Profiling of Protein Kinases in the Neoplastic Transformation of Human Ovarian Surface Epithelium

Gynecologic Oncology 82, 305–311 (2001) doi:10.1006/gyno.2001.6280, available online at http://www.idealibrary.com on

Profiling of Protein Kinases in the Neoplastic Transformation of Human Ovarian Surface Epithelium 1 Alice S. T. Wong, Ph.D.,* Sung O. Kim,† ,‡ Peter C. K. Leung, Ph.D.,* Nelly Auersperg, M.D., Ph.D.,* ,2 and Steven L. Pelech, Ph.D.† ,‡ *Department of Obstetrics and Gynaecology and †Department of Medicine, University of British Columbia, Vancouver, British Columbia V6H 3V5; and ‡Kinexus Bioinformatics Corporation, Vancouver, British Columbia, Canada Received December 8, 2000

INTRODUCTION

Objective. The aim of this study was to study the pattern of protein kinase expression in a culture model of epithelial ovarian carcinogenesis. Methods. Cultures of normal human ovarian surface epithelium (OSE), OSE from women with BRCA1 mutations, a cell culture model of preneoplastic (SV40 T-antigen-immortalized, nontumorigenic) and neoplastic (SV40-E-cadherin transfected, tumorigenic) OSE, and three ovarian cancer cell lines were used to represent OSE phenotypes of different genetic backgrounds and at different, progressive stages of neoplastic transformation. The protein kinase network signaling was studied by Western blotting, simultaneously using multiple antibodies for specific protein kinases. Results. High levels of cGMP-dependent protein kinase were found in normal and preneoplastic OSE, but were absent in neoplastic OSE. In contrast, expression of MEK6 was detected exclusively in neoplastic OSE. The expressions of casein kinase II (CK2), p38 mitogen-activated protein kinase (MAPK), cyclindependent kinase, and the phosphatidylinositol 3-kinase (PI3K) effectors Akt2 and p70 S6 kinase (S6K) were several-fold higher in neoplastic OSE than in normal OSE, whereas the expressions of the MAPKs extracellular signal-regulated kinases ERK1 and -2 were unchanged. Importantly, constitutive phosphorylation of Akt2 and p70 S6K, as found in neoplastic OSE, was also observed in overtly normal OSE from women with predisposing BRCA1 gene mutations. Conclusions. These data demonstrate that different repertoires of downstream signaling proteins, particularly those of the MEK6 –p38 MAPK–CK2 pathway and the PI3K pathway, are correlated with phenotypic manifestations of a cell culture model of OSE at progressive stages in the development of ovarian cancer. Changes in PI3K effectors are already found in overtly normal OSE from women with BRCA1 mutations. © 2001 Academic Press Key Words: protein kinase; ovarian surface epithelium; epithelial ovarian cancer. 1 Supported by grants to N.A. and S.L.P. by the National Cancer Institute of Canada with funds from the Terry Fox Run and to A.S.T.W. by a University of British Columbia Graduate Fellowship. 2 To whom correspondence should be addressed at Faculty of Medicine, Department of Obstetrics and Gynecology, The University of British Columbia, Room 2H30, 4490 Oak Street, Vancouver, B. C., Canada V6H 3V5. Fax: (604) 875-2725. E-mail: [email protected].

Ovarian cancer is the fourth or fifth most common cause of death from all cancers among women in the Western world and the leading cause of death from gynecological malignancies. Amplification, altered expression, and malfunction of a number of protein kinases and phosphatases participate in the development of ovarian epithelial neoplasms. The phosphatidylinositol 3-kinase (PI3K) pathway in particular is activated in a significant proportion of ovarian cancers. Increased PI3K activity is important in the growth and dissemination of ovarian cancer cells [1]. The PIK3CA gene, which encodes the p110␣ catalytic subunit of PI3K, and its downstream effector Akt2 are amplified in primary ovarian tumors and ovarian cancer cell lines (including OVCAR-3 which is used in the present study) [2– 4]. Overexpression of Akt2 is often associated with highgrade and late-stage tumors [3]. Mutation and/or down-regulation of the PI3K phosphatase PTEN/MMAC1 are frequently observed in ovarian endometrioid carcinomas [5]. While Ras is frequently mutated in the mucinous type of ovarian carcinomas, Src was shown to be overexpressed and activated in some late-stage tumors [6]. Akt2 mediates some of the transforming signals of Ras and Src [7]. Aberrant cell signaling through protein kinases can also result from alterations in growth factor receptors, acquired during malignant transformation. Of particular interest for ovarian cancer is the epidermal growth factor family, including the HER2/Neu receptor. HER2/Neu is amplified and overexpressed in 25–30% of ovarian and breast cancers, and this overexpression is associated with a poor prognosis. The macrophage colony stimulating factor receptor (fms) is expressed by many ovarian cancers but not by benign ovarian tumors or normal OSE. Both HER2/Neu and fms have been implicated in the altered cellular signaling found among ovarian cancers [8, 9]. In this study, we investigated changes in the expression of protein kinases during ovarian cancer development by comparing the expression of a wide range of protein kinases in ovarian cancer cell lines to its precursor, the ovarian surface epithelium (OSE). In addition to normal OSE from the general

