coregulators may play a critical role in progression of prostate cancer to AI disease. Although several coregulators have been shown to be upregulated in prostate cancer (Heemers and Tindall 2005; Culig and Bartsch 2006) (Table 4), whether these proteins contribute to prostate cancer progression remains unclear.
2.4
In Vivo Functional Studies of AR Coregulators in Prostate Cancer
There are only a few examples where a functional role for coregulators in prostate cancer has been demonstrated in animal models. PTEN is one of the best examples of this type of in vivo analysis. Prostate-specific PTEN null mice spontaneously develop prostate cancer (Sect. 2.2) (Wang et al. 2003). Studies with knockout mice (Lonard et al. 2007) have shown Zimp10 (a novel PIAS type coregulator) (Beliakoff and Sun 2006), p300 (Yao et al. 1998), ARIP4 [androgen receptor (AR)interacting protein 4] (Zhang et al. 2007), and NCoR (Hermanson et al. 2002) are all embryonically lethal, which suggests that they have a critical role in early development. Fkbp52 chaperone protein (the protein product of FKBP4)-null
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mice have defective development of the prostate (Yong et al. 2007), which implies an essential physiological role for AR-mediated signaling. Moreover, induced overexpression of DDC in LNCaP xenografts increased PSA production, which demonstrates an in vivo coactivation effect of this AR-binding protein (Wafa et al. submitted for publication). Activated ErbB-2-induced cyclin D1 expression in the mouse prostate is associated with prostate intraepithelial neoplasia (Casimiro et al. 2007). Although SRC3 has been shown to be required for prostate cancer growth (Zou et al. 2006), SRC3-null mice show a reduction of IGF (insulin-like growth factor)/ Akt signaling in prostate glands (Yan et al. 2006), which may mean that the effects of SRC3 are not exclusively via AR but involve genes in the IGF/Akt pathway. However, SRC3-null TRAMP (transgenic adenocarcinoma of the mouse prostate) mice fail to progress to poorly differentiated adenocarcinoma, which supports the notion that SRC3 is required for tumor progression (Chung et al. 2007). Disruption of SRC1 in mice results in decreased growth and development of the prostate, but knockout of SRC2 has no effect on the gland (Xu et al. 1998; Gehin et al. 2002; Mark et al. 2004). The single and combinatorial knockout targeting of these coactivators suggest that they have partial functional redundancy in vivo, whereby the absence of one SRC can be compensated for by another family member or possibly by non-SRC coactivators.
2.5
Therapeutic Targeting of Coregulators in Prostate Cancer
One of the major stumbling blocks to prostate cancer treatment is being able to predict the aggressiveness of tumors. Specific coregulators may be useful as predictive markers. Moreover, since there is no prognostic test for the clinical outcome of androgen deprivation therapy, Ross (2007) postulated that focusing on coregulators may be predictive of AR pathway status and guide prostate cancer treatment. Since aberrant modulation of AR activity by coregulators has been suggested to be involved in progression of prostate cancer, some coregulators may serve as potential therapeutic targets (Heemers and Tindall 2005). A detailed understanding of the binding motifs of coregulators to AR and computational chemistry may allow design of agents that mimic binding surfaces of protein complexes to target protein–protein interactions (target biomotifs). The aim of this approach is to block coactivator and increase corepressor binding to AR (Fletterick et al. 2005) (Fig. 4). Migliaccio et al. (2007) created a ten-amino acid peptide that inhibited the AR–Src interaction, which reduced LNCaP xenograft growth. An alternative approach would be restoration of the function of a protein lost during cancer progression. For example, DOC-2/DAB2 (differentially expressed in ovarian cancer-2/disabled 2), which is expressed in normal prostate and primary prostate cancers but not in cell lines derived from AI cancer, has been shown to disrupt AR interaction with Src (Zhoul et al. 2005). Zhou et al. (2006) attached a proline-rich sequence derived from DOC-2/DAB2 to a cell-permeable
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Fig. 4 Targeting AR coregulators in prostate cancer. Depicted are three different types of therapies targeting AR coregulators. In clinical trial, are therapies targeting chaperones (hsp) to decrease protein levels of AR and Src kinase inhibitors to prevent activation of AR. Target biomotifs to AR–coregulator interactions, aimed at blocking coactivator or increasing corepressor binding, to decrease AR transcriptional activity are under development. ARR androgen responsive region, l dihydrotestosterone, bound or unbound dihydrotestosterone
