Expression of human estrogen receptor-a and -b, progesterone receptor, and androgen receptor mRNA in normal and malignant ovarian epithelial cells (mutationysequence deletionyestrogen actionyandrogen actionyprogesterone action)

Our understanding of the roles played by sex hormones in ovarian carcinogenesis has been limited by a lack of data concerning the mode of sex hormone action in human ovarian surface epithelial (HOSE) cells, the tissue of origin of >90% of ovarian cancers. We have compared the relative abundance of estrogen receptor (ER)a, ERb, progesterone receptor (PR), and androgen receptor (AR) mRNA in four primary cultures of HOSE cells obtained from postmenopausal women to those found in late serous adenocarcinoma primary cell cultures and established ovarian cancer cell lines. We observed coexpression of ERa and ERb mRNA along with AR and PR transcripts in normal HOSE cells and disruption of ERa mRNA expression as well as dramatic down-regulation of PR and AR transcript expression in most ovarian cancer cells. In contrast, levels of ERb mRNA were unaffected by the malignant state. Additionally, a novel mutation involving a 32-bp deletion in exon 1 of ERa transcripts was detected in the SKOV3 cell line. This mutation would explain why SKOV3 was reported to be ER-positive but estrogen-insensitive. Taken together, these findings suggest that estrogens, signaling via either or both ER subtypes, may play an indispensable role in regulating normal HOSE cell functions. Therefore, loss of ERa, PR, and AR mRNA expression in HOSE cells may be responsible for neoplastic transformation in this cell type. In contrast, the roles played by ERb in normal and malignant HOSE cells remain elusive. Finally, the coexistence of mutated ERa mRNA and normal ERb transcripts in SKOV3 argues in favor of a dependency of ERb action on functional ERas. Ovarian carcinoma (OC) is the second most common and the most deadly malignancy of the female reproductive tract (for review, see refs. 1–4). Etiological factors involved in ovarian carcinogenesis remain poorly defined, and effective treatment protocols are limited (1–3). Epidemiological data suggest that endogenous and exogenous sex hormones may play important roles in the pathogenesis of the disease. In this regard, estrogens taken as oral contraceptives during premenopausal years offer protection, but when used postmenopausally as hormone replacement therapies elevate risk (1–6). The risk of developing invasive OC increases with ever-use of hormone replacement therapy and has been shown to depend on the duration of usage (5, 6). In addition to estrogens, other ovarian or adrenal steroids such as androstenedione, testosterone, and progestins have all been implicated as risk factors for OC (1–4). Androstenedione and progesterone are present at higher concentrations in the ovarian vein draining the affected ovary when compared with levels found in the vein draining the contralateral, disease-free gland (7, 8). Plasma levels of estradiol-17b (E2), progesterone, 20a-hydroxyprogesterone, testosterone, and androstenedione have all been shown to correlate with OC tumor masses (9–11). Taken together, these findings suggest that steroid hormones are likely involved in the genesis and progression of the disease, yet their mechanisms of action remain unclear. The classical estrogen receptor (ER), recently renamed ERa (12), and the progesterone receptor (PR) were found in ,50% of OC specimens, whereas androgen receptor (AR) was detected in most cases (.80%) (1–4). In recent studies, transcripts of the newly discovered ER subtype, ERb (12), were found in normal human ovaries and benign and malignant ovarian tumors (13, 14), as well as in primary cultures of normal human ovarian surface epithelial (HOSE) cells (15). Unfortunately, none of the aforementioned studies demonstrated a strong association between ERa, PR, or AR status and OC histological types or grades. Furthermore, treatments of OCs with tamoxifen, antiandrogens, or progestins produced very dismal responses (for review, see refs. 1–4). Consequently, it is widely believed that levels of sex hormone receptors have little prognostic value and are poor predictors of hormone-manipulation outcomes for OCs. A major challenge in assessing the significance of sex hormones and their receptors in ovarian carcinogenesis is the paucity of information about their expression levels in the normal HOSE. Over 90% of OCs arise from the HOSE, which shares a common embryonic origin with epithelia of Mullerian duct-derived tissues (Fallopian tube, endometrium, and endocervix) but is distinctly different from the granulosa–thecal cells of the ovary (1–4). In terms of tissue mass, this layer represents only a small fraction of the whole ovary. Thus, data generated from studies that compare levels of a molecular marker found in OCs with those observed in whole ovaries are difficult to interpret because expression pattern in HOSE could easily be masked by those in other ovarian cell types. In this regard, although previous studies have demonstrated localization of ERb in ovarian granulosal cells and ERa throughout the ovary (12, 16, 17), only recently have both ER subtypes been found in normal HOSE cells (15). However, it remains unclear as to whether their expression levels are altered after neoplastic transformation. Importantly, little is known about the relationships between the expression patterns of the two ER subtypes and those of other steroid receptors in normal and malignant HOSE cells. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. PNAS is available online at www.pnas.org. Abbreviations: HOSE, human ovarian surface epithilial; ER, estrogen receptor; PR, progesterone receptor; AR androgen receptor; OC, ovarian carcinoma; RT-PCR, reverse transcription–PCR; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. ‡To whom reprint requests should be addressed at: Department of Surgery, Division of Urology, University Campus, University of Massachusetts Memorial Medical Center, 55 Lake Avenue North, Worcester, MA 01655.