The Xenoestrogen Bisphenol A Induces Inappropriate Androgen Receptor Activation and Mitogenesis in Prostatic Adenocarcinoma Cells 1 Yelena B. Wetherill, Christin E. Petre, Kelly R. Monk, Alvaro Puga, and Karen E. Knudsen 2 Departments of Cell Biology [Y. B. W., C. E. P., K. R. M., K. E. K.] and Environmental Health [A. P.], University of Cincinnati College of Medicine, Cincinnati, Ohio 45267 Abstract Treatment for prostatic adenocarcinoma is reliant on the initial androgen dependence of this tumor type. The goal of therapy is to eliminate androgen receptor activity, either through direct inhibition of the receptor or through inhibition of androgen synthesis. Although this course of therapy is initially effective, androgen- refractory tumors ultimately arise and lead to patient morbidity. Factors contributing to the transition from a state of androgen dependence to the androgen- refractory state are poorly understood, but clinical evidence in androgen-refractory tumors suggests that the androgen receptor is inappropriately activated in these cells. Thus, the mechanisms that contribute to inappropriate (androgen-independent) activation of the androgen receptor (AR) is an area of intensive research. Here we demonstrate that bisphenol A (BPA), a polycarbonate plastic monomer and established xenoestrogen, initiates androgen-independent proliferation in human prostatic adenocarcinoma (LNCaP) cells. The mitogenic capacity of BPA occurred in the nanomolar range, indicating that little BPA is required to stimulate proliferation. We show that BPA stimulated nuclear translocation of the tumor-derived receptor (AR-T877A), albeit with delayed kinetics compared with dihydrotestosterone. This translocation event was followed by specific DNA binding at androgen response elements, as shown by electrophoretic mobility shift assays. Moreover, the ability of BPA to stimulate AR-T877A activity was demonstrated by reporter assays and by analysis of an endogenous AR target gene, prostate-specific antigen. Thus, BPA is able to activate AR-T877A in the absence of androgens. Lastly, full mitogenic function of BPA is dependent on activation of the tumor-derived AR- T877A. These data implicate BPA as an inappropriate mitogen for prostatic adenocarcinoma cells and provide the impetus to study the consequence of BPA exposure on prostate cancer. Introduction Prostatic adenocarcinoma is the second leading cause of cancer death among men in the United States (1). A major challenge in treatment of prostate cancer is the lack of ef- fective therapeutic regimens for advanced disease. The most effective treatment regimens rely on the observation that prostatic adenocarcinomas are dependent on serum andro- gen for proliferation and survival (reviewed in Refs. 2 and 3); thus, androgen ablation is a first line of therapy (4 –7). How- ever, most tumor remissions are transient, as recurrent, androgen-refractory tumors ultimately arise, leading to a high rate of patient morbidity. The events that regulate or facilitate the transition from an androgen-dependent to an androgen- refractory state are poorly understood and are the focus of intensive research. Androgen elicits its biological effect through activation of the AR, 3 a member of the nuclear receptor superfamily (8, 9). Prior to ligand binding, the AR exists in diffuse pools through- out the nucleus and cytoplasm and is held inactive through association with heat shock proteins (8, 10). In the prostate, the predominant ligand for the AR is DHT, which is reduced from testosterone through the action of 5--reductase (11). Interaction of DHT with the AR triggers dissociation of inhib- itory heat shock proteins and rapid nuclear translocation (12, 13). Activated AR forms homodimers and stimulates gene transcription from AREs in target promoters (2). Expression of one such target, PSA is dependent on association of activated AR with the PSA promoter and enhancer regions (14, 15). Because AR activity is required for and reflective of prostatic adenocarcinoma proliferation, PSA expression is monitored clinically as a key indicator of prostatic adenocar- cinoma progression (16). The importance of AR activity in prostatic adenocarcinoma is apparent with regard to both early- and late-stage disease. First-line therapies for androgen-dependent tumors aim to inhibit AR activity. These therapies are designed to either block the synthesis of AR ligands (e.g., luteinizing hormone releasing hormone agonists, 5- reductase inhibitors, inhib- itors of adrenal androgen synthesis, or bilateral orchiectomy) or directly inhibit AR transcriptional transactivation potential Received 2/20/02; revised 3/21/02; accepted 3/26/02. 1 This work was supported by NIH Training Grant ES07250-13 (to Y. B. W.; Environmental Mutagenesis and Cancer) and NIH Grant R01 CA93404-01 (to K. E. K.). 2 To whom requests for reprints should be addressed, at Department of Cell Biology, ML 0521, University of Cincinnati College of Medicine, Cin- cinnati, OH 45267-0521. Phone: (513) 558-7371; Fax: (513) 558-4454; E-mail: Karen.Knudsen@uc.edu 3 The abbreviations used are: AR, androgen receptor; DHT, dihydrotest- osterone; ARE, androgen response element; PSA, prostate-specific anti- gen; BPA, bisphenol A; FBS, fetal bovine serum; CDT, charcoal dextran treated; BrdUrd, bromodeoxyuridine; ETOH, ethanol; EMSA, electro- phoretic mobility shift assay; RT-PCR, reverse transcription-PCR; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; FACS, fluores- cence-activated cell sorter; PI, propidium iodide. 515 Vol. 1, 515–524, May 2002 Molecular Cancer Therapeutics on May 11, 2017. © 2002 American Association for Cancer Research. mct.aacrjournals.org Downloaded from