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*Ovarian Cancer
The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 11 5508-5516
Copyright © 2004 by The Endocrine Society

Overexpression of Follicle-Stimulating Hormone Receptor Activates Oncogenic Pathways in Preneoplastic Ovarian Surface Epithelial Cells

Jung-Hye Choi, Kyung-Chul Choi, Nelly Auersperg and Peter C. K. Leung

Department of Obstetrics and Gynecology, British Columbia Children’s and Women’s Hospital, British Columbia Research Institute for Children’s and Women’s Health, University of British Columbia, Vancouver, British Columbia, Canada V6H 3V5

Address all correspondence and requests for reprints to: Peter C. K. Leung, Ph.D., Department of Obstetrics and Gynecology, University of British Columbia, 2H-30, 4490 Oak Street, Vancouver, British Columbia, Canada V6H 3V5. E-mail: peleung{at}interchange.ubc.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
It has been previously demonstrated that human ovarian cancer cells express FSH receptor (FSHR). However, whether FSHR plays a role in ovarian cancer development is still ambiguous. To investigate the role of FSHR in tumor progression, we overexpressed the receptor in SV40 Tag immortalized ovarian surface epithelium (OSE) cell lines (IOSE-80PC, a postcrisis line, and IOSE-398), which are preneoplastic and nontumorigenic. We compared the expression levels of several selected oncogenes in nontransfected (80PC), vector-transfected (80PCV), FSHR-transfected IOSE (80PCF) cells, and established ovarian cancer cell lines (OVCAR-3 and SKOV-3). Significantly increased protein levels of epithelial growth factor receptor, HER-2/neu, and c-Myc, but not K-Ras, were observed in FSHR-overexpressing 80PCF cells when compared with 80PCV cells. Constitutive phosphorylation of ERK1/2 was augmented in 80PCF cells, whereas phosphorylation of the other MAPK including p38 and Jun N-terminal kinase was unchanged. Considerable constitutive phosphorylation of ERK1/2 was also observed in OVCAR-3 and SKOV-3 cell lines when compared with 80PCV. More importantly, 80PCF cells grew more rapidly than 80PC and 80PCV cells. In conclusion, we have demonstrated that FSHR was highly expressed in OVCAR-3 and 80PCF cells transfected with FSHR overexpression vector. The 80PCF cell line showed increased levels of epithelial growth factor receptor, HER-2/neu, and c-myc and constitutive activation of ERK1/2. The rate of proliferation of the 80PCF cells was increased, compared with control cell lines. These results suggest that the overexpression of FSHR may be associated with enhanced levels of potential oncogenic pathways and increased proliferation in preneoplastic ovarian surface epithelial cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FSH PLAYS AN important role in the development of follicles and production of ovarian steroids to maintain ovarian functions. It is composed of two different, noncovalently associated, carbohydrate-containing protein subunits called {alpha} and ß. The FSH receptor (FSHR) is a transmembrane receptor with a G protein-coupled signaling system and is expressed by granulosa cells in developing ovarian follicles. Actions of FSH in reproductive physiology have been well characterized and are essential for folliculogenesis and steroidogenesis (1, 2). However, the role of FSH and FSHR in ovarian surface epithelium (OSE) and ovarian epithelial cancer is not well characterized.

There is increasing evidence that the hormonal environment of the OSE can influence the incidence of ovarian cancer (3, 4, 5). The hypothesis of the present study is that the gonadotropins, FSH, and LH may be involved in the transformation and progression of normal OSE to its neoplastic counterpart. Ovarian cancer is more common in conditions with elevated gonadotropins such as postmenopausal women or women who have received treatment for induction of ovulation (3, 6, 7, 8). Reduced risk for ovarian cancer is associated with multiple pregnancies, breast-feeding, oral contraceptives, and estrogen replacement therapy, which are associated with lower levels and reduced exposure to gonadotropins (9, 10, 11).

