Steroid hormonal regulation of growth, prostate specific antigen secretion, and transcription mediated by the mutated androgen receptor in CWR22Rv1 human prostate carcinoma cells

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Abstract

CWR22Rv1 (22Rv1) is an androgen-responsive human prostate carcinoma cell line derived from a primary prostate tumor that expresses mutant (H874Y) androgen receptors (AR) and secretes low levels of prostate specific antigen (PSA). In this study, we examined the effects of various androgens and other steroid hormones on proliferation of 22Rv1 cells, PSA secretion, and transactivation. Incubation of 22Rv1 cells with various concentrations of testosterone resulted in a dose-dependent 50–80% increase in growth over 72 h. PSA release and transactivation of PRE2-tk-LUC in 22Rv1 cells were stimulated by low concentrations of natural and synthetic androgens (EC50s = 10−10 to 10−9 M) and a broad range of other classes of steroid hormones, albeit with lower potency. Uniform positive immunocytochemical staining was observed in 22Rv1 cell nuclei with mouse monoclonal antibodies to human AR. Competitive binding assays indicated that the mutant AR in 22Rv1 cytosol is more promiscuous than a wild-type AR (ARLBD: rat AR ligand binding domain). Testosterone (10−8 M)-induced PSA release and transactivation were blocked by both antiandrogens and antiprogestins with IC50s of 10−7 to 10−6 M. At high concentration (10−6 M), these antagonists showed partial agonist activity in terms of PSA secretion but not transactivation. In conclusion, the mutant AR in 22Rv1 cells binds and responds to low levels of androgens and a wide spectrum of other natural and synthetic steroid hormones, mechanisms proposed to contribute to tumor progression following androgen ablation.

Introduction

CWR22Rv1 (22Rv1) is an androgen-responsive human prostate carcinoma cell line derived from a primary prostate tumor. This cell line was isolated from a xenograft (CWR22R-2152) that was serially propagated in mice after castration-induced regression and relapse of the parental, androgen-dependent CWR xenograft (Sramkoski et al., 1999). 22Rv1 cells form tumors in nude mice and secrete prostate-specific antigen (PSA) into the circulation of tumor-bearing mice. PSA, a kallikrien-like serine protease that is synthesized by luminal epithelial cells of the human prostate, is the most important tumor marker for diagnosis and management of prostate disease, especially adenocarcinoma of the prostate. Regulation of PSA is mediated by the androgen receptor (AR), and androgens increase PSA mRNA in LNCaP human prostate carcinoma cells (Young et al., 1991). The AR binds to androgen response elements (AREs) in the regulatory regions of target genes to promote transcription. At least three AREs have been identified in the PSA gene, at positions −170, −394, and approximately −4200 (Gsur et al., 2002). The 22Rv1 cell line is of particular interest because it represents one of the few human prostate cancer lines expressing AR.

In the clinical situation, most prostate cancers initially present with an intact AR signaling pathway and respond to androgen ablation therapy, but later relapse to hormone independent growth. In androgen resistant prostate cancer, which no longer depends on the presence of the physiological androgen ligands, alternative pathways may develop for activation of AR. More than 90% of the patients who fail to respond to androgen ablation therapy actually overexpress AR, and among these, overexpression is associated with AR mutations in 10–40% of the cases (for review, see Litvinov et al., 2003). Development of refractoriness of prostate tumors to androgen ablation therapy may occur through mutations in AR which lead to increased sensitivity to very low levels of androgens and/or to increased promiscuity of the AR which allows inappropriate activation by other steroid hormones or androgen antagonists (Feldman and Feldman, 2001).