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population, overtly normal OSE from women with proven BRCA1 mutations were included to investigate preneoplastic or predisposing changes in the OSE. To examine intermediate stages in the neoplastic progression of OSE, we introduced transforming genes into normal OSE [10]. We also examined three ovarian carcinoma lines: OVCAR-3, SKOV-3, and CaOV-3. This experimental model represents OSE at different, progressive stages of neoplastic progression. We used multiple antibodies for specific protein kinases on Western blots. This method enabled us to detect simultaneously a wide range of protein kinases of several signaling pathways and define their interrelated changes. We and others have shown that hepatocyte growth factor (HGF) induces cellular proliferation in normal and neoplastic OSE [11, 12]. In this study, HGF was used to investigate the signaling pathways that are responsible for this effect and to identify changes in protein kinase expression and phosphorylation. MATERIALS AND METHODS Cells and Cell Lines Institutional approval for experimentation with human tissues was obtained prior to this study. OSE was obtained from ovarian biopsies and at laparoscopy from 10 women from the general population with no family histories of breast/ovarian cancer (NFH-OSE) having surgery for nonmalignant gynecologic diseases. Another group of 6 women underwent prophylactic oophorectomies because of predisposing BRCA1 mutations. Histopathological analysis confirmed the absence of neoplasms in all specimens. OSE fragments were scraped off the ovary and cultured intact without prior dissociation. They were characterized by their distinct morphology and keratin expression [13]. The human ovarian adenocarcinoma lines CaOV-3, OVCAR-3, and SKOV-3 (ATCC Nos. HTB75, -77, and -161) were included in the study. To examine intermediate changes in the neoplastic progression of OSE, we introduced transforming genes into normal OSE. IOSE-29 was obtained by immortalization of normal OSE with SV40 large T antigen. These cells have an extended life span in culture, but are nontumorigenic in athymic and SCID mice [14, 15]. Cotransfection of mouse E-cadherin and neomycin-resistance (SV2neo) genes into IOSE-29 cells produced the IOSE-29EC cell line [10] which expressed characteristics associated with ovarian neoplasia and formed tumors resembling serous adenocarcinomas in SCID mice. From one of these tumors, we generated the ovarian tumor cell line IOSE-29EC/T4 [15]. Parental cells sham-transfected with SV2neo were designated IOSE-29neo [10]. This cell culture model represents OSE cells at different, progressive stages of ovarian carcinogenesis. Cell Culture Cells were routinely grown and maintained in Medium 199/ MCDB 105 (1:1) (Sigma, St. Louis, MO) with 50 ␮g/ml

