[CANCER RESEARCH 61, 7310 –7317, October 1, 2001] Androgen Receptor Mediates the Reduced Tumor Growth, Enhanced Androgen Responsiveness, and Selected Target Gene Transactivation in a Human Prostate Cancer Cell Line 1 Bekir Cinar, Kenneth S. Koeneman, Magnus Edlund, Gail S. Prins, Haiyen E. Zhau, 2 and Leland W. K. Chung 2 Departments of Biochemistry and Molecular Genetics [B. C.], Cell Biology [L. W. K. C.], and Urology and the Molecular Urology and Therapeutics Program [B. C., M. E., H. E. Z., L. W. K. C.], University of Virginia School of Medicine, Charlottesville, Virginia 22908; Department of Urology, Southwestern Medical School, Dallas, Texas 75390 [K. S. K.]; and Department of Urology, University of Illinois, Chicago, Illinois 60612 [G. S. P.] ABSTRACT The growth and development of the prostate gland are regulated by the androgen and the androgen receptor (AR). Despite our molecular under- standing of the roles of the AR regulating; a downstream target gene transcription, the direct or indirect (stromally mediated) actions of the androgen in controlling prostate cell growth and differentiation are still unclear. In this report, an invasive; and metastatic human prostate tumor cell line, androgen-repressed human prostate cancer cell line (ARCaP), either transduced with wild-type human AR (hAR) or a control neomycin- resistant plasmid DNA, was used to evaluate the direct role of AR in regulating prostate tumor cell growth and gene transcription. Results showed that: (a) introduction of wild-type hAR to ARCaP cells restored positive androgen regulation of prostate tumor cell growth in vitro through an enhanced cell-cycle progression from G 0 /G 1 to S and G 2 -M phases; (b) hAR was shown to transactivate glucocorticoid-responsive element but not prostate-specific antigen promoter-directed reporter gene expression; and (c) hAR-transduced ARCaP cells exhibited reduced growth, invasion, and migratory behavior in vitro and tumor growth in vivo. These results suggest that the introduction of hAR into the invasive human prostate cancer ARCaP cell line restored its androgen-regulated cell growth, decreased the rate of tumor growth, and selectively activated AR target gene expression. These cellular functions in response to andro- gen are commonly associated with increased differentiation of prostate epithelial cells. INTRODUCTION Steroid hormones have long been thought important in the etiology of cancers from hormonally responsive tissues like prostate (1–3). The biological activities of steroid hormones are mediated by their specific receptors, namely androgen, estrogen, progesterone, glucocorticoid, and mineralocorticoid receptors, which are members of the nuclear receptor superfamily (4 – 6). All of the members of this family have a similar structural organization with a highly variable NH 2 -terminal transactivation domain, followed by a well-conserved DNA-binding domain consisting of two zinc finger DNA-binding motifs, a hinge region consisting of nuclear localization signals, and a moderately conserved COOH-terminal ligand-binding domain. Steroid receptors regulate the transcriptional activity of target genes by binding to the cognate DNA sequence of hormone response elements, followed by the initiation, assembly, and stabilization of the preinitiation complex in the region of the target gene promoter (7–13). Despite clear evidence that the regulation of gene expression by steroid hormones is mediated by the interaction of steroid hormone receptors and their respective cofactors on the cis-DNA element of target gene promoters, only very limited and somewhat controversial information is available on how cell growth is regulated by specific molecular interactions between steroid hormones and cellular target genes. One of the difficulties in elucidating the direct action of steroid hormone receptors controlling target cell growth is that steroid hor- mone receptors may act on target cells indirectly, with the action mediated by stromal cells (14 –18). Steroid hormone action on target tumor epithelial cells may be mediated by a wide spectrum of “cross- talk” between soluble growth factors, insoluble matrix proteins, and steroid hormone receptors in the stromal and/or epithelial cells (19 – 22). Another difficulty is the lack of appropriate tumor cell models that provide a suitable framework to decipher how steroid hormones may regulate target gene expression and cell growth in vitro. Steroid hormones and their receptors may affect the growth of hormone- sensitive tumors by a number of mechanisms including the direct regulation of epithelial cells alone, stromal-epithelial communication, tumor angiogenesis, and host immune reactivity around tumor epithe- lium (14 –25). Most data reported in the literature citing steroid hormone regulation of target tissue growth were obtained by in vivo evaluation. The goal of this study is to evaluate the possible direct role of AR 3 regulating both prostate tumor epithelial cell growth and gene expres- sion. The hAR gene encodes 919 amino acids with a M r 110,000 protein (26 –29) that is a ligand-activated transcription factor (30 –32) and recognizes the ARE as a homodimer (33–37). AR is essential in regulating the development and maintenance of male reproductive systems by controlling prostate cell proliferation, apoptosis, differen- tiation, and senescence (38 – 43). Liganded AR is also pivotal to the onset of PCa and its subsequent progression to androgen independ- ence (44 – 48). Using animal models bearing human prostate tumor xenograft, Thalmann et al. (49, 50) and others (51, 52) have shown that on androgen withdrawal, PCa can progress from an androgen- dependent to an AI state and eventually become refractory to hormo- nal therapy. Our laboratory developed a novel PCa cell model, ARCaP. The ARCaP cell line was derived from the ascites fluid of a PCa patient with advanced metastatic disease and characterized to express the low level of AR and PSA (53). ARCaP cell growth in vitro and tumor growth in vivo were also repressed by androgen (53). In this paper, we seek to define how AR may regulate PCa growth and target gene transactivation. By introducing hAR to ARCaP cells, we observed that AR plays a critical role in promoting target gene transactivation, mediating androgen-induced cell growth, and inhibit- ing tumor cell migration, invasion in vitro, and tumor growth in vivo. Received 2/28/01; accepted 7/26/01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by National Cancer Institute Grants CA-82739 and CA-76620 (to H. E. Z. and L. W. K. C., respectively), and also in part by DK-40890 (to G. S. P.). B. C. was supported by a scholarship from the Department of Higher Education Council, Ankara, Turkey. 2 To whom requests for reprints should be addressed, at Molecular Urology and Therapeutics Program, Emory University Winship Cancer Institute, 1365B Clifton Rd., N.E., Suite B4100, Atlanta, GA 30322. E-mail: Iwchung@emory.edu (for L. W. K. C.) or E-mail: haiyen_zhau@emory.org (for H. E. Z.). 3 The abbreviations used are: AR, androgen receptor; AI, androgen-independent; ARCaP, androgen-repressed human prostate cancer cell line; PSA, prostate-specific antigen; hAR, human AR; hAR wt, wild-type human AR; ARE, androgen-responsive element; DOTAP, N-[1-(2,3-dioleoyloxyl)propyl]-N,N,N-trimethylammoniummethyl sul- fate; GRE, glucocorticoid response element; PCa, prostate cancer; GAPDH, glyceralde- hyde-3-phophate dehydrogenase; EtOH, ethanol; RT-PCR, reverse transcription PCR; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DCC, dextran- coated charcoal; FBS, fetal bovine serum; his, histidine; 4-OH-FL, 4-hydroxy flutamide. 7310 Research. on September 22, 2015. © 2001 American Association for Cancer cancerres.aacrjournals.org Downloaded from