The ovarian surface epithelium (OSE) is the precursor of common epithelial ovarian carcinomas. In the present study, we examined the molecular mechanisms and possible physiological basis for the propensity of OSE cells to undergo epithelio-mesenchymal transition (EMT) in response to environmental influences. We hypothesized that EMT may be a homeostatic mechanism that permits displaced OSE to assume a stromal phenotype within the ovarian cortex. We report that EGF in conjunction with hydrocortisone is the EMT-inducing factor of OSE as shown by changes to a fibroblast-like morphology and growth pattern. EGF increased cell motility, enhanced the activities of secreted pro-matrix metalloproteinase (MMP)-2 and -9, and enhanced expression and activation of Erk and integrin-linked kinase (ILK). Increased ILK expression correlated with the activation of PKB/Akt, the phosphorylation of GSK-3β, and the increased expression of cyclin E and cdk2 kinase. EGF withdrawal resulted in a more epithelial morphology and reversal of the EGF-induced activation of signaling pathways and pro-MMP activity. In contrast, treatment of EGF-treated cells with specific inhibitors of phosphatidylinositol 3-kinase, Mek, or ILK inhibited the inhibitor-specific pathways. The inhibitors caused suppression of EGF-induced migration and pro-MMP-2/-9 activities but did not lead to any change in EGF-induced mesenchymal morphology. ILK small interfering RNA inhibited Akt phosphorylation and reduced pro-MMP-2/-9 activities but had no effect on Erk activation or cell morphology. These results indicate that the EGF-induced morphological and functional changes in OSE cells are controlled by distinct signaling mechanisms working in concert. EMT of OSE cells displaced by ovulation likely permits their survival and integration with a fibroblast-like identity within the stroma. Failure to do so may lead to the formation of epithelium-derived inclusion cysts, which are known preferential sites of malignant transformation.
- epidermal growth factor
the ovarian surface epithelium (OSE) is a single, flat to cuboid layer of cells supported on the ovarian surface by a basement membrane and tunica albuginea. The cells are held together laterally by desmosomes and tight junctions (31). Surface epithelial cells are continuous with the mesothelium of the ovarian ligament and the peritoneum. This single layer of epithelial cells contributes to ovulation by lysis and reconstruction of the ovarian cortex and is of major importance in gynecological pathology because it is thought to be the source of 90% of ovarian neoplasms, the epithelial ovarian carcinomas (5, 28, 30). OSE-derived epithelial inclusion cysts are the preferred sites for the origin of epithelial ovarian cancer. These observations emphasize the need to determine the mechanisms regulating the postovulatory fate of OSE and to better understand the initiation of its neoplastic transformation as well as the biology of ovulation.
Sequential interaction of cellular signaling pathways is operative during ovarian follicular development, ovulation, and the postovulatory luteinization period (34). During the preovulatory phase, preferential growth of the ovulatory follicle brings it into close apposition with the OSE (31). The OSE produces lysosomal enzymes and urokinase plasminogen activator (uPA) and tissue-type plasminogen activator just before ovulation (18), and mice deficient in the plasminogen activator gene functions have been shown to have lower ovulatory efficiency (20). uPA stimulates the release of TNF-α from thecal endothelium, which progressively induces matrix metalloproteinase (MMP) expression and other inflammatory responses (29). Hence, collagenolysis precedes ovulation and is accompanied by the exfoliation and displacement of a discrete region of OSE close to the dome of the ruptured ovulatory follicle. In the postovulatory phase, some of the exfoliated, displaced OSE cells undergo apoptosis (27), whereas others undergo repair and mend the surface injury (26). In addition, the OSE might undergo epithelio-mesenchymal transition (EMT) in response to ovulation-induced inflammatory mediators and migrate from the ovarian surface into the stroma, or they might become trapped in the ruptured follicle, where they would be exposed to EGF and other EMT-inducing factors derived from the follicular fluid, blood, platelets, and luteal cells. Hence, the process of repair and wound healing of scarred OSE and the reshaping of displaced OSE to a mesenchymal migratory form occur consecutively as part of the postovulatory ovarian remodeling process. Although EMT presumably permits OSE to become incorporated into the ovarian stroma, epithelial inclusion cysts are derived from OSE that does not undergo EMT within the stromal environment (4). Histopathological examination suggests that early malignant changes frequently occur in OSE-lined crypts and epithelial inclusion cysts rather than on the ovarian surface (5), suggesting a potential link between the process of ovulation and the initiation of ovarian carcinoma. Furthermore, the risk of developing epithelial ovarian cancer increases with the frequency of ovulation (4). It is reasonable to assume, therefore, that EMT of OSE might reduce the chances of OSE-derived malignant transformation. This phenomenon must be distinguished from EMT of advanced epithelial ovarian carcinomas, in which EMT likely enhances invasiveness in a manner similar to that observed in other types of cancer.