gentamicin (Gibco, Grand Island, NY), supplemented with 10 –15% fetal bovine serum (Hyclone, Logan, UT) for OSE and with 5% newborn calf serum (NCS) for immortalized cell lines, E-cadherin-transfected IOSE, and ovarian cancer cell lines at 37°C in a humidified incubator with 5% CO 2:95% air. All were subcultured with 0.06% trypsin/0.01% EDTA in Ca 2⫹-, Mg 2⫹-free Hanks’ balanced salt solution. Before Western blot analysis, subconfluent cells were cultured overnight in serum-free medium which renders them stationary or with reduced (2%) NCS which permits limited cell division. There was no difference in the expression of the protein kinases reported here between cultures maintained under these two conditions, indicating that there were no cell-cycle-dependent changes in the protein levels of these kinases. All kinase determinations were therefore carried out in 2% NCS, as the more physiologically appropriate of the two media. To investigate the protein–serine kinases that might be relevant to the mitogenic actions of HGF and to identify altered protein kinases in OSE, cells were treated with 20 ng/ml recombinant HGF (R&D systems, Minneapolis, MN) at 37°C for 10 min. Antibodies Affinity-purified rabbit polyclonal antibodies for extracellular signal-regulated kinase (ERK1 and ERK2), ribosomal S6 kinase (Rsk1), Rsk2, cGMP-dependent protein kinase (PKG), cyclin-dependent kinase (CDK1), casein kinase II (CK2), Akt1 (PKB␣), Akt2 (PKB␤), p70 S6 kinase (p70 S6K), and glycogen synthase kinase 3␤ (GSK3␤) were raised against the synthetic peptides [16]. Most of the aforementioned polyclonal rabbit antibodies, as well as those for MEK6, p38 mitogenactivated protein kinase (MAPK), and integrin-linked kinase (ILK) were obtained from StressGen Biotechnologies Corp. (Vancouver, B.C., Canada). Secondary antibody of goat antirabbit IgG conjugated to horseradish peroxidase was purchased from Calbiochem (San Diego, CA). Western Blot Analysis Cells were lysed in buffer (20 mM MOPS, pH 7.2, 5 mM EGTA, 2 mM EDTA, 0.1 mM sodium orthovanadate, 1 mM sodium fluoride, 24 mM ␤-glycerophosphate, and 1% v/v Triton X-100) containing freshly added protease inhibitors (1 mM PMSF, 25 ␮g/ml leupeptin, 1.4 ␮g/ml pepstatin A, 2 ␮g/ml aprotinin, and 0.5 mM DTT). Cell lysates containing 50 ␮g protein as measured by the Bio-Rad Bradford protein assay were separated on 12.5% SDS–PAGE gels. Proteins were transferred onto nitrocellulose membranes. Blots were then blocked for 30 min with 5% w/v nonfat dried milk and probed for 3 h with primary antibodies against specific kinases. In some experiments, we used a Bio-Rad 20 lane multiblotter for detection of at least 15 different proteins simultaneously on Western blots. After extensive washing, immune complexes were detected with goat anti-rabbit (1:20,000) IgG conjugated with horseradish peroxidase for 1 h followed by an enhanced

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chemiluminescence (ECL) detection system (Amersham, Arlington Heights, IL). Changes in phosphorylation, indicative of kinase activation, were deduced from band shifts [16, 17]. The expressions of total ERK1 and ERK2 MAPK, which were unchanged among of the subgroups, confirmed standardized loading of the cell lysates in the Western blots. Densitometry and Statistical Analysis Images from ECL autoradiograms were captured using NIH Image and Adobe software. To determine whether differences in specific protein kinase expression at different stages of ovarian neoplastic progression were present, the Kruskal–Wallis test was applied. Differences were considered significant at P ⬍ 0.05. All statistical evaluations were performed using the Prism statistical software package (GraphPad Software, San Diego, CA). RESULTS The expression and reductions of SDS–PAGE mobility of a variety of protein kinases were studied immunologically in normal, preneoplastic (SV40 large T-antigen-immortalized, nontumorigenic) and neoplastic (IOSE-29EC, IOSE-29EC/T4, and ovarian cancer cell lines) OSE. Such reductions in the SDS– PAGE mobilities of many protein kinases, including ERK1, ERK2, Akt1, Akt2, p70 S6 kinase, Rsk1, and Rsk2, are known to reflect their phosphorylation and activation of their catalytic activities [12, 16, 17]. The immunoreactivities were quantified by densitometry on autoradiographs of immunoblots and their mean intensities are summarized in Table 1. As shown in Fig. 1, PKG was found at high endogenous levels in normal OSE and at low levels in preneoplastic OSE and was absent in neoplastic OSE (P ⬍ 0.001). In contrast to PKG expression, MEK6 was absent in normal and preneoplastic OSE and detected consistently in neoplastic OSE (P ⬍ 0.001). There was a trend for higher MEK6 protein expression levels in ovarian cancer cell lines than in the neoplastic SV40-E-cadherin transfected OSE. The expression of p38 MAPK, a downstream effector of MEK6, was also upregulated and expressed at 2-fold increased values in (pre)neoplastic OSE (P ⬍ 0.05). As observed in a variety of tumors, levels of CK2 were substantially higher (2- to 3-fold) in preneoplastic and neoplastic OSE than in normal OSE (P ⬍ 0.05). CDK1 was expressed at relatively low levels in normal OSE, and its expression level was increased by more than 10-fold in preneoplastic and neoplastic OSE (P ⬍ 0.001). Expression of GSK3␤ was 2- to 3-fold higher in preneoplastic, tumorigenic IOSE-29EC and IOSE-29EC/T4 and the ovarian cancer cell lines CaOV-3 and OVCAR-3, but not in SKOV-3. Classical PKC-␣, ␤, which were abundant in SV40-E-cadherin transfected OSE cells, were expressed at a 5-fold reduced levels in normal OSE and all three ovarian cancer cell lines (P ⬍ 0.05). Expression of Rsk1 was modestly higher in preneoplastic and neoplastic OSE than in normal OSE, and this difference was not significant (P ⬍ 0.05)