Treatment with FSH results in the growth stimulation in normal, immortalized OSE, and some ovarian cancer cells in a dose- and time-dependent manner in vitro (12, 13, 14, 15, 16, 17, 18). The treatment of the epithelial ovarian cancer line HRA with FSH significantly increases the levels of protein kinase C{alpha} mRNA and protein, suggesting that the stimulation of protein kinase C{alpha} transcription is involved in the FSH-induced cell proliferation (17). In ovarian cancer cell line OVCAR-3 cells, gonadotropins stimulate estradiol secretion and increase steroid-dependent growth stimulation (18). Recent studies demonstrated that the IL-6/signal transducer and activator of transcription-3 signaling pathway may mediate FSH-stimulated immortalized OSE cell proliferation (19). However, only a few studies demonstrated the expression of FSHR in normal OSE and/or ovarian cancer cells (12, 13, 14), and, to our knowledge, few reports have directly demonstrated an involvement of FSHR in ovarian epithelium tumorigenesis. Considering that FSH might play a role in ovarian cancer development (15, 20, 21, 22, 23, 24, 25), we sought to investigate the molecular events associated with FSHR expression in ovarian epithelial cells. It is hypothesized that overexpression of FSHR may alter intracellular signaling pathways and activate oncogenic pathway in normal and immortalized OSE cells because FSHR plays a critical role in the early phase of ovarian cancer development (13, 26). Thus, in the present study, we investigated the following: 1) the expression of FSHR at the mRNA and protein levels in preneoplastic immortalized OSE (IOSE) and two ovarian carcinoma cell lines, OVCAR-3 and SKOV-3; these ovarian cancer cell lines are highly malignant and invasive in vivo, but OVCAR-3 cells adhere and form tightly cohesive epithelial colonies in culture, whereas SKOV-3 cells display a spindle-shaped dispersed phenotype (27); 2) the activation of oncogenic pathways and MAPKs in FSHR-overexpressing IOSE cells; and 3) the proliferative rate of FSHR-overexpressing IOSE cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell transfections

The expression vector of human FSHR (pcDNAHFSHR) was generously provided by Dr. T. Minegishi (Gumma Medical School, Gumma, Japan). The cDNA coding for the full human FSH receptor was cloned into the EcoRI site of the pcDNA 3.1 (Invitrogen Life Technology, San Diego, CA) expression vector. The stable transfection was performed in the IOSE-80PC (passage 50–57) and the IOSE-398 (passage 13–18) cell line. One or two micrograms of the constructed plasmid pcDNAHFSHR were transfected into IOSE-80PC and IOSE-398 cells by FuGENE 6 (Roche Applied Science, Laval, Québec, Canada) according to the manufacturer’s suggested protocol when the cells were around 50% confluent on 6-well plates. The transfected cells were grown in a selection medium including G418 (Invitrogen, 200 µg/ml) and changed every 3 d for 3 wk to obtain G418-resistant individual colonies (28).

Cell culture

Normal human OSE cells were scraped from the ovarian surface during laparoscopies from premenopausal women (n = 3) for nonmalignant disorders and cultured as previously described (29). The use of these tissues was approved by the Committee for Ethical Review of Research on the use of human subjects, University of British Columbia. All women provided informed written consent. The nontumorigenic SV40 Tag-immortalized OSE-derived lines, IOSE-80PC (a postcrisis line) and IOSE-398, were cultured as previously described (30) in medium 199:MCDB 105 (Sigma-Aldrich Corp., St. Louis, MO) containing 10% fetal bovine serum (FBS; Hyclone Laboratories Ltd., Logan, UT), 100 U/ml penicillin G, and 100 µg/ml streptomycin (Life Technologies, Inc., Rockville, MD) in a humidified atmosphere of 5% CO2-95% air and passaged with 0.06% trypsin (1:250)/0.01% EDTA in Mg2+/Ca2+-free Hanks’ balanced salt solution when confluent. For monolayer culture, the cell lines were maintained on tissue culture dishes (Falcon; Becton Dickinson, Franklin Lakes, NJ). The IOSE-80PC cell line was generously provided by Dr. A. Godwin (Fox Chase Cancer Center, Philadelphia, PA). The OVCAR-3 and SKOV-3 cells, ovarian adenocarcinoma cancer cell lines, were purchased from the American Type Tissue Collection (Manassas, VA).