Studies of the early events of ovarian carcinogenesis have been hampered by the minute amount of recoverable OSE from the human ovary and also by the limited life span of OSE cells in culture conditions, in which their epithelial phenotype is maintained. EGF has been described as a potent mitogen for human OSE (39). Human OSE demonstrate significant expression of EGF receptors (EGFRs) in vivo and in culture (6). At ovulation, OSE cells are exposed to EGF released from platelets within blood clots and subsequently produced by stromal and luteal cells (23). In addition, TGF-α binds to and activates EGFR in ovarian stromal and thecal cells (16). Hydrocortisone (HC) by itself is not mitogenic for OSE cells but enhances the effect of EGF (39).
EMT, which is characterized by the dissociation of epithelial cells from epithelial sheets to migratory fibroblast-like cells, is an important event in embryonic morphogenesis, gynecological physiology, and malignant transformation (7, 9, 44). However, EMT by cancer cells does not follow an orderly program and is different from physiological and developmental EMT (46). EMT induced by growth factors and cytokines requires reprogramming of epithelial cells to be reshaped for locomotion and invasion (24). In addition to increased motility, the process can also induce proteolytic digestion of basement membranes on which epithelia reside (24). Local expression of growth factors such as TGF-β, EGF, IGF, and FGF-2 can initiate and facilitate this process by binding receptors with intrinsic ligand-inducible kinase activity (17). The TGF-β-induced response involves an increase in EGFRs on the epithelial cell surface, and EGF or other EGFR ligands can assist in completing the response by initiating a diverse range of signaling pathways, with the major ones being the Ras-Erk, Rac-JNK-p38 MAPK, PLC-γ1, phosphatidylinositol 3-kinase (PI3-kinase), and downstream Akt pathways (12, 13, 48). Depending on the cell type, this activation may result in a number of biological responses, including mitogenesis, motility, protein secretion, and differentiation.
To understand the physiology of OSE in the postovulatory microenvironment, it is important to elucidate the response of these cells to stimuli present in that environment. EGF was previously shown to induce several components of EMT in OSE (36). In the present study, we therefore examined further phenotypic characteristics and the molecular pathways regulating EGF-HC-induced EMT in OSE. We report that EGF-HC-induced EMT encompasses increased motility and pro-MMP-2/-9 activity in OSE. Furthermore, we show that this EMT is dependent on the consecutive activation of Erk and integrin-linked kinase (ILK) pathways, that these pathways act in concert, and that the inhibition of either of these pathways has no effect on the complementary pathway. As a result, there is an inhibition of EGF-induced functions without any change in EGF-induced morphology. These results support the hypothesis that EMT of OSE cells that are trapped in the postovulatory follicle may be important in preventing the formation of epithelial inclusion cysts and thus may provide protection from the initiation of neoplastic transformation.
MATERIALS AND METHODS
Cells and culture methods.
Appropriate ethical permits were obtained as required by the University of British Columbia (Vancouver, BC, Canada). The participating patients signed consent forms. All women who participated in the study were premenopausal and between ages 29 and 49 yr (mean age, 36 yr). No correlation with clinical parameters could be established, because information about the women's hormonal status, menstrual cycle stage, or use of oral contraceptives was not available. The cell culture method used was described previously (36). Briefly, fragments of OSE were scraped from the ovarian surface during laparoscopic procedures for nonmalignant gynecological conditions. The fragments were incubated in 1.0 ml of culture medium [medium 199-MCDB 105 Medium (Sigma, St. Louis, MO)-10% FBS] in 35-mm-diameter culture dishes precoated with FBS at 37°C in a 5% CO2 atmosphere. After 2 h, an additional 1.0 ml of medium was added to the culture dishes. At confluence, cells were subcultured with 0.06% trypsin and 0.01% EDTA. In certain cases, OSE was grown in the presence of 10 ng/ml EGF and 1.0 μg/ml HC.
