Proc. Natl. Acad. Sci. USA Vol. 96, pp. 5722–5727, May 1999 Medical Sciences 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) KIN-MANG LAU*, SAMUEL C. MOK†, AND SHUK-MEI HO*‡ *Department of Biology, Tufts University, Medford, MA 02155; and †Laboratory of Gynecological Oncology, Department of Obstetrics, Gynecology, and Reproductive Biology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115 Communicated by Elwood V. Jensen, Karolinska Institute, Huddinge, Sweden, March 15, 1999 (received for review September 15, 1998) b a ABSTRACT Our understanding of the roles played by sex estradiol-17 (E2), progesterone, 20 -hydroxyprogesterone, hormones in ovarian carcinogenesis has been limited by a lack testosterone, and androstenedione have all been shown to of data concerning the mode of sex hormone action in human correlate with OC tumor masses (9–11). Taken together, these ovarian surface epithelial (HOSE) cells, the tissue of origin of findings suggest that steroid hormones are likely involved in >90% of ovarian cancers. We have compared the relative the genesis and progression of the disease, yet their mecha- abundance of estrogen receptor (ER)a,ERb, progesterone nisms of action remain unclear. receptor (PR), and androgen receptor (AR) mRNA in four The classical estrogen receptor (ER), recently renamed ERa primary cultures of HOSE cells obtained from postmeno- (12), and the progesterone receptor (PR) were found in ,50% pausal women to those found in late serous adenocarcinoma of OC specimens, whereas androgen receptor (AR) was de- primary cell cultures and established ovarian cancer cell lines. a b tected in most cases ( 80%) (1–4). In recent studies, tran- We observed coexpression of ER and ER mRNA along with scripts of the newly discovered ER subtype, ERb (12), were AR and PR transcripts in normal HOSE cells and disruption a found in normal human ovaries and benign and malignant of ER mRNA expression as well as dramatic down-regulation ovarian tumors (13, 14), as well as in primary cultures of of PR and AR transcript expression in most ovarian cancer normal human ovarian surface epithelial (HOSE) cells (15). cells. In contrast, levels of ERb mRNA were unaffected by the Unfortunately, none of the aforementioned studies demon- malignant state. Additionally, a novel mutation involving a strated a strong association between ERa, PR, or AR status 32-bp deletion in exon 1 of ERa transcripts was detected in the and OC histological types or grades. Furthermore, treatments SKOV3 cell line. This mutation would explain why SKOV3 was reported to be ER-positive but estrogen-insensitive. Taken of OCs with tamoxifen, antiandrogens, or progestins produced together, these findings suggest that estrogens, signaling via very dismal responses (for review, see refs. 1–4). Conse- either or both ER subtypes, may play an indispensable role in quently, it is widely believed that levels of sex hormone regulating normal HOSE cell functions. Therefore, loss of receptors have little prognostic value and are poor predictors ERa, PR, and AR mRNA expression in HOSE cells may be of hormone-manipulation outcomes for OCs. responsible for neoplastic transformation in this cell type. In A major challenge in assessing the significance of sex contrast, the roles played by ERb in normal and malignant hormones and their receptors in ovarian carcinogenesis is the HOSE cells remain elusive. Finally, the coexistence of mutated paucity of information about their expression levels in the ERa mRNA and normal ERb transcripts in SKOV3 argues in normal HOSE. Over 90% of OCs arise from the HOSE, which favor of a dependency of ERb action on functional ERas. shares a common embryonic origin with epithelia of Mullerian duct-derived tissues (Fallopian tube, endometrium, and endo- Ovarian carcinoma (OC) is the second most common and the cervix) but is distinctly different from the granulosa–thecal most deadly malignancy of the female reproductive tract (for cells of the ovary (1–4). In terms of tissue mass, this layer review, see refs. 1–4). Etiological factors involved in ovarian represents only a small fraction of the whole ovary. Thus, data carcinogenesis remain poorly defined, and effective treatment generated from studies that compare levels of a molecular protocols are limited (1–3). Epidemiological data suggest that marker found in OCs with those observed in whole ovaries are endogenous and exogenous sex hormones may play important difficult to interpret because expression pattern in HOSE roles in the pathogenesis of the disease. In this regard, could easily be masked by those in other ovarian cell types. In estrogens taken as oral contraceptives during premenopausal this regard, although previous studies have demonstrated years offer protection, but when used postmenopausally as localization of ERb in ovarian granulosal cells and ERa hormone replacement therapies elevate risk (1–6). The risk of throughout the ovary (12, 16, 17), only recently have