RNA extraction, RT-PCR procedure, and Southern blot analysis

Total RNA was prepared from cultured cells using the RNaid kit (Bio/Can Scientific, Mississauga, Ontario, Canada) according to the manufacturer’s suggested procedure. RNA integrity was confirmed by using agarose gel electrophoresis and ethidium bromide staining. The total RNA concentration was determined by spectrophotometric analysis at A260/280. cDNA was synthesized from 2.5 µg total RNA by reverse transcription at 37 C for 2 h using a first-strand cDNA synthesis kit (Amersham Pharmacia Biotechnology, Oakville, Ontario, Canada). The synthesized cDNA was used as a template for PCR amplification. A semiquantitative PCR amplification was carried out with denaturing for 1 min at 94 C, annealing for 60 sec at 55 C, extension for 90 sec at 72 C, and a final extension for 15 min at 72 C using a thermal cycler (DNA thermal cycler; Perkin-Elmer, Norwalk, CT). The primers were designed to amplify FSHR mRNA based on the published sequences of human FSHR (24). In addition, amplification of human glyceraldehyde phosphate dehydrogenase (GAPDH) was performed using specific primers (31) to rule out the possibility of RNA degradation and was used to control the variation in mRNA amount in PCR. The primers of FSHR are composed of sense, 5'-GAGAG CAAGG TGACA GAGAT TCC-3' (exon 1, nucleotides 97–120), and antisense, 5'-CCTTT TGGAG AGAAT GAATC TT-3' (exon 5, nucleotide 439–417). The sequences of GAPDH amplification are sense, 5'-ATGTT CGTCA TGGGT GTGAA CCA-3', and antisense, 5'-TGGCA GGTTT TTCTA GACGG CAG-3'. The PCRs were performed in 25 µl PCR mixture containing 1 x PCR buffer, 0.2 mM each dNTP, 1.6 mM MgCl2, 50 pmol specific primers, 1 µl cDNA template, and 0.25 U Taq polymerase. Twelve microliters of PCR products were analyzed by agarose (1%) gel electrophoresis and visualized by ethidium bromide staining; the sizes were estimated by comparison with DNA molecular weight markers. After electrophoresis, Southern blot analysis was performed to detect a specific signal with digoxigenin (DIG)-labeled probes for FSHR or GAPDH as previously described (30, 32). In addition, the PCR products isolated from gel were cloned into pCRII vector using the TA cloning kit (Invitrogen) and were sequenced by the dideoxy nucleotide chain termination method using the T7 DNA polymerase sequencing kit (Amersham Pharmacia Biotechnology).

Antibodies

The antibody of FSHR was kindly provided by Dr. J. A. Dias (Wadsworth Center, David Axelrod Institute for Public Health, Albany, NY). The antibodies of epithelial growth factor receptor (EGFR), c-myc, K-Ras, HER-2/neu, and the phosphorylated Jun N-terminal kinase (p-JNK) were purchased from Santa Cruz Biotechnology Ltd. (Santa Cruz, CA). The antibodies of pan- and phosphorylated p38 MAPK (p-p38) and pan- and phosphorylated ERK1/2 and pan-JNK were purchased from Biosource International, Inc. (Camarillo, CA).

Immunoblot assay

The cells were seeded at a density of 2 x 105 cells in 35-mm culture dishes and cultured in a humidified atmosphere of 5% CO2-95% air at 37 C. The cells were washed twice with ice-cold PBS and lysed in ice-cold radioimmunoprecipitation assay buffer [150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Tris (pH 7.5), 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 100 µg/ml aprotinin]. The extracts were placed on ice for 15 min and centrifuged to remove cellular debris. Protein amount of supernatants was determined using a Bradford assay (Bio-Rad Laboratories). Thirty micrograms of total protein were run on 10% SDS-PAGE gels and electrotransferred to a nitrocellulose membrane (Amersham Pharmacia Biotech). The membrane was immunoblotted using specific primary antibodies at 4 C overnight. After washing, the signals were detected with horseradish peroxidase-conjugated secondary antibody for 1 h and visualized using the enhanced chemiluminescence chemiluminescent system (Amersham Pharmacia Biotechnology). Quantitation of the immunoblots was performed on Scion Image 4.0.2 (Scion Corp., Frederick, MD). Briefly, intensities of interested protein bands were scanned and quantified by density plot (14).

In vitro growth assay

The nontransfected, control vector-transfected, and FSHR-transfected IOSE-80PC cells were plated in medium 199:MCDB 105 containing 10% FBS, 100 U/ml penicillin G, and 100 µg/ml streptomycin in a humidified atmosphere of 5% CO2-95% air in 24-well plates at a concentration of 1 x 105 cells/ml, maintained in logarithmic growth by passaging them every 2–3 d, and incubated for 1, 2, 4, and 6 d. The cells were washed in PBS and passed through a 22-gauge needle to generate a single cell suspension. Cell numbers were counted by the trypan blue exclusion method.