Preparation of cell lysates.
OSE growing in standard medium or treated with EGF-HC were lysed for 30 min on ice in lysis buffer (1% Nonidet P-40, 50 mM HEPES, pH 7.4, 150 mM NaCl, 2 mM EDTA, 2 mM PMSF, 1 mM orthovanadate, 1 mM NaF, 10 μg/ml aprotinin, and 10 μg/ml leupeptin). Cell extracts were centrifuged at 10,000 g for 20 min, with the resulting supernatant being the cell lysates used in assays. In some cases, cells were inhibited with PD-98059 (10 or 20 μM; Sigma), LY-294002 (20 or 40 μM; Calbiochem, La Jolla, CA), or KP-392 (50 or 100 μM; Kinetek Pharmaceuticals, Vancouver, BC, Canada) for 24 h before the preparation of cell lysates. The relative protein concentration of the cell lysates was determined using a commercial protein assay kit with BSA standards according to the manufacturer's instructions (Bio-Rad Laboratories, Hercules, CA).
Western blot analysis.
Cell lysates containing equal amounts of protein were subjected to SDS-PAGE on 10% gels under nonreducing conditions and the transferred onto Immobilon-P nitrocellulose membranes (Millipore, Billerica, MA). Antibodies used to probe Western blots were anti-ILK MAb (Becton-Dickinson), phospho-Erk, total Erk, phospho-Akt, total Akt, phospho-GSK-3β, total GSK-3β (Cell Signaling Technology, Beverly, MA), cyclin E (Upstate Biotechnology, Lake Placid, NY), and β-actin (Sigma). Bands were visualized using peroxidase-labeled secondary antibody and an ECL detection system (Amersham, Little Chalfont, UK) according to the manufacturer's instructions.
Preparation of conditioned medium.
Cells were allowed to grow in 25-cm2 flasks in standard medium or in EGF-HC-containing medium until they were 80–90% confluent. Serum-free conditioned medium was prepared as described previously (2). Protein content in the conditioned medium was estimated using a commercial protein assay kit with BSA standards according to the manufacturer's instructions (Bio-Rad Laboratories).
Pro-MMP-2 and pro-MMP-9 activities in the conditioned medium of OSE were analyzed using 10% SDS gelatin zymography (1 mg/ml final concentration) under nonreducing conditions as described previously (2). Gelatinolytic activity attributed to pro-MMP-2 and pro-MMP-9 was confirmed by activation with 4-aminophenylmercuric acetate (2 mM), a known activator of MMP, or 1:10 phenanthroline (2 mM), an inhibitor of MMP activation described previously (2).
Four cases of low-passage OSE were grown in standard medium with or without EGF-HC for two to five passages. Cells were grown to confluence and then placed into low serum (0.1%) for 24 h before wounding. Inhibitors were added 2 h before wounding, and monolayers were wounded with the tip of a sterile 200-μl pipette. DMSO was used as a control vehicle. The wound was marked, and measurements were performed using an ocular micrometer. Ten representative fields were marked and measured. Wounds were measured again 6 h after wounding. Data were analyzed using one-way ANOVA, followed by the Bonferroni test. All data were considered significantly different at P < 0.05.
Small interfering RNA (siRNA) were designed and synthesized as described previously (45). A control nonsilencing (NS) RNA (ACG UGA CAC GUU AGA AdTT) and ILK-FSF (21-bp ILK gene specifically targeting the kinase domain CAU UGG GAG AAC UUG ACA dTT) were transiently transfected into OSE growing in medium containing EGF-HC (2 passages) using 6 μl of Lipofectamine reagent (Invitrogen, Groningen, The Netherlands) according to the manufacturer's guidelines. To ensure optimal efficiency of transfection and better incorporation of siRNA, cells were passaged once at 48 h and ILK expression was checked 4 days posttransfection (45).
Morphological changes induced by EGF-HC.