peptide delivery system, which restored the function of DOC-2/DAB2 and resulted in suppression of androgen-induced growth of prostate cancer cells. Moreover, AZD0530, a dual Src/v-Abl (Abelson murine leukemia viral oncogene homologue) kinase inhibitor is in multicenter phase II clinical trials for a variety of malignancies, which include prostate cancer (Chang et al. 2007). Molecular chaperones of AR, such as hsp, have also been targeted to abrogate AR activity. hsp90 is a therapeutic target in prostate cancer clinical drug trials, where inhibition of hsp90 has been shown to lead to increased degradation of AR (Neckers and Ivy 2003; Harashima et al. 2005). In addition, a multicenter phase I clinical study is underway targeting hsp27 (ClinicalTrials.gov identifier NCT00487786). High-throughput functional screening of known drugs is also being pursued to identify coregulator targets. Estebanez-Perpina et al. (2007) assessed more than 55,000 compounds for their ability to inhibit AR AF-2/SRC2 LXXLL binding. They found three off-patent aspirin derivatives already approved for human use and two types of thyroid hormones that could block binding of SRC2. In addition, they discovered a previously unrecognized allosteric regulatory site (binding function-3)
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near the AF2 of AR, which could be a pharmaceutical target. Selective AR modulators (SARMs) are being designed to modulate coregulator recruitment (Gao and Dalton 2007). Targeting of a wide variety of classic and nonclassic AR coregulators, as part of combinatorial AR targeted therapy, may be efficacious in the treatment of advanced prostate cancer (Singh et al. 2006).
3 Conclusions The limiting factor in the survival of a patient with prostate cancer is the rate of progression to AI disease (Albertsen et al. 2005; Kent and Hussain 2003; Martel et al. 2003). In order to impact on mortality rates, prostate cancers that will rapidly progress to the AI stage must be identified and the AI phenotype must be prevented, delayed, or treated effectively. AR is a critical regulator of prostate cancer development and AI progression. Since recurrent AI prostate cancer cells generally retain AR activity, inhibition of AR function must be a central focus of treatment. Because of this importance of AR in prostate cancer, a plethora of studies have tried to identify specific coregulator proteins that modify AR action to allow growth in spite of therapeutic intervention. Coactivators of AR can contribute to aberrant AR activation under androgen-deprived conditions and can be predictive for aggressive prostate cancers. Moreover, loss of AR corepressor function may contribute to deregulated AR activity during prostate cancer progression. Hence, in addition to the targeting of AR directly and ligand-independent/ligand-reduced signaling pathways that activate the receptor, AR coregulator proteins may provide targets for therapy. Since numerous coregulators have been shown to modulate AR activity and since compensatory mechanisms of action exist, it is probably necessary to simultaneously target many of these receptor-binding proteins in order to achieve maximal suppression of AR activity. With the ability to target coregulators using new strategies and drugs that effectively shut down AR signaling, more effective treatment options may arise to improve the clinical outcome of patients with AI prostate cancer.
References Aaltomaa S. et al. 2005. Reduced alpha- and beta-catenin expression predicts shortened survival in local prostate cancer. Anticancer Res 25:4707–4712. Aarnisalo P. et al. 1998. CREB-binding protein in androgen receptor-mediated signaling. Proc Natl Acad Sci U S A 95:2122–2127. Albertsen P. C. et al. 2005. 20-Year outcomes following conservative management of clinically localized prostate cancer. JAMA 293:2095–2101. Alen P. et al. 1999. The androgen receptor amino-terminal domain plays a key role in p160 coactivator-stimulated gene transcription. Mol Cell Biol 19:6085–6097.
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Interaction of the Androgen Receptor Ligand-Binding Domain with the N-Terminal Domain and with Coactivators Jan Trapman
Abstract The ligand-binding domain of the androgen receptor not only binds ligands, but also contains a ligand-induced protein interaction surface, the cofactor-binding groove. The groove interacts with short amphipatic a-helices in cofactors composed of an FxxLF motif, or with LxxLL motifs at a lower affinity. Moreover, the cofactor-binding groove interacts with an FxxLF motif in the N-terminal domain of the androgen receptor. The groove is able to adapt its shape in complexes with interacting peptides. In the peptide motifs, an F at +1 seems essential for high-affinity binding. L+4 can be replaced by several other hydrophobic amino acid residues without losing activity. Although F at +5 has the highest activity, it can be substituted by tryptophane or tyrosine. Studies of the spatial and temporal distribution of the androgen receptor in the living cell indicates consecutive protein interactions, including intramolecular and intermolecular androgen receptor domain interactions and cofactor binding, depending on the cellular localization.