[3H]Thymidine incorporation assay

[3H]Thymidine incorporation assay was performed to analyze the effect of FSH on proliferative index in nontransfected and FSHR-transfected IOSE cell lines (14, 30). The cells were plated in 24-well plates at 2 x 104 cells/well in 0.5 ml medium 199:MCDB105 supplemented with 10% FBS and antibiotics and incubated for 48 h. Human recombinant FSH was purchased from National Hormone and Pituitary Program (Harbor-University of California-Los Angeles Medical Center, Torrance, CA), and epithelial growth factor (EGF) was obtained from Sigma-Aldrich. Before treatment with FSH and EGF, the cells were starved in serum-free media for 4 h. After starvation, the cells were incubated with 100 ng/ml FSH and/or 10 nM EGF in serum-free media for 24 h. One mirocurie [3H]thymidine (5.0 Ci/mmol; Amersham Pharmacia Biotech.) was added 4 h before the cells were harvested. At the end of the incubation period, the culture medium was removed, and cells were washed three times with PBS, followed by precipitation with 0.5 ml 10% trichloroacetic acid for 20 min at 4 C. The precipitate was washed in methanol twice and solubilized in 0.5 ml 0.1 N sodium hydroxide, and the incorporated radioactivity was measured in the Tri-Carb liquid scintillation analyzer (model 2100TR; Packard Instrument Co., Meriden, CT) as previously described (32).

Data analysis

Data are shown as the means of three individual experiments with triplicate and are presented as the mean ± SD. In the trypan blue and thymidine incorporation assays, the values are expressed as the percentage of growth, compared with a control, and are the mean ± SD of three individual experiments in triplicate. Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test or Dunnett’s test. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Stable transfection in IOSE cells

To generate the stable cell lines of FSHR overexpression, IOSE-398 and IOSE-80PC cells were transfected with FSHR expression vector (pcDNAHFSHR) containing G418-resistance. Consequently, stable cell lines that survived in the G418-containing media were selected for further characterization. The mRNA expression of FSHR in transfectants was investigated by RT-PCR and Southern blot analysis. A predicted PCR product of FSHR was obtained as 369 bp and confirmed by Southern blot analysis using DIG-labeled FSHR probe (Fig. 1AGo). Human granulosa luteal cells (hGLCs) and immortalized granulosa cells (SVOG-4o) were used for a positive control to express FSHR (33). As seen in Fig. 1AGo, the IOSE-80PC and IOSE-398 transfected with FSHR cDNA expression vector (80PCF and 398F, respectively) showed a high level of FSHR mRNA when compared with vector-transfected IOSE-80PC or IOSE-398 cells. hGLCs constitutively expressed FSHR mRNA as well. In parallel with FSHR mRNA level, expression level of FSHR protein was further examined by immunoblot analysis. The expression of FSHR protein was significantly enhanced in 80PCF, compared with nontransfected cells (Fig. 1BGo). Subsequently, further experiments were then performed using nontransfected IOSE-80PC (80PC), control vector-transfected IOSE-80PC (80PCV) and FSH-overexpressing IOSE-80PC (80PCF).



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  		FIG. 1. Overexpression of FSHR mRNA and protein in IOSE cell line. IOSE cell lines, OSE-80PC (80PC) and IOSE-398 (398), were transfected with FSHR overexpression vector using FuGENE 6 reagent, and stable cell lines were produced (80PCF and 398F, respectively). A, The mRNA expression of FSHR was investigated by RT-PCR and Southern blot analysis. A predicted PCR product of FSHR was obtained as 369 bp and confirmed by Southern blot analysis using DIG-labeled probe and sequence analysis (data not shown). The amplification of GAPDH (373 bp) was performed to rule out the possibility of RNA degradation and was used to control the variation in mRNA concentration in the PCR. B, The increased level of FSHR protein was demonstrated in FSHR-overexpressing 80PCF cells by Western blot analysis. The hGLCs and immortalized hGLCs (SVOG-4o) were used as a positive control. a, P < 0.05 vs. nontransfected control. C, Expression of FSHR in FSHR-overexpressing cell line. The expression levels of FSHR protein were measured in nontransfected (80PC), vector-transfected (80PCV), FSHR-transfected (80PCF), OVCAR-3 (OV), and SKOV-3 (SK) cells. Data are shown as the means of three individual blots and are presented as the mean ± SD. a, P < 0.05 vs. 80PC.

