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Endocrinology, doi:10.1210/en.2007-0361
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*Ovarian Cancer
Endocrinology Vol. 148, No. 11 5195-5208
Copyright © 2007 by The Endocrine Society

Synergistic Effects of Epidermal Growth Factor and Hepatocyte Growth Factor on Human Ovarian Cancer Cell Invasion and Migration: Role of Extracellular Signal-Regulated Kinase 1/2 and p38 Mitogen-Activated Protein Kinase

Hong Y. Zhou1, Yuen L. Pon1 and Alice S. T. Wong

School of Biological Sciences, The University of Hong Kong, Hong Kong

Address all correspondence and requests for reprints to: Dr. Alice S. T. Wong, University of Hong Kong, School of Biological Sciences, 4S-14 Kadoorie Biological Sciences Building, Pokfulam Road, Hong Kong. E-mail: awong1{at}hku.hk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Ovarian cancer is the primary cause of death from gynecological malignancies with a poor prognosis characterized by widespread peritoneal dissemination. However, mechanisms of invasion and metastasis in ovarian cancer remain poorly understood. Epidermal growth factor (EGF) and hepatocyte growth factor (HGF) are often both overexpressed and contribute to the growth of ovarian cancer by activating autocrine pathways. In the present study, we investigated the mechanisms of invasive activity of EGF, HGF, and their synergistic effects in human ovarian cancer cells. Here our data suggest that EGF and HGF may use unique and overlapping signaling cascades leading to the invasive phenotype. We revealed that HGF-mediated cell migration and invasion required the coordinate activation of the phosphatidylinositol 3-kinase/Akt and extracellular signal-regulated kinase 1/2. Although EGF-dependent invasive phenotype appeared to have similar requirements for phosphatidylinositol 3-kinase, this growth factor used the alternative p38 MAPK pathway for cell invasion. A significant role of p38 MAPK was further supported by the observation that expression of dominant negative p38 MAPK likewise inhibited EGF-dependent invasiveness and cell motility. We also showed that EGF cooperated with HGF to promote a highly invasive phenotype via the increased secretion of matrix metalloproteinase (MMP)-9. The coincident induction of MMP-9 was functionally significant because inclusion of MMP-9 inhibitor or an anti-MMP-9 neutralizing antibody abolished EGF- and HGF-induced cellular invasion. These findings provide insights into the mechanism of the malignant progression of ovarian cancer.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
EPITHELIAL OVARIAN CANCER remains the leading cause of gynecological cancer deaths in developed countries. Poor prognosis is in part due to the fact that almost 70% of women are diagnosed with advanced disease, i.e. has spread to the upper abdomen (stage III) or beyond (IV) (1). Therefore, a better understanding of the mechanisms that promote the invasion and metastasis of ovarian cancer cells is critical for reducing the mortality and morbidity caused by the disease.

Growth factors have been proven to play important roles in cell migration associated with metastasis. Among the many growth factors that can contribute to such a phenotypical modulation of ovarian cancer cells, epidermal growth factor (EGF) and hepatocyte growth factor (HGF) possess the most potent mitogenic and motogenic effects. Overexpression of EGF receptor by ovarian tumors is associated with more aggressive clinical behavior and correlates with a poor prognosis (2, 3). Increased expression of EGF receptor and its ligands has been detected in tumors, ascitic fluid, and urine of ovarian cancer patients (4, 5, 6). Expression of EGF in ovarian tumor tissues has been demonstrated immunohistochemically, with RIA, and RT-PCR (7, 8, 9). Serum concentration of EGF in patients with ovarian cancer was 1.1–84.9 fmol/ml (6). EGF is known to enhance cell migration, invasion, and proteolytic activity of ovarian cancer cells in vitro (10, 11).