As described previously (36), the response to EGF-HC in terms of growth pattern and morphological changes varied among OSE from different women; in the majority of cases (60–70%), however, the cells demonstrated EMT in primary culture when EGF-HC was present. In this study, eight cases of OSE that underwent EMT in response to EGF-HC were selected and the molecular pathways responsible for these changes were investigated. As described previously, OSE cultures without EGF-HC formed cobblestone-like monolayers and became stationary at confluence. In contrast, OSE cells treated with EGF-HC tended to stratify and disperse, demonstrating reduced contact inhibition of growth, and formed multiple layers, resulting in increased saturation densities. In the absence of EGF-HC, the epithelial phenotype of the cells was retained until senescence, whereas in the presence of EGF-HC, cells developed a fibroblast-like morphology (Fig. 1, A and B). We reported previously (36) that keratin was expressed by OSE cultured without EGF-HC, but there was a loss of keratin expression within 1 wk of EGF-HC treatment.
EGF-HC-induced EMT is associated with enhanced motility.
The ability of EGF-HC to induce a motile phenotype in OSE was evaluated using a wounding assay in which the motility of cells was estimated on the basis of the cells' location at the edge of an artificially produced wound to colonize the wounded area. Under these conditions, wound repair occurred in low growth factor medium (0.1% FBS) and was dependent solely on cell migration. As shown in Fig. 2B, EGF promoted wound repair of OSE cells within 6 h, whereas in the absence of EGF, no closure of the wound was observed (Fig. 2A). In some cases, the magnitude of cell motility in the presence of EGF was enhanced 10-fold within 6 h (Fig. 2C; subject 485). These results suggest that EGF plays an important role in mediating the motility of OSE.
EGF-HC-induced EMT is associated with enhanced activities of secreted pro-MMP-2/-9.
Because deregulation of cell motility contributes to invasive phenotypes, we next investigated whether EGF-HC-induced EMT in OSE is associated with the induction of MMP activity, a key event in the disruption of epithelial basement membranes for the migratory OSE. The expression of many members of the MMP family is regulated by growth factor receptors (24), and MMPs are implicated in cell migration and invasion (11). Analysis of MMP levels in a gelatin zymographic assay using conditioned medium collected from OSE not treated with EGF revealed relatively more 72-kDa pro-MMP-2 than 92-kDa pro-MMP-9 (Fig. 3). In the presence of EGF, the activity of pro-MMP-2 was consistently enhanced (Fig. 3; paired samples: lane 1 vs. lane 4, lane 2 vs. lane 5, and lane 3 vs. lane 6) with increases of pro-MMP-9 activity in four of six cases (Fig. 3; data shown for 3 subjects: lane 1 vs. lane 4, lane 2 vs. lane 5, and lane 3 vs. lane 6), indicating that the migratory and invasive potential of OSE may rely on pro-MMP secretion.
Activation of Erk and ILK pathways induced by EGF-HC correlates with EMT.
EGF is known to activate several signaling pathways, such as the PI3-kinase pathway (47), which can activate the downstream serine/threonine Erk phosphorylation cascade as well as the complementary ILK pathway, resulting in the downstream phosphorylation of Akt and GSK-3β kinase (32). To investigate the potential involvement of Erk and ILK in EGF-induced EMT, we examined the activation and expression of Erk, ILK, and downstream signaling components in OSE induced by EGF-HC for two or three passages. As shown in Fig. 4, treatment of OSE with EGF induced activation of Erk with enhancement of total Erk expression (Fig. 4, A and B; paired samples: lane 1 vs. lane 4, lane 2 vs. lane 5, and lane 3 vs. lane 6). Concomitant with Erk activation and enhanced expression, EGF also induced increased expression of ILK (Fig. 4C) and activated the downstream Akt pathway (Fig. 4D). There was no change in the expression of total Akt (Fig. 4E), and ILK activation of Akt resulted in the phosphorylation of GSK-3β (Fig. 4F) with a resultant increase in the expression of cyclin E and associated cyclin-dependent kinase (cdk)2 kinase (in 2 cases) (Fig. 4, G and H). These results suggest that EGF-induced EMT in OSE is associated with the activation of both the Erk and ILK pathways.
Reversibility of EGF-HC-induced EMT by EGF-HC withdrawal.