FIG. 1. Expression of protein kinases (PKG, MEK6, p38 MAPK, CK2, CDK1, GSK3␤, PKC-␣,␤, and Rsk1) of normal, preneoplastic (IOSE-29), and neoplastic (IOSE-29EC, IOSE-29EC/T4, OVCAR-3) OSE, with or without HGF stimulation (20 ng/ml), assessed by Western blots. Note that PKG is expressed only in OSE and IOSE-29, whereas MEK6 is found only in IOSE29EC, IOSE-29EC/T4, and OVCAR-3. Expression of p38 MAPK, CK2, CDK1, and GSK3␤ increases with the neoplastic progression of OSE. High levels of PKC-␣,␤ are found in IOSE-29EC and IOSE-29EC/T4. With the exception of Rsk in all but OVCAR-3, there was no change in the expression of these kinases with or without HGF stimulation. n.d., not determined.

(Fig. 1). Akt1 was expressed at high levels in tumorigenic lines IOSE-29EC and IOSE-29EC/T4 (Fig. 2a). Expression of other downstream regulators of PI3K, Akt2 (Fig. 2b), and p70 S6K (Fig. 2c) were 2- to 5-fold higher in neoplastic SV40-E-cadherin transfected OSE and in some, but not all, ovarian cancer cell lines than in normal and preneoplastic OSE (P ⬍ 0.05). There was no difference in the expression of ERK1 and ERK2 among the subgroups (P ⬎ 0.05) (Fig. 2d). Phosphorylation of Rsk1, Akt1, Akt2, ERK1 and ERK2, and p70 S6K was implied from mobility reductions on Western blots [16, 17]. These kinases were rapidly phosphorylated by HGF (Figs. 1, 2), and the HGF-induced mobility shifts were completely or partially reversed by the use of inhibitors of specific protein kinases (i.e., PD98059 for Mek1, LY294002 for PI3K, rapamycin for FRAP/mTOR; data not shown). In fact, in the presence of LY294002, the Akt1, Akt2, and p70 S6K bands migrated with faster mobilities than was evident in the NFH-OSE cells in the absence of HGF (data not shown). Upon HGF treatment, Akt1, Akt2, and p70 S6K underwent further reductions in mobility, which was indicative of further phosphorylation of these protein kinases in response to HGF. Some of the dephosphorylated form of Akt1 was found in neoplastic SV40-E-cadherin transfected OSE and ovarian can-