Another important growth factor for ovarian cancer is HGF. HGF is a stromal-derived factor that plays a major role in organ formation during embryogenesis and in tissue homeostasis in the adult. Inappropriate activation of the HGF pathway leads to invasive growth in cancer. High levels of HGF are found in ovarian cancer ascitic fluids (4.2 ng/ml; range 2.3–11.2 ng/ml), and the fluid of benign (3.8 ng/ml; range 0.09–15.3 ng/ml) and malignant ovarian cysts (13.0 ng/ml; range 4.1–16.0 ng/ml) (12, 13). Moreover, the receptor for HGF, encoded by the Met protooncogene, is frequently (40–68%) overexpressed in ovarian carcinomas (14, 15). Recently, we have demonstrated that HGF is a potent stimulator of motility and invasion of ovarian cancer cells, and is mediated through the phosphatidylinositol 3-kinase (PI3K)/Akt/p70S6K signaling pathway (16). These findings suggest that ovarian cancer can progress and metastasize through aberrant activation of Met-dependent signaling.

Because ovarian cancer cells are exposed to both EGF and HGF at the malignant lesions, we postulated that these two growth factors may exert synergistic effects on the phenotypical modulation of ovarian tumor cells. In this study we found that EGF and HGF were cooperative in inducing ovarian tumor cell migration, in vitro invasion, and matrix metalloproteinase (MMP)-9 production through the coordinate activation of unique and overlapping signaling pathways.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials and reagents
Human recombinant EGF was obtained from Sigma-Aldrich (St. Louis, MO), and HGF was obtained from R&D Systems, Inc. (Minneapolis, MN). Matrigel was purchased from Becton Dickinson (Palo Alto, CA). LY294002, PD98059, SB203580, MMP-9 inhibitor (SB-CT3), and anti-MMP-9 were purchased from Calbiochem (San Diego, CA). Antitissue inhibitor of metalloproteinase (TIMP)-1, and human MMP-2 and MMP-9 standards were from Chemicon International, Inc. (Temecula, CA), and anti-FLAG was from Sigma-Aldrich. Anti-Akt, extracellular signal-regulated kinase (ERK) 1/2, anti-p38 MAPK, anti-phospho-Akt, anti-phospho-ERK1/2, and anti-phospho-p38 MAPK were obtained from Cell Signaling Technologies, Inc. (Beverly, MA). Small interfering RNA (siRNA) oligonucleotides targeting Akt, ERK1/2, and p38 MAPK, and nonspecific siRNA controls were obtained from Dharmacon Research (Lafayette, CO). The FLAG-tagged cDNA encoding the dominant- negative p38 MAPK (DN-p38) was a kind gift from Dr. J. Han (The Scripps Institute, La Jolla, CA) (17).

Cell culture and treatment
The human ovarian epithelial carcinoma cell lines SKOV-3 and CaOV-3 (generously provided by Dr. N. Auersperg, Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, British Columbia, Canada) were grown in 1:1 medium 199:105 (Sigma-Aldrich) containing 5% fetal bovine serum, and supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin, at 37 C in a humidified incubator with 5% CO2. All experiments were performed using cells within five passages to ensure their genetic stability. The cells were plated in serum-containing medium overnight before treatment with EGF and/or HGF. In additional experiments, inhibitors or other chemical reagents were added 30 min before EGF and/or HGF treatment. To express DN-p38, cells were grown on 35-mm dishes and transfected 1.5 µg plasmid DNA using Lipofectamine reagent (Life Technologies, Inc., Gaithersburg, MD) according to the manufacturer’s instructions. Twenty-four hours later, the cells or conditioned media were collected for Western blotting, gelatin zymography, or biological assays.

Western blot analysis
Equal amount of protein was subjected to SDS-PAGE and transferred to nitrocellulose. Western blot analysis was done using appropriate primary antibodies at optimal dilution. The immunocomplex was detected with horseradish peroxidase-conjugated IgG as secondary antibody (Bio-Rad Laboratories, Hercules, CA), and blots were developed with an enhanced chemiluminescence kit (Amersham Biosciences, Little Chalfont, UK).

Analysis of cell scattering and Matrigel invasion
The scattering assay was performed as previously described (18). Briefly, the cells were in 24-well plates at a density of 3000 cells in each well. After 5 d, small, cohesive, and discrete colonies were formed, and then treated with EGF and/or HGF in the presence or absence of various inhibitors. Twenty-four hours later, the cells were washed in PBS, fixed with methanol, and stained with crystal violet (Sigma-Aldrich). Scattered colonies were judged by a typical change in morphology, characterized by cell-cell dissociation, and the acquisition of a migratory, fibroblast-like phenotype. Scattering activity was measured in the total number of scattered colonies from 50 colonies under a light microscope.