1 Introduction The structure of the ligand-binding domains (LBD) of essentially all nuclear receptors (NR) is composed of twelve a-helices, folded in a three-layered antiparallel sandwich conformation (Li et al. 2003). Ligands bind to a buried ligandbinding pocket formed by the cooperative action of specific amino acid residues scattered over many helices. The LBD is not only indirectly involved in transcription regulation by ligand binding, but also more directly, via the ligand-induced
J. Trapman Department of Pathology, Josephine Nefkens Institute, Erasmus Medical Center, PO Box 2040, 3000 CA Rotterdam, The Netherlands, E-mail:
[email protected]
D. Tindall and J. Mohler (eds.), Androgen Action in Prostate Cancer, DOI: 10.1007/978-0-387-69179-4_16, # Springer Science + Business Media, LLC 2009
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activation function 2 (AF-2). Upon binding of an agonistic ligand, the position of helix 12 (H12) in the LBD is shifted in such a way that it directly interacts with the ligand. The agonist-induced repositioning of H12 not only completes the formation of the ligand-binding pocket, but also generates a hydrophobic protein interaction interface at the LBD surface, the cofactor-binding groove (Greschik and Moras 2003). The agonist-induced cofactor-binding groove facilitates the binding of many NR coactivating proteins. Antagonists induce an unfavorable conformation of the groove, which prohibits binding of coactivators. Corepressors appear to bind in the same cofactor groove or an overlapping binding site. Many NR coactivators bind to the LBD via LxxLL motifs, the so-called NR boxes, where L represents a leucine and x any amino acid (McKenna and O’Malley 2002; Rosenfeld et al. 2006). These peptide motifs form small amphipatic ahelices. Amino acid residues flanking the leucines modulate the specificity and affinity of LxxLL motifs. Cofactors might contain one or more LxxLL motifs. Best studied in this regard are the members of the p160 (SRC) family of cofactors, which possess three or four NR boxes. One p160 cofactor can bind simultaneously to a NR homodimer or heterodimer. Although the LBD of the androgen receptor (AR) has high sequence homology with the LBDs of other NRs and folds in a similar manner, the affinity for LxxLL motifs is relatively low. Instead, the AR favors FxxLF-like motifs (F is phenylalanine) for high-affinity interactions. The unique properties of the AR cofactorbinding groove induce specificity to interacting proteins (Fig. 1). Interestingly, it facilitates also the interaction between an FxxLF motif in the AR N-terminal domain (NTD) with the LBD, which plays a role in intramolecular and intermolecular interaction between the LBD and NTD (AR N/C interaction). AF-2 function of AR is weaker than that of other NRs. In this chapter the characteristics of the interaction of FxxLF- and LxxLL-like motifs with the AR LBD are described. Moreover, the in vivo relevance of interaction of AR LBD with the NTD and with coactivators is discussed.
Cofactors
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Fig. 1 Schematic presentation of protein–protein interactions of the AR LBD. The AR LBD can interact with cofactors and with the NTD via FxxLF- and LxxLL-like amphipatic a-helix peptide motifs. NTD N-terminal domain, DBD DNA-binding domain, LBD ligand-binding domain
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2 Peptides Interacting with the Cofactor-Binding Groove of the Androgen Receptor Yeast two-hybrid protein interaction assays have identified many protein fragments that can interact with AR in a ligand-dependent fashion (Heinlein and Chang 2002; Heemers and Tindall 2007). For most of these proteins the mode of interaction and the functional relevance of the interaction remains unknown. For five interacting proteins, ARA54, ARA70, Rad9, Gelsolin, and PAK6, it has been shown that their high, ligand-induced affinity for the AR LBD is mediated by a specific peptide motif (He et al. 2002; Hu et al. 2004; Wang et al. 2004; van de Wijngaart et al. 2006). None of the interacting peptides is composed of LxxLL motifs. Instead, they are composed of short amphipatic a-helices with phenylalanine residues at positions +1 and +5. Yeast protein–protein interaction assays showed not only interaction of AR LBD with other proteins, but also binding between the AR LBD and its NTD (Doesburg et al. 1997). This interaction is mediated by an FxxLF motif in the NTD (He et al. 2000; Steketee et al. 2002). The primary structures of the FxxLF motifs in interacting proteins and in AR NTD are summarized in Table 1. ARA54, ARA70, Rad9, and AR NTD possess an FxxLF motif that interacts with AR LBD (He et al. 2000, 2002; Steketee et al. 2002; Hu et al. 2004; Wang et al. 2004). Recently, the interacting motif of Gelsolin was established as FxxFF, and that of PAK6 as FxxMF (van de Wijngaart et al. 2006). Phage display has been used to select randomly for interacting peptide sequences with high affinity for the AR LBD (Hsu et al. 2003; Hur et al. 2004; Chang et al. 2005). Large libraries have been screened for peptides that interacted with AR LBD in the presence but not in the absence of ligand, and the interacting motifs were characterized. Peptides that interact with high affinity are mainly composed of FxxLF, FxxLY, or FxxLW motifs. Peptides showing high affinity with a different amino acid residue than phenylalanine on position +1 are hardly detected. No highaffinity peptides with LxxLL motifs were reported. It is unclear why in many cases at +4 a leucine was found, considering the more flexible position of this amino acid residue (see later).