Kinase

ERK1/ERK2

Rsk1

Rsk2

PKC-␣,␤ ILK

Akt1

❚❚❚❚ ❚❚❚❚❚❚■ ❚❚❚❚❚❚❚ ❚❚❚❚❚❚■❙ ❚❚❚❚❚❚■ ❚❚❚❚❚❙ ❚❚❚❚❚❚■❚❚ ❚❚

❚❚❚❚❚■❚ ❚❚❚❚❚■ ❚❚❚❙ ❚❚❚❚❚■❚ ❚❙

❚❚❚ ❚❚❚ ❚❚

❚❚❚ ❚❚❚ ❚❚❚

GSK3␤

❚❚❚ ❚❚❚❙

❚❙ ❚❙ ❚❙ ❚❙ ❚❙ ❚❙ ❚

❚❙ ❚ ❚ ❚❙ ❚ ❚❙ ❚❙

Akt2

❚❚❚❚❚❚■❙ ❚❚❚❚❚❚■❚❚ ❚❚❚❚ ❚❚❚❚❚❚ ❚❚❚❙

❚❚❚❚ ❚❚❚❚ ❚❚❚❚ ❚❚❚

❚❚ ❚❚ ❚❚❙ ❚❚ ❚❚❙ ❚❚ ❚❚❙ ❚❚❙ ❚❚ ❚❚❙ ❚❚❙ ❚❚❙ ❚❚ ❚❚❙ ❚❚❚

p70 S6K

N/S N/S N/S N/S

❚❚ ❚❚ ❚ ❚

❚❚❚❚❚

❚❚❚❚❚ ❚❚❚❚❚❚❚ ❚❚❚❚❚❚

❚❚❚❚

PKG

❚❚❚❚❚❚ ❚❚❚❙ ❚❚❚❚❚❚■❚ ❚❚❚❚❚❚■

N/S N/S N/S N/S

N/S N/S N/S

N/S N/S N/S

MEK6

❚❚❚❚❚❚■❚❚ ❚❚❚❚❚❚■❚ ❚❚❚❚❚❚■ ❚❚❚❚❚❚■ ❚❚❚❚❚❚■

❚❚❚❚❚❚❚ ❚❚❚❚❚❚■ ❚❚❚❚❚❚■ ❚❚❚❚❚❚■

❚❚❚❚

❚❚❚❚❚ ❚❚❚❚❚ ❚❚❚❚❚

❚❚❚❚❙

p38 MAPK

❚❚❚❚❚❚■❚❙ ❚❚❚❚❚❚■❚

❚❚❚❚❚❚■❚❚❙ ❚❚❚❚❚❚■❚❚

❚

❚❙

CDK1

❚❚❚❚

❚❚❚❚❚❚ ❚❚❚❚❚❚❚❚

❚❚❚

❚❚ ❚❚

CK2

Note. Empty spaces represent kinase/cell type combinations that were not tested. p., passage; N/S, no signal. The numbers following the designations NFH-OSE (no family history, general population) and BRCA1⫹ (family history, carry BRCA1 mutations) identify individual cases. The length of the bars is proportional to densitometric readings of individual cases of normal OSE, while in neoplastic and preneoplastic OSE the bars represent means with standard deviations of less than 5% of three separate experiments.

Normal OSE NFH-OSE-250 p.0 ❚❚❚❚❚ ❚❚❚❚❚❚■ NFH-OSE-299 p.1 NFH-OSE-300 p.1 NFH-OSE-190 p.2 NFH-OSE-277 p.2 ❚❚❚❚❙ ❚❚❚❚❚❙ ❚❚❚❚❚❚❙ ❚❙ ❚❚❙ ❚❚❚❚ NFH-OSE-281 p.2 ❚❚❚❚ ❚❚❚❚❚❚ ❚❚❚❚ NFH-OSE-288 p.2 ❚❚❚❚❙ ❚❚❚❚❚❚ ❚❚❚❚❙ NFH-OSE-294 p.2 NFH-OSE-135 p.3 ❚❚❚❚❚ ❚❚❙ NFH-OSE-242 p.3 BRCA1⫹ 233 p.1 BRCA1⫹ 236 p.1 BRCA1⫹ 293 p.1 BRCA1⫹ 204 p.2 ❚❚❚❚ ❚❚ ❚❚❙ ❚❚❚❚❚ BRCA1⫹ 215 p.2 ❚❚❚❚❙ ❚❙ ❚❚❙ ❚❚❚❚❚ BRCA1⫹ 286 p.3 ❚❚❚❚ ❚❚❚❚ Preneoplastic, nontumorigenic SV40 large T-antigen-immortalized OSE IOSE-29 ❚❚❚❚❚ ❚❚❙ ❚❚❚ ❚❚❚❚ IOSE-29neo ❚❚❚❚❚ ❚❚❚❚❚❚ ❚❚❚❚ ❚❚❚❙ ❚❚❚❚❚❚ ❚❚❚❚❙ IOSE-120 ❚❚❚❚ ❚❚❚ ❚❚❚ ❚❚❚❚❙ IOSE-120neo ❚❚❚❚ ❚❚❚❙ ❚❚❚❚ Neoplastic, tumorigenic SV40-E-cadherin transfected OSE and ovarian cancer cell lines IOSE-29EC ❚❚❚❚❚ ❚❚❚❚❚❚■ ❚❚❚❚❚❚❙ ❚❚❚❚❚❚■❚❚ ❚❚❚❙ ❚❚❚❚❚■❙ IOSE-29EC/T4 ❚❚❚❚❚ ❚❚❚❚❚❚■❙ ❚❚❚❚ ❚❚❚❚❚❚■ ❚❚❚❚❚ ❚❚❚❚❚❚❚ CaOV-3 ❚❚❚❚❚ ❚❚❚❚❚❚■ ❚❚❚❚ N/S ❚❚❚ OVCAR-3 ❚❚❚❚ ❚❚❚❚❚❚■ ❚❚❙ N/S ❚❚❚❚❚ ❚❚❚❙ SKOV-3 ❚❚❚❚❙ ❚❚❚❚❚❚■❙ ❚❚❚❚❙ N/S ❚❚❚❙ ❚❚❚

