Expression of Estrogen Receptor (ER)-a and ER-b in Normal and Malignant Prostatic Epithelial Cells: Regulation by Methylation and Involvement in Growth Regulation

The aim of the current study is to demonstrate normal and malignant prostatic epithelial cells (PrECs) as targets for receptor-mediated estrogenic and antiestrogenic action. Using an improved protocol, we have successfully isolated and maintained highly enriched populations of normal PrECs from ultrasound-guided peripheral zone biopsies, individually determined to be morphologically normal. Semiquantitative reverse transcription-PCR analyses were used to determine whether transcripts of estrogen receptor (ER)-a and those of ER-b were expressed in our normal PrEC primary cultures, in a commercially available PrEC preparation (PrEC; Clontech), in an immortalized PrEC line established from a benign prostatic hyperplasia specimen (BPH-1), and in three prostatic cancer cell lines (LNCaP, PC-3, and DU145). Expression levels of ER-a and ER-b transcripts were related to those of two estrogen-responsive genes [progesterone receptor (PR) and pS2], at the message levels, to gain insights into the functionality of the ER subtypes in PrECs. Interestingly, only transcripts of ER-b, but not those of ER-a, were found in our primary cultures of normal PrECs, along with both PR and pS2 mRNA. These data strongly suggest that estrogen action was signaled exclusively via ER-b in normal human PrECs. In contrast, PrEC (Clontech) and BPH-1 cells expressed both ER-a and ER-b transcripts and no PR nor pS2 mRNA in PrEC and only a minimal level of PR mRNA in BPH-1. Among the three prostate cancer cell lines, LNCaP expressed ER-b mRNA along with transcripts of PR and pS2, DU145 expressed messages of ER-b and PR, and PC-3 cells exhibited ER-a, ER-b, and pS2 mRNA. Thus, unlike normal PrECs, expression patterns of these genes in malignant PrECs are more variable. Treatment of prostate cancer cells with demethylation agents effectively reactivated the expression of ER-a mRNA in LNCaP and DU145 and that of pS2 message in DU145. These findings provide experimental evidence that ER-a gene silencing in prostate cancer cells, and perhaps also in normal PrECs, are caused by DNA hypermethylation. To evaluate the potential of using antiestrogens as prostate cancer therapies, we have assessed the growth-inhibitory action of estrogens (estradiol and diethylstilbestrol) and antiestrogens (4-hydroxy-tamoxifen and ICI-182,780) on PC-3 and DU-145 cells. In PC-3 cells, which express both ER subtypes, estrogens as well as antiestrogens are effective inhibitors. In contrast, in DU145 cells, which express only ER-b, antiestrogens, but not estrogens, are growth inhibitors. By comparison, ICI 182,780 is the more effective cell growth inhibitor. Importantly, the ICI 182,780induced antiproliferative effects were reversed by cotreatment of DU145 cells with an ER-b antisense oligonucleotide, hence lending additional support to a central role played by ER-b in mediating growth-inhibitory action of antiestrogens. INTRODUCTION Charles Huggins pioneered the use of the synthetic estrogen, DES, in the treatment of advanced PCa in the early 1940s (1). The action of DES was thought to be mediated via a blockade of the pituitarytesticular axis, which effectively lowered circulating levels of androgen and caused tumor regression (2). However, recent investigations have demonstrated that DES exerts direct growth-inhibitory effects on prostatic cancer cells via induction of mitotic arrest or apoptosis (3–6). Unfortunately, because of serious adverse effects induced by the estrogenicity of DES (feminization, exacerbation of heart failure, vascular complications, gynecomastia, and impotence), the xenoestrogen has lost its attractiveness as a mainstay treatment for advanced PCa (7). Clinical use of TAM, a nonsteroidal estrogen mixed agonist/ antagonist, was introduced in the 1980s as an alternative to DES in the treatment of PCa. It was better tolerated than DES but only produced low response rates (8–12). It was therefore concluded that further investigation of TAM in advanced PCa treatment was not warranted. Of late, two recent developments have rekindled interests in using antiestrogens as therapeutics for PC: (a) pure estrogen antagonists (e.g., ICI-164,384 and ICI-182,780; Ref. 13) and selective estrogen receptor modulators, such as raloxifene (14), have become available for clinical trials. Experimental evidence suggests that their mechanisms of action may differ significantly from those of estrogens (15–18) and therefore may yield more favorable outcomes; and (b) a newly discovered ER subtype (ER-b) was found to be expressed at high levels in the epithelial compartments of the rat prostate (19–23). Although ER-b shares high homology with the classical ER (ER-a), the two ER subtypes may regulate different sets of cellular functions (18, 24). Recent findings from an ER-b knockout mouse suggest that ER-b may suppress proliferation and prevent hyperplasia in the rodent prostate (25). Taken together, these new findings raise an intriguing possibility that ER-b is expressed in normal and/or malignant human PrECs and plays a role in mediating estrogen action in these cell types. Knowledge of the distribution of ER-b in normal and malignant human PrECs is limited at this time. A recent study reported a lack of ER-b expression in human prostate tissues (26), whereas several preliminary reports noted expression of this receptor subtype in basal epithelial cells of the human (27–29). In this study, we reported the development of an effective method to obtain and culture “pure” or highly enriched populations of normal PrECs from needle biopsies of the peripheral zone of the human prostate. Expression levels of ER-a and ER-b transcripts in our primary cultures of normal PrECs were compared with those found in a PrEC preparation obtained from a commercial source (PrEC; Clontech), in an immortalized PrEC cell line established from a BPH specimen (BPH-1; Ref. 30), and in three prostatic cancer cell lines (DU145, PC-3, and LNCaP). Expression Received 11/24/99; accepted 4/13/00. 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 Research was supported in part by United States Army Grant DAMD17-98-1-8606 and NIH Grants CA15776 (to S-M. H.). 2 Present address: New England College of Osteopathy, Biddeford, ME 04005. 3 To whom requests for reprints should be addressed, at Division of Urology, Department of Surgery, Rm S4-746, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655. E-mail: [email protected]. 4 The abbreviations used are: DES, diethylstilbestrol; PCa, prostatic adenocarcinoma; TAM, tamoxifen; ER, estrogen receptor; E2, 17b-estradiol; PrEC, prostatic epithelial cell; BPH, benign prostatic hyperplasia; PR, progesterone receptor; 4OH-TAM, 4-hydroxyTAM; ICI, ICI-182,780; ODN, oligodeoxynucleotide; FBS, fetal bovine serum; P/S, penicillin/streptomycin; RT-PCR, reverse transcription-PCR; GAPDH, glyceraldehyde-3phosphate dehydrogenase; AR, androgen receptor.