 
Expression of FSHR in the transfectants and ovarian cancer cell lines

To verify the expression levels of FSHR protein in nontransfected, FSHR-transfected IOSE, and ovarian cancer cell lines, immunoblot analysis was performed in 80PC; 80PCV; 80PCF; and ovarian cancer cell lines, OVCAR-3 and SKOV-3 cells. As seen in Fig. 1CGo, low levels of FSHR protein were demonstrated in nontransfected 80PC and vector-transfected 80PCV cell lines. It is of interest that the FSHR protein highly expressed up to a 7-fold increase in 80PCF and OVCAR-3 but not in SKOV-3 cells. The high level of FSHR protein was also observed in another human ovarian adenocarcinoma cell line, CaOV-3 cells (data not shown).

Expression of potential oncogenes in FSHR overexpressing 80PCF

To investigate whether oncogenes that play a critical role in tumor formation and progression of ovarian cancer are regulated by overexpression of FSHR, we determined the expression levels of oncogenes including EGFR, HER-2/neu, c-myc, and K-Ras in the nontransfected, vector-transfected, FSHR-transfected IOSE cell lines and two ovarian carcinoma cell lines, OVCAR-3 and SKOV-3, as a positive control. The expression levels of EGFR, c-myc, and HER-2/neu proteins were up-regulated by overexpression of FSHR in 80PCF cells (Fig. 2Go). In addition, the oncogenes including EGFR, K-Ras, and HER-2/neu were significantly overexpressed in OVCAR-3 and SKOV-3 cell lines. The OVCAR-3 cells expressed a high level of c-myc, but the expression level of c-myc is significantly lower in the SKOV-3 cell line. In contrast, no significant increased expression level of K-Ras protein was observed after FSHR overexpression. Therefore, overexpression of FSHR in preneoplastic IOSE-80PC enhanced EGFR, HER-2/neu, and c-myc proteins, which may be related with neoplastic transformation of OSE.



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  		FIG. 2. Effect of overexpressed FSHR on the expression levels of EGFR, HER-2/neu, c-myc, and K-Ras oncogenic pathways. The expression levels of EGFR, c-myc, K-Ras, and HER-2/neu were examined in nontransfected (80PC), vector-transfected (80PCV), FSHR-transfected (80PCF), OVCAR-3 (OV), and SKOV-3 (SK) cells. Data are shown as the means of three individual blots and are presented as the mean ± SD. a, P < 0.05 vs. 80PC.

 
Regulation of MAPKs in FSHR overexpressing 80PCF

To examine the effects of FSHR overexpression on MAPK phosphorylation in nontransfected and FSHR-transfected IOSE cell lines, we performed immunoblot analysis with specific antibodies that can detect total JNK, p38, and ERK1/2 and their phosphorylated forms. No significant difference was detected in the expression levels of phosphorylated p38 (p-p38) and p-JNK in 80PC, 80PCV, and 80PCF cell lines. Interestingly, phosphorylation of ERK1/2 was enhanced in the FSHR-overexpressing 80PCF cell line and two adenocarcinoma cell lines, OVCAR-3 and SKOV-3, compared with nontransfected 80PC and vector-transfected 80PCV cells (Fig. 3Go). These results suggest that overexpression of FSHR in IOSE-80PC cells may activate ERK1/2, which plays an important role in proliferation and is regulated by external stimuli such as hormones and growth factors in ovarian cancer (14).



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  		FIG. 3. Effect of overexpressed FSHR on the phosphorylation of p38, JNK, and ERK1/2 MAPK pathways. The p-JNK, p-p38, and phosphorylated ERK normalized by total JNK, p38, and ERK, respectively, were analyzed in nontransfected (80PC), vector-transfected (80PCV), FSHR-transfected (80PCF), OVCAR-3 (OV), and SKOV-3 (SK) cells (A). The phosphorylation of ERK is shown as the means of three individual experiments and presented as the mean ± SD. a, P < 0.05 vs. 80PC as a control (B).