Cell type

TABLE 1 Expression Profile of Protein Kinases in Normal, Preneoplastic, and Neoplastic OSE

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FIG. 2. Effects of HGF stimulation on kinase phosphorylation of representative samples of OSE from the general population (NFH-OSE) and from women with BRCA1 mutations (BRCA1⫹) and preneoplastic (IOSE-29) and neoplastic OSE (IOSE-29EC, IOSE-29EC/T4, and OVCAR-3), assessed by phosphorylation-induced reductions of kinase mobilities on Western blots. The bottom (faster migrating) bands generally represent the unphosphorylated (or less phophorylated) form of the kinases, whereas the upper (slower migrating) bands represent the phosphorylated forms. Treatment with 20 ng/ml HGF resulted in the phosphorylation of (a) Akt1, (b) Akt2, and (c) p70 S6K and (d) the p44 ERK1 and p42 ERK2 MAPKs in all subgroups. Note that phosphorylated forms of Akt2 and p70 S6K are present even in the absence of HGF stimulation in OSE from women with BRCA1 mutations and preneoplastic and neoplastic OSE, but not in NFH-OSE. The expression levels of Akt2 and p70 S6K are increased in neoplastic OSE.

cer cell lines, but not in normal and preneoplastic OSE (Fig. 2a). Akt2 and p70 S6K were not only overexpressed but also constitutively phosphorylated, as indicated by the increase in upper (slower migrating) bands, in both preneoplastic and neoplastic OSE (Figs. 2b and 2c). Such phosphorylation was also found in OSE from all women with BRCA1 mutations, but only in some of the NFH-OSE cases [12]. In summary, there were no differences in expression levels, but there were differences in phosphorylation between OSE from the general population and cancer-prone individuals. PKG expression was highest in NFH-OSE, reduced in immortalized nontumorigenic lines, and undetectable in the tumorigenic lines, whereas MEK6 was found only in tumorigenic lines. The expression of p38 MAPK, CK2, CDK1, and GSK3␤ were increased with the neoplastic progression of OSE. High levels of PKC-␣,␤ were found in IOSE-29EC and IOSE-29EC/T4. Some differences in the regulation of protein kinases among the ovarian cancer cell lines CaOV-3, SKOV-3, and OVCAR-3 were observed, despite the similarities in the expression of most protein kinases. While Akt2 and GSK3␤ were present at high levels in OVCAR-3 and at intermediate levels in CaOV-3, their expression was limited in SKOV-3 (Fig. 3). Among the three cancer lines, OVCAR-3 was the most similar to the experimental tumorigenic lines IOSE-29EC and IOSE-29EC/ T4. DISCUSSION In this report, we focused on the tracking of a wide range of protein kinases of multiple signaling pathways in epithelial