both ER developing invasive OC increases with ever-use of hormone subtypes been found in normal HOSE cells (15). However, it replacement therapy and has been shown to depend on the remains unclear as to whether their expression levels are duration of usage (5, 6). In addition to estrogens, other ovarian altered after neoplastic transformation. Importantly, little is or adrenal steroids such as androstenedione, testosterone, and known about the relationships between the expression patterns progestins have all been implicated as risk factors for OC of the two ER subtypes and those of other steroid receptors in (1–4). Androstenedione and progesterone are present at normal and malignant HOSE cells. higher concentrations in the ovarian vein draining the affected ovary when compared with levels found in the vein draining Abbreviations: HOSE, human ovarian surface epithilial; ER, estrogen the contralateral, disease-free gland (7, 8). Plasma levels of receptor; PR, progesterone receptor; AR androgen receptor; OC, ovarian carcinoma; RT-PCR, reverse transcription–PCR; GAPDH, The publication costs of this article were defrayed in part by page charge glyceraldehyde-3-phosphate dehydrogenase. ‡To whom reprint requests should be addressed at: Department of payment. This article must therefore be hereby marked ‘‘advertisement’’ in Surgery, Division of Urology, University Campus, University of accordance with 18 U.S.C. §1734 solely to indicate this fact. Massachusetts Memorial Medical Center, 55 Lake Avenue North, PNAS is available online at www.pnas.org. Worcester, MA 01655. 5722 Downloaded by guest on September 23, 2021 Medical Sciences: Lau et al. Proc. Natl. Acad. Sci. USA 96 (1999) 5723 To fill this data gap, in the present study, we have compared hyde-6-phosphate dehydrogenase (GAPDH), a housekeeping the expression of ERa,ERb, PR, and AR transcripts in gene. Genomic DNA was eliminated by digestion with DNase primary cultures of normal HOSE cells, obtained from post- I. One microgram of total RNA was reverse-transcribed by menopausal women, to those found in primary ovarian cancer using the GeneAmp RNA PCR kit (Perkin–Elmer). Subse- cell cultures and in established OC cell lines. This approach has quently, 2 ml of the resulting cDNA samples was used in each allowed us to observe that (i)ERa and ERb mRNA, as well PCR. as AR and PR transcripts, are coexpressed in normal HOSE The primer sequences for GAPDH, ERa,ERb, PR, and AR cells, (ii) whole exon-deletion variants of ERa and ERb are are given in Table 1 (20–22). Three pairs of primers were used commonly found in HOSE and ovarian cancer cells, and (iii) to detect ERa wild-type and variant mRNAs, and two pairs of PR and AR mRNA expression are significantly down- primers were used in the identification of wild-type and exon regulated in ovarian cancer cells that exhibit altered ERa, but 5 deletion variant of ERb (Table 1). Hot start PCR using normal ERb, message expression. A previously unknown ERa AmpliTaq Gold DNA polymerase (Perkin–Elmer) was used in mutation, leading to a 32-bp deletion in exon 1, has been all amplification reactions. The enzyme was activated by identified in an ovarian cancer cell line, SKOV3. This cell line preheating the reaction mixtures at 95°C for 6 minutes before a has been shown to express ER but is estrogen- and anties- thermal cycling. This protocol was chosen to minimize non- trogen-resistant (18). Taken together, these findings implicate specific product amplification. The routine PCR program was regulation of normal HOSE cell functions by estrogens, and 30 cycles of 1 min at 94°C, 1 min at 60°C (annealing temper- possibly by progestins and androgens. Additionally, the emer- ature), and 1 min at 72°C. mRNA-specific modifications gence of sex hormone resistance, via down-regulation or included (i) an annealing temperature of 58°C used to amplify mutational inactivation of receptors, may be a key feature of ERb cDNA, (ii) an annealing temperature of 55°C in the ovarian epithelial transformation. amplification of ERa and AR cDNAs, (iii) a cycle number of 35 for ERa cDNA amplification, (iv) GAPDH cDNA ampli- MATERIALS AND METHODS fied for 22–26 cycles, and its levels served as loading control. After PCR, the products were resolved on 2% agarose gel with Cell Cultures and Cell Lines. Four primary cultures of ethidium bromide. The images was captured under UV tran- normal HOSE cells (HOSE27, HOSE20, HOSE17, and sillumination. In initial experiments, after amplification, PCR HOSE13), one primary culture of normal mesothelial cells products were excised, purified, and subjected to direct se- (MesO13), four primary cultures of ovarian carcinoma cells quencing to verify amplification of the correct sequences. (OVCA420, OVCA429, OVCA432, and OVCA433), and Serial dilutions of total RNA and cDNA were used in early three ovarian cancer cell lines (DOV13, SKOV3, and CAOV3) experiments to establish the range of linearity between signal were used in this study.