Table 1 Composition of peptide motifs in proteins interacting with AR LBD +4 +1+5 AR NTD KTYRGAFQNLFQSVRE ARA54 DPGSPCFNRLFYAVDV ARA70 RETSEKFKLLFQSYNV RAD9 TPPPKKFRSLFFGSIL Gelsolin GGETPLFKQFFKNWRD PAK6 SLKRRLFRSMFLSTAA
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3 The Structure of the Cofactor-Binding Groove of the Androgen Receptor Computer modeling, comparing the primary structure of AR and ER LBD, predicted that FxxLF motifs have a similar interaction with the cofactor-binding groove of AR as LxxLL motifs have with the cofactor groove of the estrogen receptor (ER) (Dubbink et al. 2004) (Fig. 2). However, the cofactor-binding groove in AR is much deeper than that of ER and many other NRs. This deep groove facilitates docking of the bulky F residues in the groove. It also explains the weaker binding of many LxxLL motifs to the groove, because important hydrophobic interactions between the peptide motif and the amino acid residues lining the groove in the LBD cannot take place. Like other NRs the AR LBD contains two highly conserved amino acid residues, K720 in H3 and E897 in H12, which form a ‘‘charge clamp’’ that stabilizes the interaction with the a-helical structure of peptide motifs. Crystallographic studies of ligand-activated AR LBD complexed with peptide motifs provided detailed information about the structure of the coactivator-binding groove in the AR LBD and of the interactions with peptide motifs (He et al. 2004; Hur et al. 2004; Estebanez-Perpina et al. 2005). Not unexpected, the L-shaped AR cofactor-binding groove is formed by amino acid residues in helices H3, H4, H5, and H12. F+1 in the peptide motif binds to a hydrophobic part of the groove that is formed by L712, V716, M734, I737, Q738, M894, and I898. The F+5 binding site is formed by V712, K720, F725, V730, Q733, M734, and I737. L+4 interacts with V713, V716, and M894.
K720 F+5 V716
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Fig. 2 Model of the cofactor-binding groove complexed with an FxxLF peptide. In the groove charge clamp amino acid residues, and amino acid residues interacting with L+4 are indicated. Note that F+1 and F+5 of the peptide are buried in the groove and that L+4 lies on an edge of the groove (See Color Insert)
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The coactivator groove is not a rigid structure, but it can adapt its form slightly upon peptide binding. Side chains of amino acid residues that line the coactivatorbinding groove rearrange upon binding of the peptide motif. The largest conformational changes were observed for K720, M734, M894, and E897. As expected, in FxxLF peptides F+1 and F+5 are directed to the AR LBD surface, whereas the amino acid residues at positions +2 and +3 are exposed to the solvent. L at +4 binds to a shallow hydrophobic depression and is partly solvent exposed (see Fig. 2). From the structure of the AR LBD complexed with an FxxLF peptide motif it can be predicted that substitutions of F+1 or F+5 are restricted, whereas position +4 in the peptide is more flexible. Crystal structures indicate that all interacting peptides bind in slightly different conformations to the coactivator groove. For some peptides it has been established clearly that flanking amino acid residues contribute to peptide binding. Differential binding was also deduced from functional studies. Mutations were introduced in the charge clamp residues K720 and E897. K720A and E897A substitutions both affect FxxLF interaction, but E897 is less important for interaction with some LxxLL motifs, due to the shifted position in the coactivator-binding groove (Dubbink et al. 2004; He et al. 2004; Hur et al. 2004; Estebanez-Perpina et al. 2005).