 
Growth stimulation after FSHR overexpression

To investigate the role of FSHR in 80PCF, the doubling time of 80PCF cells was determined by trypan blue exclusion assay. The same number (1 x 105 cells/ml) of nontransfected, vector-transfected, and FSHR-transfected IOSE-80PC cells were seeded, and the cell numbers were counted after growth for 1, 2, 4, and 6 d. Significant growth stimulation was observed from 2 d in FSHR overexpressing 80PCF, compared with 80PC and 80PCV cells as seen in Fig. 4Go. The stimulatory effect in FSHR-overexpressing 80PCF was also observed in [3H] thymidine incorporation assay (Fig. 5Go). These results indicate that FSHR-overexpressing 80PCF proliferated up to 2-fold more rapidly, compared with 80PC and 80PCV cell lines.



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  		FIG. 4. Effect of overexpressed FSHR on cell growth. The cell growth pattern was compared in nontransfected (80PC), vector-transfected (80PCV), and FSHR-transfected (80PCF) by trypan blue exclusion method. Data are shown as the means of three individual experiments performed in triplicate and are presented as the mean ± SD. a, P < 0.05 vs. 80PC.

 


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  		FIG. 5. Effect of FSH and EGF in nontransfected 80PCV and FSHR-overexpressing 80PCF cells. The cells were treated with FSH (100 ng/ml) and EGF (10 nM) for 48 h, and a [3H]thymidine incorporation assay was performed as described in Materials and Methods. Data are shown as the means of three individual experiments performed in triplicate and are presented as the mean ± SD. a, P < 0.05 vs. untreated control; b, P < 0.05 vs. EGF-treated only; c, P < 0.05 vs. untreated 80PCV.

 
Effects of FSH and EGF in FSHR-overexpressing 80PCF

To evaluate the effects of FSH and EGF in FSHR-overexpressing 80PCF and 80PCV cells, the cells were treated with FSH (100 ng/ml) in the presence or absence of EGF (10 nM) for 48 h. The dose and time of FSH treatment (100 ng/ml for 48 h) were selected based on our previous study (14), and [3H]thymidine incorporation assay was performed. Treatment with FSH did not result in a significant increase in the thymidine uptake in 80PCV, whereas it induced a considerable increase in DNA synthesis in 80PCF cells (Fig. 5Go). The EGF treatment alone resulted in significant growth stimulation in both 80PCV and 80PCF cells, but it appears that the effect of EGF on the stimulation of cell growth is much higher in 80PCF when compared with 80PCV cells. It is of interest that treatment with FSH plus EGF potentiated EGF-induced cell proliferation in both 80PCV and 80PCF cells as seen in Fig. 5Go.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In addition to its well-documented functions in ovarian physiology, FSH, one of the pituitary glycoprotein hormones, has been suggested to play a role in ovarian cancer development. An increased occurrence of ovarian cancer with exposure to high levels of gonadotropins during postmenopause or infertility therapy has been suggested by epidemiological studies (5, 22, 23). Despite an involvement of FSH in ovarian tumorigenesis (20, 21), limited information such as mRNA levels is available regarding the expression of FSHR in normal and neoplastic OSE cells (12, 21, 35, 36). Recent results indicate that the levels of FSHR increased from presumed precursor lesions, ovarian epithelial inclusions, to benign ovarian epithelial tumors (OETs) and borderline OETs, whereas its levels decreased from borderline ovarian tumors to ovarian carcinomas (26). Although it is currently unclear whether OETs develop from OSE and/or ovarian epithelial inclusions to cystadenomas, borderline tumors and carcinomas, these results suggest that not only serum FSH but also FSHR in ovarian epithelium may play a significant role in ovarian OET development. In this regard, it is important to confirm the protein expression level of FSHR in normal and neoplastic ovarian epithelium cells and to discern particular molecular changes after the binding of FSH to its receptor. To understand the role of FSHR and its overexpression in ovarian cancer, we evaluated altered signaling pathways after FSHR overexpression in 80PCF transfected with human FSHR cDNA. The FSHR protein was highly expressed in OVCAR-3, compared with IOSE-80PC cells, and scarcely expressed in SKOV-3 cells. The SKOV-3 cells represent a late stage of ovarian carcinogenesis, and a growth of ovarian cancer at the malignant stage becomes gonadotropin independent. Thus, it is not surprising that the level of FSHR is low in SKOV-3 cells, and it appears that the growth of SKOV-3 cells may be independent on FSH action. The FSHR-overexpressing 80PCF cells showed a significantly high level of FSHR mRNA and protein levels, compared with the 80PC and 80PCV cell lines, and the expression levels of FSHR mRNA and protein of 80PCF cells were comparable with those of OVCAR-3 cells.