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ovarian carcinogenesis using normal OSE and ovarian cancer cell lines and an experimental model that represents progressive stages in ovarian neoplastic transformation. Our data indicate that, in our experimental model, the expression of various signaling proteins of the MEK6 –p38 MAPK–CK2 pathway and the PI3K pathway correlated with phenotypic manifestations of OSE at progressive stages in the development of ovarian cancer. Changes in PI3K effectors are already found in overtly normal OSE from women with BRCA1 mutations and thus may represent an early step in the development of ovarian cancer. Signal threshold is an important concept in signaling specificity. In many systems, a twofold decrease in the level of a signaling protein can be sufficient to abrogate signaling, and conversely a twofold increase can initiate signaling [18]. Some of our most profound changes in the expression profile of protein kinases in ovarian carcinogenesis were observed for the cGMP-dependent protein kinases (PKG) and MEK6. These results indicate that the downregulation of PKG and upregulation of MEK6 show a very significant correlation with the development of ovarian cancer. The mammalian ovary is under constant stimulation by pituitary gonadotropins, which can exert their effects by activating cGMP synthesis through nitric oxide [19]. PKG is the primary mediator of cGMP events in eukaryotes. PKG has been implicated in the stimulation of smooth muscle contraction and cellular proliferation, and the latter activity has been shown to be mediated through the small GTP-binding protein Ras [20 –22]. Thus, PKG may play a role in the contractile and wound repair activity of OSE following ovulatory ruptures. Altered expression of MEK6 in neoplastic OSE was associated with an increased expression of p38 MAPK and CK2, indicating that the MEK6 –p38 MAPK–CK2 pathway, which is an important component of the stress response required for the homeostasis of a cell, may play an important role in the neoplastic progression of OSE. CK2 has been associated with proliferation and cell transformation, because CK2 activity and protein levels are commonly elevated in solid human tumors and transformed cell lines [23–28]. p38 MAPK can directly activate CK2 by an allosteric mechanism, and this interaction regulates cell cycle progression, DNA repair, and apoptosis through p53 [29]. Recently, we also reported that p38 MAPK can directly bind to the ERK1 and ERK2 MAP kinases and

FIG. 3. Differential expression of (a) Akt2 and (b) GSK3␤ among the ovarian cancer cell lines CaOV-3, OVCAR-3, and SKOV-3. Note that levels of Akt2 and GSK3␤ are high in OVCAR-3, but these kinases are expressed at low levels in SKOV-3. HGF activated Akt2 in OVCAR-3, but not in CaOV-3 and SKOV-3.

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prevent their phosphorylation and activation by MEK1 and MEK2 [30]. We speculate that the increased production of Mek6, p38, and CK2 may, in fact, reflect a futile attempt of the transformed cell lines to compensate for a loss of p53 function. While PKG is associated with normal OSE functions, changes in MEK6, p38 MAPK, and CK2 expression and/or activation appear to be a common occurrence with oncogenic transformation. In this study, we demonstrated that Akt2 and p70 S6K were not only overexpressed but also showed phosphorylation-induced reduction of PAGE mobility in neoplastic SV40-Ecadherin transfected OSE and ovarian cancer cell lines, even in the absence of HGF stimulation. These findings are consistent with the frequent activation of PI3K and Akt2 observed in ovarian cancers, including the ovarian cancer cell lines OVCAR-3 and SKOV-3 that were included in this study [4, 31]. Importantly, such changes were also found in preneoplastic SV40-immortalized OSE and overtly normal OSE from all women with BRCA1 mutations, but only in some of the cases of OSE from the general population [12]. These data support other evidence which indicates that changes in the PI3K cascade contribute to the propensity of OSE to undergo neoplastic transformation. These observations also indicate that the effects of cytokines and growth factors, which activate the PI3K pathway in normal OSE, are altered in OSE from women with BRCA1 mutations [12]. These changes in cancer-prone individuals, in conjunction with other phenotypic alterations [22– 34], may indicate increased autonomy and represent very early or predisposing steps to ovarian carcinogenesis. Because an increase in PI3K activity contributes to Akt activation in human primary ovarian carcinomas and Akt indirectly activates p70 S6K, we speculate that an increased phosphorylation/ activation of p70 S6K in OSE may also be the result of an upregulation of PI3K. The knowledge about the role of Akt1 in ovarian cancer is limited. High levels of Akt1, in addition to Akt2, were found in neoplastic, SV40-E-cadherin transfected OSE. In contrast to Akt2, basal, unphosphorylated Akt1 was observed in tumorigenic, SV40-E-cadherin transfected OSE and ovarian cancer cell lines, but not in normal and preneoplastic OSE. This is one of the first indications that Akt1 and Akt2, members of the Akt family, might be regulated by different mechanisms. The regulation of protein kinases by SV40 and E-cadherin is not fully understood. We show here that introducing E-cadherin into SV40 large T-antigen-immortalized OSE caused changes in a variety of protein kinases to a pattern resembling that of ovarian cancer cell lines. These changes include the loss of PKG expression, the gain of MEK6 expression, and the expression of high levels of Akt2. These observations support our hypothesis that E-cadherin can contribute in an important way to ovarian carcinogenesis [10, 15]. Elevated levels of CDK1 and p38 MAPK, Akt2, and p70 S6K, which were present in neoplastic OSE, were already found in SV40-immortalized OSE but not in normal OSE. Therefore, these