Invasive activity was quantified using a Boyden chamber (8-µm pore size) coated with Matrigel (50 µl/well) (lot 75124; BD Biosciences, Palo Alto, CA), as reported previously (19). Approximately 1 x 105 cells per well were seeded onto the Matrigel-coated wells and incubated in serum free medium with EGF and/or HGF for 24 h. In selected experiments, the proteinase dependence of invasion was determined by quantifying invasion in the presence of the MMP-9 inhibitor SB-3CT or anti-MMP-9. Noninvading cells were removed from the upper chamber with a moistened cotton swab. Cells that had migrated through the membrane to the lower surface were fixed with ice-cold methanol, stained with 0.5% crystal violet, and counted in five different fields under a light microscope. Each experiment was done in duplicate wells and repeated three times, and the results were expressed as mean ± SD.

Gelatin zymography
Aliquots (80 µg) of cell-conditioned media were loaded on SDS-PAGE gels containing 0.1% gelatin under nonreducing conditions. After electrophoresis, gels were washed with renaturing buffer [50 mM Tris-HCl (pH 7.4), 2% Triton X-100)] for 30 min to remove the sodium dodecyl sulfate, and subsequently incubated overnight at 37 C in developing buffer containing 50 mM Tris-HCl and 5 mM CaCl2 (pH 7.4). The proteinase activity was visualized by staining the gel with 0.5% Coomassie brilliant blue R-250 in 30% (vol/vol) methanol/10% (vol/vol) acetic acid for 6 h and, subsequently, destaining of the gel until bands became clear.

Statistical analysis
Data were expressed as mean ± SD of three independent experiments. Statistical analysis was done using one-way ANOVA followed by Tukey’s least significant difference t test for post hoc analysis (GraphPad Software, San Diego, CA), and P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
EGF promotes HGF-mediated cell scattering and Matrigel invasion
Initially, the dose response of EGF or HGF alone was tested in the range shown in Fig. 1Go. EGF induced a modest dose-dependent increase in invasion of both SKOV-3 and CaOV-3, which was noted at 12.5 pM (0.075 ng/ml) and 25 pM (0.15 ng/ml) for SKOV-3 and CaOV-3, respectively, and reached a peak at 1600 pM (9.6 ng/ml). HGF treatment resulted in a more rapid increase in invasion relative to EGF throughout the tested concentrations. The maximal invasion was observed at 100 pM (8.5 ng/ml). To assess the potential synergistic effect of EGF and HGF, invasion was analyzed using suboptimal doses with equal molar concentration of each growth factor. Interestingly, the combination of EGF and HGF significantly promoted cell invasion (~5-fold) (P < 0.05) even at a concentration (12.5 pM) at which EGF alone had no significant effect, and HGF slightly increased the invasive activity. The increased doses of both EGF and HGF (25 pM) tended to enhance further the invasion (~11-fold) that was significantly greater than induced by either individually (2-fold for EGF and 3-fold for HGF compared with untreated control cells) or additively in both SKOV-3 and CaOV-3 cell lines (P < 0.005) (Fig. 2AGo). Because migratory capacity is a prerequisite for cell invasion through the basement membrane (Matrigel), we asked whether EGF promotes not only invasive phenotype but also cell motility in these cells. As evidenced by cell scattering, a response that represents both increased cell migration and disruption of cell-cell adhesive junctions (18), the migratory response of SKOV-3 and CaOV-3 cells was induced by EGF (Fig. 2BGo). A similarly strong enhancement (>10-fold increase) was observed when EGF was treated with HGF, suggesting that EGF-dependent signals could synergize with HGF.


Figure 1
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  		FIG. 1. Effect of either HGF or EGF on invasion of ovarian cancer cells. SKOV-3 and CaOV-3 cells were untreated or treated with increasing concentrations of EGF or HGF, as indicated and assayed for their ability to migrate through 8-µm porous filters coated with Matrigel. Micrographs show invaded cells on the lower side of the filters. The results shown are the mean number of invading cells per field (average of five fields per filter) from three independent experiments performed in duplicate. Error bars indicate SD of the mean. Bar, 100 µm. *, P < 0.05; **, P < 0.005 vs. untreated controls.