A single AR mutation which changed codon 874 from CAT for histidine to TAT for tyrosine (H874Y) was described in the original CWR22 xenografts (Tan et al., 1997). These authors investigated effects of several steroid hormones on transactivation of a mouse mammary tumor virus long terminal repeat-luciferase reporter vector (MMTV-LUC) mediated by recombinant H874Y transiently transfected into CV-1 cells. Their results suggested that this mutation, like the T877A mutation in LNCaP cells, leads to reduced specificity of AR. More recently, two aberrant forms of AR protein have been found in 22Rv1 cells and in the relapsed tumor from which the cell line was isolated (Chlenski et al., 2001, Tepper et al., 2002, van Bokhoven et al., 2003). This cell line does not appear to contain any normal size AR protein (110–112 kDa) by Western blot analysis (Tepper et al., 2002, van Bokhoven et al., 2003; Attardi and Tepper, unpublished results). The larger form of the AR (114 kDa) contains a tandem duplication of exon 3, resulting in the addition of 39 amino acids in the DNA-binding domain, and also harbors the original H874Y mutation described in the CWR xenograft (Tepper et al., 2002, van Bokhoven et al., 2003). This form presumably accounts for the responsiveness of 22Rv1 cells to steroid hormone agonists and antagonists as the second smaller form (75–80 kDa) represents a carboxy-terminally truncated variant that lacks the ligand binding domain. Chlenski et al. (2001) proposed that the ∼75 kDa AR protein may arise from premature termination of translation or specific proteolysis as RT-PCR analysis did not reveal a product which could encode a truncated AR. The level of AR gene expression in 22Rv1 cells was ∼16-fold higher than in the CWR22 xenograft, and AR protein levels, analyzed by Western blots, were also increased (Sirotnak et al., 2002).

The antiprogestin, mifepristone, was shown to have growth inhibitory and antitumor activity in both nude mice bearing human prostate tumors (El Etreby et al., 2000a) and prostate cancer cell lines (Lin et al., 1995, El Etreby et al., 2000b). We were interested in confirming and extending these results to examine the effects of unique antiprogestins (e.g. CDB-2914 and CDB-4124, see Attardi et al., 2002) and/or antiandrogens on growth of 22Rv1 prostate tumors in nude mice. However, prior to examining the effects of steroid hormone agonists and antagonists on growth of these tumors in vivo, in the present study, we carried out a detailed characterization of the ability of a variety of steroid hormone agonists and antagonists to bind to the mutated AR in the 22Rv1 cell line and to induce growth, PSA secretion, and transcriptional activity in vitro. Transcription assays were carried out in the context of the 22Rv1 prostate cancer cell rather than in an unrelated cell type. Our results indicate that the mutant AR in 22Rv1 cells displays increased sensitivity to androgens and decreased ligand selectivity, thereby supporting the hypothesis that these factors play a role in the development of androgen-independent prostate cancer.

Section snippets

Chemicals

Testosterone (T), 5α-dihydrotestosterone (DHT), estradiol (E2), progesterone (P4), cortisol (cort), methylprednisolone (MP), dexamethasone (dex), and medroxyprogesterone acetate (MPA) were purchased from Steraloids (Newport, RI); mifepristone, 4-hydroxytamoxifen (OH tamoxifen), and methyltestosterone (methylT), from Sigma (St. Louis, MO); methyltrienolone (R1881) and promegestone (R5020), from Perkin-Elmer Life Sciences Inc. (Boston, MA); and ICI 182,780 and nilutamide, from Tocris-Cookson Ltd.

Regulation of cell proliferation and PSA secretion

Fig. 1A shows that, although 22Rv1 cells grow in the absence of androgens, testosterone stimulated 22Rv1 cell proliferation in a dose-dependent manner after a 72 h treatment. The EC50 for growth stimulation was 4.5±2.1×10−10 M (n = 6), and the magnitude of the increase was 50–80%. Late passage 22 Rv1 cells (passage > 100), which grew more rapidly than earlier passage cells (passage = 30–40), continued to show a growth response to testosterone. Growth of 22Rv1 cells was also stimulated by 30–80%

Discussion

Feldman and Feldman (2001) described five pathways that may lead to androgen independence of prostate cancer: (1) hypersensitivity to androgens (due to AR amplification, increased AR sensitivity, or increased androgen levels); (2) promiscuity of AR (decreased specificity of ligand binding); (3) use of ligand-independent mechanisms for activation of AR (e.g. growth factors, receptor tyrosine kinases, AKT); (4) bypass of the androgen signaling cascade entirely; (5) presence and survival of a

Acknowledgements

The authors would like to acknowledge the excellent technical assistance of Lisa Radler, Devi Weier, Trung C. Pham, Margaret Krol, Eileen Curreri, Jessica Luke, and Bruce Till. This work was supported by NICHD contract NO1 HD-2-3338 awarded to BIOQUAL Inc.

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    A portion of this work was presented in abstract form at the 84th Annual Meeting of the Endocrine Society, June 2002.

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