The maintenance of mesenchymal characteristics by OSE was strictly dependent on the presence of EGF-HC in the culture medium. When the cells growing in medium 199-MCDB 105 (1:1)-containing EGF was subcultured in EGF-free medium, the fibroblast-like morphology reverted to an epithelial phenotype within two passages (36). The keratin content of the cells remained low for 24 h after EGF withdrawal; if the cells were maintained without EGF for 1 wk, however, their growth slowed, their shape became more epithelial, and keratin levels increased approached those of parental cells in EGF-free medium (36). Consistent with these changes, EGF withdrawal also resulted in a significant loss of Erk activation (paired samples: lane 1 vs. lane 4, lane 2 vs. lane 5, and lane 3 vs. lane 6; adjusted mean volume of bands: +EGF = 25 ± 1.8 and −EGF = 6.5 ± 2; P < 0.003) (Fig. 5A) with a modest but significant decrease in the expression of ILK (adjusted mean volume of bands: +EGF = 17 ± 1.04 and −EGF = 13.7 ± 0.43; P < 0.05) and downstream activation of Akt (adjusted mean volume of bands: +EGF = 35 ± 4 and −EGF = 27 ± 2.5; P < 0.05) with a resulting decrease in cyclin E expression (adjusted mean volume of bands: +EGF=6.9 ± 0.42 and −EGF = 4.1 ± 0.25; P < 0.008) (Fig. 5, C, D, and F). Changes in signaling pathways correlated with a decrease in secreted pro-MMP-2 activity (Fig. 5H). The decrease in pro-MMP-9 activity was observed in four of six cases. These results suggest that EGF-induced EMT in OSE is reversible.
Inhibition of Erk and ILK pathways does not affect EGF-HC-induced morphology but inhibits cell motility and secreted pro-MMP-2 activity.
To evaluate whether EGF-induced EMT is dependent on both the Erk and Akt pathways, we determined the effects of phosphatidylinositol (PI) 3-kinase inhibitor (LY-294002), Mek inhibitor (PD-98059), and ILK inhibitor (KP-392) on the morphology and functional responses of cells treated with EGF for two passages. Treatment of EGF-induced cells with PD-98059 (10 μM) and LY-294002 (20 μM) for 24 h resulted in the inhibition of Erk activation without any change in total Erk expression (Fig. 6, A and B). On the one hand, KP-392 (50 μM), a specific inhibitor of ILK activity, had no effect on Erk activity (Fig. 6A), but both KP-392 and LY-294002 inhibited Akt phosphorylation (Fig. 6C). On the other hand, in the presence of PD-98059 (10 μM), no change in Akt phosphorylation was observed (Fig. 6C) and the expression of total Akt remained unchanged (Fig. 6D). These results suggest that EGF-induced EMT in OSE cells generates signals through the upstream PI3-kinase with an effect on the overlapping downstream pathways.
The effect of pharmacological inhibition of PI3-kinase, Erk, and Akt phosphorylation and/or activation had no effect on the morphological phenotype of EGF-induced epithelial cells. Within 24 h in the presence of the inhibitors, the cells did not revert to an epithelial phenotype and maintained their fibroblast-like morphology (data not shown). In contrast, significant inhibition in cell motility was observed within 6 h in EGF-treated cells in the presence of PD-98059 (20 μM), LY-294002 (40 μM), or KP-392 (100 μM) (Fig. 7A). Inhibition of cell motility by LY-294002 and KP-392 was also observed in uninduced cells, indicating that PI3-kinase and ILK pathways are involved in the endogenous motility of OSE. Concomitant with the inhibition of cell motility, the activity of secreted pro-MMP-2, but not pro-MMP-9, was diminished by the inhibitors (Fig. 7B).
Knockdown of ILK expression does not affect EGF-HC-induced morphology but inhibits secreted pro-MMP-2/-9 activities.
To determine whether ILK expression is essential for EGF-induced EMT in OSE cells, we used double-stranded siRNA (ILK-FSF) to knock down ILK expression in EGF-treated cells. Suppression of ILK expression resulted in the inactivation of Akt with a loss of Akt expression (Fig. 8, A–C). There was no change in Erk activation or expression (Fig. 8, D and E). At the same time, there was no reversion of fibroblast-like morphology to epithelium-like cells, and the cells remained attached and spread (Fig. 8, F and G). Suppression of ILK expression, however, inhibited secreted pro-MMP-2/-9 activities (Fig. 8H), again suggesting that the morphological change induced by EGF in OSE is not regulated solely by the ILK pathway.