Epithelial ovarian carcinomas, which comprise approximately 90% of human ovarian cancer, arise in the OSE, whereas the rest originate in granulosa cells or, occasionally, in the stroma or germs cells (37). The OSE is composed of a single layer of flat to cuboidal epithelial cells with few distinguishing features. Besides, there are no good animal models that develop ovarian epithelial carcinoma, and it is difficult to isolate and maintain normal human OSE under experimental conditions. Thus, in contrast to neoplasms in other organs in which the normal tissue of origin is well defined, the physiology and susceptibility to oncogenic influences of the OSE are poorly understood (38, 39). Nevertheless, some recent studies contribute to our understanding of the biology of OSE and carcinogenesis. The OSE in mature women expresses a combined epitheliomesenchymal phenotype; epithelial features include keratin, mucin, desmosomes, apical microvilli, and a basal lamina, but, like mesenchymal or stromal cells, OSE contains vimentin and N-cadherin and lacks the epithelial differentiation markers CA125 and E-cadherin (38, 39).

With neoplastic progression, the tendency of OSE to undergo epitheliomesenchymal conversion diminishes, and the cells become increasingly committed to complex epithelial phenotypes, which include the appearance of E-cadherin (40, 41), the receptor for hepatocyte growth factor (c-met) (42), and secretory products including mucins (MUC1, MUC2, MUC3, and MUC4) and CA125 (43, 44). The genetic changes such as augmentation, changed expression, and mutations in a few protooncogenes and tumor suppressor genes play a role in the progression of ovarian epithelial cancer. Thus, c-myc (45), K-Ras (46), ERBB2 (47), EGFR (48), and cFMS (the receptor for colony-stimulating factor 1) (49) are frequently amplified in ovarian carcinoma. In the case of tumor suppressor genes, BRCA1/BRCA2 (50), p53 (49), phosphatase and tensin homolog deleted from chromosome 10 (51), and ras homolog gene family, member 1 (NOEY2, ARHI) (52) are frequently mutated in ovarian cancer. In the present study, we have demonstrated that EGFR, K-Ras, and HER-2/neu, which are overexpressed in ovarian cancer, are highly expressed in two ovarian cancer cell lines, OVCAR-3 and SKOV-3, compared with preneoplastic IOSE cell lines, whereas c-myc was highly expressed in OVCAR-3 but not in SKOV-3 cells. It is of interest that FSHR-overexpressing 80PCF cells showed high levels of EGFR, c-myc, and HER-2/neu. These results suggest that overexpression of FSHR resulted in an increased expression of EGFR, c-myc, and HER-2/neu. The mechanism by which levels of EGFR, c-myc, and HER-2/neu are enhanced through overexpression of FSHR warrants further investigation.

MAPKs are a group of serine/threonine kinases that are activated in response to a diverse array of extracellular stimuli and mediate signal transduction from the cell surface to the nucleus (53, 54). These MAPKs are divided into three subgroups: ERK1/2, JNKs, and p38. It is well known that MAPK cascade is activated by two distinct classes of cell surface receptors, receptor tyrosine kinases and G protein-coupled receptors (54, 55, 56, 57, 58). The signals transmitted through this cascade lead to activation of a set of molecules that regulate cell growth, division, and differentiation. The most extensively studied members of the cascade are the ERK1 (p44 MAPK) and ERK2 (p42 MAPK). In ovarian cancer cells, MAPKs are regulated by cisplatin (59), paclitaxel (60), endothelin-1 (61), GnRH (62), and FSH (14). Moreover, in a previous study, we have shown that FSH stimulated the activation of MAPK cascade and phosphorylated Elk-1 in human OSE cells, which was responsible for proliferation by FSH (14). In the present study, we observed that the constitutive phosphorylation of ERK1/2 was higher in FSHR-overexpressing 80PCF cells than 80PC and 80PCV cells, whereas there was no difference in the expression levels of p-JNK and p-p38. Although there is evidence that the MEK-p38 MAPK-CK2 pathway participates in ovarian epithelial carcinogenesis (63), we failed to observe any change of pan- and phospho-p38 level in FSHR-overexpressing 80PCF cells, compared with the control. These results suggest that the ERK1/2 pathway is probably a downstream cascade of FSHR-induced signaling pathways, and FSHR accelerates the phosphorylation of ERK1/2 but does not increase the constitutive protein level of ERK1/2.