changes, which accompany ovarian carcinogenesis, seem insufficient to render the cells tumorigenic. We also compared the regulation of protein kinases among the three human ovarian cancer cell lines CaOV-3, SKOV-3, and OVCAR-3. Although they all derived from serous ovarian adenocarcinomas, each line possesses some distinguished characteristics which reflect the heterogeneity found among ovarian carcinomas. For instance, OVCAR-3 expresses estrogen and androgen receptors, which are absent in the other two lines, and overexpression of HER-2/neu is observed only in SKOV-3. We show here high expression levels of Akt2 and GSK3␤ in OVCAR-3 compared with SKOV-3, indicating that PI3K is overexpressed in OVCAR-3, but not in SKOV-3. These observations agree with the report that OVCAR-3 has a greater copy number of the PIK3CA gene than line SKOV-3 [4]. Together, our data demonstrate changes in the expression and regulation of signaling proteins involved at different stages in the development of ovarian cancer. Although there is still much to understand concerning both the molecular and the cellular events which accompany changes in protein kinase regulation, the data present interesting and valuable information for future research. The accumulating knowledge in this area may lead to the identification of predictive markers for early detection and, perhaps, to the development of specific therapeutic modulators of protein kinases as a potential management strategy of ovarian cancer patients. REFERENCES 1. Hu L, Zaloudek C, Mills GB, Gray J, Jaffe RB. In vivo and in vitro ovarian carcinoma growth inhibition by a phosphatidylinositol 3-kinase inhibitor (LY294002). Clin Cancer Res 2000;6:880 – 6. 2. Cheng JQ, Godwin AK, Bellacosa A, Taguchi T, Franke TF, Hamilton TC, Tsichlis PN, Testa JR. AKT2, a putative oncogene encoding a member of a subfamily of protein–serine/threonine kinases, is amplified in human ovarian carcinomas. Proc Natl Acad Sci USA 1992;89:9267–71. 3. Bellacosa A, de Feo D, Godwin AK, Bell DW, Cheng JQ, Altomare DA, Wan M, Dubeau L, Scambia G, Masciullo V. Molecular alterations of the AKT2 oncogene in ovarian and breast carcinomas. Int J Cancer 1995;64: 280 –5. 4. Shayesteh L, Lu Y, Kuo WL, Baldocchi R, Godfrey T, Collins C, Pinkel D, Powell B, Mills GB, Gray J W. PIK3CA is implicated as an oncogene in ovarian cancer. Nat Genet 1999;21:99 –102. 5. Ali IU, Schriml LM, Dean M. Mutational spectra of PTEN/MMAC1 gene: a tumor suppressor with lipid phosphatase activity. J Natl Cancer Inst 1999;91:1922–32. 6. Wiener JR, Nakano K, Kruzelock RP, Bucana CD, Bast RC Jr, Gallick GE. Decreased Src tyrosine kinase activity inhibits malignant human ovarian cancer tumor growth in a nude mice model. Clin Cancer Res 1999;5:2164 –70. 7. Liu A-X, Testa JR, Hamilton TC, Jove R, Nicosia SV, Cheng JQ. AKT2, a member of the protein kinase B family, is activated by growth factors, v-Ha-ras, and v-src through phosphatidylinositol 3-kinase in human ovarian epithelial cancer cells. Cancer Res 1998;58:2973–77. 8. Bast RC Jr, Xu F, Yu Y, Fang XJ, Wiener J, Mills GB. Overview: the molecular biology of ovarian cancer. In Sharp F, Blackett T, Berek J, Bast R, editors: Ovarian cancer 5. Oxford, Isis Medical Media, 1998:87–97.

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