In most epithelia, the acquisition of a motile function is correlated with a dramatic change in cell physiology (21). Migrating cells no longer express epithelial characteristics and acquire mesenchymal properties (21). Profound changes such as EMT have been reported previously, including a cellular process manifested during embryonic and organ morphogenesis (14), wound healing (43), tumor progression (35), and reproductive tract physiology (9). The cellular features of EMT are a loss of epithelium-like polygonal morphology, apicobasolateral cell polarity and adhesive contacts, development of a fibroblast-like shape, reorganization of cytoskeletal filaments, increased cell motility, and induction of proteases for ECM degradation requisite for migration and invasion. Our findings demonstrate that EGF regulates many of the key processes that lead to EMT in OSE, and that these events might play a role in the survival of displaced OSE during the postovulatory process (5).
It has long been known that EGF-HC greatly increases the proliferation potential of cultured human OSE and instigates changes from epithelial to mesenchymal cell shapes, growth patterns and cell dispersion, increased collagen type III deposition, and loss of keratin, the phenotype representative of the process (36, 39). In this study, we have demonstrated that EGF-induced EMT changed signaling pathways, enhanced cell motility, and induced activities of secreted pro-MMP-2/-9. EMT changes were induced in all of the eight cases studied, and 80–100% of cells underwent EMT within days of EGF treatment compared with untreated samples, which retained an epithelial morphology and phenotype until senescence under similar experimental conditions. An ∼3.5–10-fold increase in cell motility was observed in OSE cells obtained from different women exposed to EGF compared with untreated epithelial cells. In addition to enhanced motility, EGF also induced the activity of secreted pro-MMP-2/-9, but no change in secreted uPA expression was detected (data not shown). These results support recent evidence suggesting the involvement of MMPs in cell migration. During wound closure, there is elevated expression of several MMPs, including MMP-1, MMP-2, MMP-3, and MMP-9 (37). Moreover, MMP expression is enhanced in basal cells of the migrating epithelium sheet in vivo, and chemical inhibitors for MMP have been shown to inhibit EGF-induced cell migration (22).
To dissect the mechanisms involved in EGF-HC-induced EMT, we have examined the status of the components of the MAPK and ILK pathways. Activation and increased expression of Erk was observed after two passages of the cells in EGF medium. The increase in Erk expression in the continuous presence of EGF-HC may be indicative of increased proliferation of OSE cells, as shown previously by increased population doubling capacity (36). EGF also induced increased expression of ILK and activated the downstream Akt pathway with enhanced phosphorylation of GSK-3β and a resultant increase in the expression of cyclin E and associated cdk2 kinase. These results are consistent with alteration in the expression of cell cycle regulators by ILK overexpression in epithelial cells (33).
Downregulation of the Erk pathway using a Mek inhibitor, PD-98059, or a PI3-kinase inhibitor, LY-294002, for 24 h demonstrated consistent inhibition of Erk activity in EGF-treated cells. No inhibition in Erk activity was observed by KP-392 treatment. On the other hand, LY-294002 and KP-392 inhibited Akt activity, but no effect induced by PD-98059 was observed. These data suggest that EGF initiates multiple signaling pathways and that specific inhibition of one pathway has no effect on complementary compensatory pathway.
The effect of the pharmacological inhibition of PI3-kinase, Erk, and Akt phosphorylation/activation during a 24-h period had no effect on the morphology of EGF-induced cells, but significant inhibition in cell motility was observed within 6 h. The inhibition of cell motility was concurrent with the inhibition of the activity of secreted pro-MMP-2. The lack of reversal of morphology by Erk, ILK and PI3-kinase inhibitors is likely due to the short duration of exposure of cells to these inhibitors and suggests that the reversal of the functional phenotype by EGF withdrawal precedes morphological changes. The reversal of EGF-deprived cells from mesenchymal to more epithelial shapes is accelerated by subculturing (36), which suggests that the morphological response is a slow process and is accelerated by changes in the adhesive mechanisms associated with subculturing. Consistent with these results, suppression of ILK expression by siRNA inhibited Akt activation and expression and pro-MMP-2/-9 secretion but had no effect on Erk activation and expression and mesenchymal morphology. Suppression of activation of Akt by knocking down ILK expression has been observed in other cell lines, but no suppression of Akt expression was observed under such condition (45). ILK has been shown to interact with Akt (32). It is possible that the expression of Akt is dependent on the level of expression of ILK, and in certain cellular systems such as OSE, drastic suppression of ILK expression by siRNA may adversely affect interacting Akt expression. The role of ILK and Erk in regulating EGF-induced EMT in human OSE cells is shown in Fig. 9.