 

Figure 2
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  		FIG. 2. EGF promotes HGF-mediated cell scattering and Matrigel invasion. A, SKOV-3 and CaOV-3 cells were untreated or treated with 12.5 and 25 pM EGF, HGF, or EGF together with HGF and assayed for their ability to migrate through 8-µm porous filters coated with Matrigel. Micrographs show invaded cells on the lower side of the filters. The results shown are the mean number of invading cells per field (average of five fields per filter) from three independent experiments performed in duplicate. B, The effect on cell scattering was determined by microscopic evaluation and photography. Scattered colonies were scored, and the bar graph summarizes the mean percentage of scattering activity from three independent experiments performed in duplicate. Error bars indicate SD of the mean. Bar, 100 µm. *, P < 0.05; **, P < 0.005 vs. untreated control.

 
p38 MAPK, but not ERK1/2, activity is required for EGF-dependent invasion and motility
In prior studies, we have demonstrated that HGF signals through PI3K/Akt pathway to regulate the invasive phenotype and motility in ovarian cancer cells (16, 19). We then set out to examine whether PI3K/Akt activity is involved in EGF-induced invasiveness and/or motility by treating cells with 25 µM LY294002 or 200 nM wortmannin before EGF treatment. To understand fully the intracellular signaling pathway responsible for the action of EGF, the dose of 25 pM (Fig. 1Go), in which we could detect a significant invasive activity in both cell lines, was used for all subsequent experiments. Treatment with LY294002 (Fig. 3AGo) or wortmannin (data not shown) effectively impeded EGF-induced invasion. The inhibitor alone had no effect (Fig. 3AGo). In contrast, inhibition of PI3K/Akt did not interfere with EGF stimulation of cell scattering (Fig. 4AGo). To define further the role of PI3K/Akt in EGF-enhanced cell invasion/migration, we chose to deplete Akt using siRNA. The efficacy of silencing Akt was determined by immunoblotting (Fig. 3BGo). Most importantly, reduced Akt expression with siRNA also markedly attenuated EGF-induced invasion compared with cells transfected with nonspecific siRNA (Fig. 3AGo). In contrast, Akt siRNA at the same concentration did not affect cell scattering (Fig. 4BGo). These results illustrate differences in the role of PI3K/Akt in EGF-dependent invasive and migratory response in ovarian cancer cells.


Figure 3
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  		FIG. 3. Inhibition of PI3K inhibits EGF-induced cell invasion. A, SKOV-3 and CaOV-3 cells were untreated or pretreated with 25 µM LY294002 (LY) for 30 min or cotransfected either 10 nM Akt-specific siRNA or nonspecific (NS) siRNA. Pretreated cells were then assayed for their ability to invade through the Matrigel-coated filters for 24 h in the presence or absence of EGF (25 pM). Micrographs show invaded cells on the lower side of the filters. The results shown are the mean number of invading cells per field (average of five fields per filter) from three independent experiments performed in duplicate. B, Whole cell lysates were analyzed for the level of Akt by Western blot analysis. Bar, 100 µm. *, P < 0.05 vs. untreated controls.

 

Figure 4
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  		FIG. 4. Signaling requirement for EGF-induced cell scattering. A, SKOV-3 and CaOV-3 cells were pretreated with 25 µM LY294002 (LY), 50 µM PD98059 (PD), or 10 µM SB203580 (SB) for 30 min, and then incubated with 25 pM EGF for 24 h. B, In some cases, cells were cotransfected with 10 nM nonspecific (NS) siRNA, Akt siRNA, ERK1/2 siRNA, or p38 MAPK (p38) siRNA before EGF treatment. The effect on cell scattering was determined by microscopic evaluation and photography. Scattered colonies were scored, and the bar graph summarizes the mean percentage of scattering activity from three experiments performed in duplicate. Error bars indicate the SD of the mean. C, Whole cell lysates were analyzed for the level of ERK1/2 and p38 by Western blot analysis. Bar, 100 µm. *, P < 0.05 vs. untreated controls or nonspecific siRNA.