To evaluate the contribution of the FSHR to cell growth, we carried out a cell growth assay and demonstrated that FSHR-overexpressing 80PCF cells showed rapid growth when compared with nontransfected and vector-transfected cell lines. It is known that aberrant oncogene magnification and inappropriate constitutive expression of growth factors and their receptors may lead to uncontrollable growth of transformed cells (34). In the present study, treatment with FSH did not result in a significant increase in cell growth of 80PCV, whereas it induced a considerable increase in DNA synthesis of 80PCF cells. Although the expression levels of FSHR mRNA and protein are enhanced in 80PCF cells, the effect of FSHR overexpression on the cell growth is modest. It appears that the effect of EGF on the stimulation of cell growth is much higher in 80PCF when compared with 80PCV cells, indicating that elevated EGFR derived from FSHR overexpression may enhance the effect of EGF on the cell growth of 80PCF. In addition, it is of interest that treatment with FSH plus EGF potentiated EGF-induced cell proliferation in both 80PCV and 80PCF cells. Thus, these results suggest that the mitogenic effect due to FSHR overexpression may be derived from enhanced oncogenic pathways including EGFR, c-myc, and HER-2/neu. In addition, the activated ERK1/2 pathway is also involved in increased cell proliferation in FSHR-overexpressing 80PCF cells. Regarding the effect of FSH in the FSHR-overexpressing cell line, 80PCF, treatment with FSH induced a considerable increase in cell growth of 80PCF cells, whereas it failed to increase the thymidine uptake in 80PCV cells, suggesting that overexpression of FSHR may amplify the role of FSH in cell growth and intracellular signaling pathways via FSHR. The effect of EGF on the stimulation of cell growth is much greater in 80PCF when compared with 80PCV cells, suggesting that this effect of EGF in the cell growth is enhanced through FSHR-induced EGFR amplification. Furthermore, treatment with FSH plus EGF potentiated EGF-induced cell proliferation in both 80PCV and 80PCF cells, indicating that there is cross-talk between FSH-FSHR and EGF-EGFR.

In conclusion, the overexpression of FSHR increased the expression of EGFR, c-myc, and HER-2/neu and activated ERK1/2 MAPK in IOSE cells. In addition, the overexpression of FSHR accelerated cell proliferation in these cells. These results may support a pivotal role of FSHR in ovarian cancer development in terms of neoplastic conversion and growth potential.


   Acknowledgments
 
The expression vector of human FSHR (pcDNAHFSHR) and FSHR monoclonal antibody were generously provided by Drs. T. Minegishi (Gumma Medical School, Gumma, Japan) and J. A. Dias (Wadsworth Center, David Axelrod Institute for Public Health, Albany, NY), respectively. We are thankful to Dr. A. Godwin (Fox Chase Cancer Center, Philadelphia, PA) for providing IOSE-80PC cell line.


   Footnotes
 
This work was supported by the Canadian Institutes of Health Research. P.C.K.L. is the recipient of a Distinguished Scholar Award from the Michael Smith Foundation for Health Research. J.-H.C. is the recipient of a graduate studentship awarded from the British Columbia Research Institute of Children’s and Women’s Health.

Abbreviations: DIG, Digoxigenin; EGF, epithelial growth factor; EGFR, EGF receptor; FBS, fetal bovine serum; FSHR, FSH receptor; GAPDH, glyceraldehyde phosphate dehydrogenase; hGLC, human granulosa luteal cell; IOSE, immortalized OSE; OET, ovarian epithelial tumor; OSE, ovarian surface epithelium; p-JNK, phosphorylated Jun N-terminal kinase; p-p38, phosphorylated p38 MAPK.

Received January 9, 2004.

Accepted August 19, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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