EGF withdrawal resulted in a significant loss of Erk activation, with a modest but significant decrease in ILK expression and downstream inhibition of activation of Akt. These changes correlated with the change in mesenchymal-to-epithelial morphology and a decrease in secreted pro-MMP-2/-9 activity, suggesting that the maintenance of the mesenchymal morphology and phenotype were strictly dependent on the continuous presence of EGF or the mesenchyme-inducing agent. The reversibility of the mesenchymal phenotype to an epithelial characteristic by the withdrawal of the mesenchyme-inducing agent is consistent with the reversibility of EMT reported in NBT-II rat bladder epithelial carcinoma cells (8) and menstrual effluent-induced mesothelial cells (9).
The role of Erk and ILK pathways in mediating EMT-associated normal and cancer cell migration and induction of MMP is well documented (3, 15, 19, 21, 40, 41). EMT resulting from overexpression of ILK in renal tubular epithelial cells resulted in the loss of E-cadherin expression, the induction of MMP-2 secretion, and the promotion of cell migration and invasion (21). ILK-mediated EMT of mammary epithelial cells resulted not only in the loss of E-cadherin and keratin expression but also in the induction of vimentin expression, as well as in the promotion of invasion (41). EGF also has been shown to induce mesenchyme-associated motility and related loss of E-cadherin expression and gain of vimentin expression in PMC42-LA breast carcinoma cells (1). Under these conditions, keratin expression was maintained in EGF-induced PMC42-LA cells. OSE cells do not express E-cadherin (42) but do express keratin and vimentin (25). Although the status of vimentin expression in EGF-induced OSE is not known, a loss in keratin expression has been demonstrated (36). These results indicate that in OSE cells, EMT mimics the same signaling pathways described for normal and malignant cells but is significantly different in the expression of EMT-associated intercellular adhesion markers. This difference may be attributed to variations in the induction of transformation or to differences in tissue-specific lineage that may execute EMT by regulating members of the same family of adhesion molecules differently to achieve similar cellular functions. Thus EMT in normal and malignant cells cannot be identified on the basis of the alteration of a single marker; nevertheless, a signature of common or related molecular changes can be predicted.
Evidence of EMT in human OSE cells was presented previously (5). The relevance of EGF-induced EMT in OSE cells in vivo is still under investigation. In previous studies, we observed EMT in OSE cells from both pre- and postmenopausal women (38, 39). In both age groups, the conversion of OSE to fibroblast-like cells would permit the cells to transdifferentiate to a structural and functional phenotype rather than inducing death or differentiation into epithelial inclusion cysts. In the premenopausal group, however, the integrity of the DNA of some of the displaced OSE cells during ovulation is compromised and cannot be amended by repair mechanisms (30). Such cells may lose responsiveness to environmental mediators and may fail to undergo EMT, resulting in clonal expansion and the formation of inclusion cysts, contributing to malignant transformation. It is likely that the high frequency of ovarian cancer in postmenopausal women is due to the accumulation of epithelial inclusion cysts during the course of women's lives. This hypothesis is consistent with studies indicating that with malignant transformation, OSE cells become committed to an epithelial phenotype and are less likely to undergo EMT (5). A small study of women with a family history of ovarian cancer demonstrated resistance of OSE cells to EMT in response to external stimuli compared with cells from women with no such family history (10). The results of these studies suggest that EMT is an process essential to OSE physiology and that the failure to undergo such a process may contribute to neoplastic progression. The present study may provide a molecular basis linking OSE to ovarian cancer and may render a platform from which to explore the early changes in the process of ovarian carcinogenesis.
This work was supported by the Broken Hill Proprietary Billiton Trust Funds Australia (to N. Ahmed and M. A. Quinn) and by a grant from National Cancer Institute of Canada, with funds from the Canadian Cancer Society (to N. Auersperg). N. Ahmed was the recipient of a Yamagiwa-Yoshida Memorial International Union Against Cancer Fellowship in Dr. Shoukat Dedhar's laboratory during the course of the study.
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