4 Mutational Analysis of Peptides Binding to the Coactivator Groove To study the role of individual amino acid residues the peptide motifs have been mutated extensively. First, mutational analysis of peptide motifs by alanine scanning of the AR FQNLF motif showed that not only the phenylalanine and leucine residues are essential for binding to AR LBD, but also that flanking amino acids modulate this interaction (He et al. 2000; Steketee et al. 2002). Similar findings were observed by alanine scanning of the FxxFF motif in Gelsolin and the FxxMF motif in PAK6 (van de Wijngaart et al. 2006). If F+1 and F+5 in the AR NTD FxxLF motif, FQNLF, were randomly mutated, none of the novel peptide motifs displayed a stronger interaction than the original FxxLF peptide (Dubbink et al. 2004). However, substitution of F+5 by M or W retained part of the activity. Substitution of F+1 by other amino acids resulted in inactive peptides, indicating that this amino acid residue is very important for highaffinity AR binding. Next, L+4 in the AR FQNLF motif was substituted by a complete panel of amino acids (van de Wijngaart et al. 2006). FQNFF and FQNMF had the same activity as the original peptide; all others interacted weakly or did not interact at all. This observation is in complete agreement with the structural data. Finally, the importance of Q+2 and N+3 in the AR FQNLF motif has been investigated by random mutagenesis (Dubbink et al. 2004). Many, but not all, amino acids can substitute Q and N without loss of activity. In general, an E at
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+2 and a charged or polar amino acid residue at +3, R,K, or Q, are preferred, probably because the stability of the amphipatic a-helical peptides is increased (Dubbink et al. 2006). Less is known about the preferred properties of amino acids flanking the FxxLF motif. Based on comparison of the composition of interacting peptides and limited mutation analysis, positively charged amino acids flanking F+1 seem preferred for stronger interaction (He and Wilson 2003). The optimal properties of amino acids flanking F+5 are not yet very clear. Complementary data came from F/L swapping experiments of FxxLF and LxxLL-based peptide motifs (Dubbink et al. 2006). Peptide interaction with AR LBD was systematically studied by substituting the phenylalanine residues by leucines in three FxxLF peptides, AR NTD, ARA54, and ARA70. In all three motifs, substitutions of F+1 and F+5 by leucine residues completely abrogated interaction with the AR LBD, clearly showing that in these motifs the bulky F+1 and F+5 are essential for interaction. Also, leucine residues at +1 and +5 were substituted with phenylalanine residues in nine LxxLL motifs, NR boxes of SRC1 and TIF2(SRC2) and the LxxLL peptides, D11 and D30 peptides (Dubbink et al. 2004, 2006). D11 and D30 were originally identified by phage display as highaffinity ER-binding peptides (Chang et al. 1999). The original LxxLL peptides showed variable interaction with AR LBD. Differential effects were observed for the L/F swaps. Binding of already interacting LxxLL motifs was unchanged or increased upon L to F substitutions. Interestingly, certain noninteracting LxxLL motifs turned into strong interacting peptides (SRC-1 NB-I and -IV); however, other inactive peptides remained inactive by L/F swaps. Mutational analysis of the AR LBD confirmed the essential role of K720 in interaction with FxxLF and LxxLL motifs. K717 and R726 modified this interaction in a peptide-specific manner. E897A substitution not only had less effect on LxxLL peptides, but also on some FxxLF peptides, extending previous observations that many peptides show different conformations if docked in the coactivator groove and that sequences flanking the core motif determine the specific mode of interactions. FxxLF peptides have also been assessed for interaction with LBDs of other NRs (Dubbink et al. 2006). Many NRs seem unable to interact with FxxLF peptides. Most information is available for the progesterone receptor (PR). L/F swapping indicates that PR LBD can interact with both LxxLL and FxxLF peptides; however, LxxLL interaction is clearly preferred above FxxLF. The obvious question is why the AR contains such a unique cofactor-binding site and why it contains an FxxLF motif in the NTD. Such questions remain to be answered. It has been shown that peptide binding can delay ligand dissociation; however, the effect is limited (He et al. 2000; Dubbink et al. 2004). It can be speculated that the combination FxxLF in AR NTD and a specific cofactor-binding groove in AR LBD protects the AR from unfavorable protein interactions by N/C interaction. Subsequently, the FxxLF motif in AR NTD and the cofactor-binding groove might be independently important for AR activity and specificity during tra