 
The MAPK pathway is critical for the activation of invasion and motility in many tumor cell types (20). Therefore, we asked if they play a role in EGF-mediated in vitro invasiveness and motility in ovarian cancer cells. As shown in Fig. 4AGo, inhibition of p38 MAPK activity with 10 µM SB203580 blocked the EGF-increased cell scattering, whereas inhibiting MEK1 with 50 µM PD98059 had no effect. Similar results were also obtained with a different MEK1 inhibitor U0126 and with a different p38 MAPK inhibitor SB202190 (data not shown). Moreover, treatment with either inhibitor alone did not affect cell motility (Fig. 4AGo). In contrast to EGF, the signaling activity required for HGF-mediated migratory response is markedly different. Whereas PD98059 inhibited HGF-stimulated dispersal of more than 90% of SKOV-3 and CaOV-3 cells, SB203580 pretreatment failed to inhibit HGF-induced cell scatter (Fig. 5AGo). To establish that ERK1/2 is a downstream substrate of MEK1 in the signaling activated by HGF, we blocked ERK1/2 activity with siRNA (Fig. 4CGo). As shown, the simultaneous addition of ERK1/2 siRNA, but not nonspecific siRNA, suppressed HGF-induced cell scattering (Fig. 5BGo). On the other hand, ERK1/2 siRNA did not block any EGF-enhanced cell migration (Fig. 4BGo). Consistent with the effects of SB203580, both invasion (data not shown) and cell motility (Fig. 4BGo) induced by EGF were clearly reduced in the presence of p38 MAPK siRNA. No inhibition was observed for nonspecific siRNA. In contrast to EGF stimulation, p38 MAPK siRNA showed negligible effect on HGF-induced scattering response (Fig. 5BGo). Thus, HGF-dependent invasive phenotype is primarily dependent on ERK1/2 activation, whereas EGF-induced invasion and motility are p38 MAPK dependent.


Figure 5
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  		FIG. 5. Effects of PD98059 on HGF-induced cell scattering. A, SKOV-3 and CaOV-3 cells were pretreated with 25 µM LY294002 (LY), 50 µM PD98059 (PD), or 10 µM SB203580 (SB) for 30 min, and then incubated with 25 pM HGF for 24 h. B, In some cases, cells were cotransfected with 10 nM nonspecific (NS) siRNA, Akt siRNA, ERK1/2 siRNA, or p38 MAPK (p38) siRNA before HGF treatment. The effect on cell scattering was determined by microscopic evaluation and photography. Scattered colonies were scored, and the bar graph summarizes the mean percentage of scattering activity from three experiments performed in duplicate. Error bars indicate the SD of the mean. Bar, 100 µm. *, P < 0.05 vs. untreated controls or nonspecific siRNA.

 
Expression of DN-p38 prevents EGF-induced invasive response
In addition, to investigate the role for p38 MAPK in EGF-dependent cellular invasion and migration, we transiently transfected SKOV-3 and CaOV-3 cells with the FLAG-tagged DN-p38, in which the dual phosphorylated motif, Thr-Gly-Thr, was mutated to Ala-Gly-Phe (17). The expression of DN-p38 was confirmed by Western blotting with an anti-FLAG antibody (Fig. 6AGo). Cell scattering was clearly reduced in DN-p38 transfectants, as shown in Fig. 6BGo. Invasive phenotype was also inhibited by transfection with the DN-p38 construct (Fig. 7Go). DN-p38 transfection had no effect on HGF-dependent cell motility and invasion (Figs. 6BGo and 7Go). The results demonstrate that activation of p38 MAPK pathway is critical for EGF-, but not HGF-, induced ovarian cancer cell migration and invasion.


Figure 6
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  		FIG. 6. Effect of DN-p38 on cell scattering. A, Cells transiently transfected with a FLAG-tagged DN-p38 were treated with 25 pM EGF or 25 pM HGF for 24 h. p38 MAPK expression was determined by Western blotting with FLAG-specific antibodies. β-Actin was included to confirm equal loading. B, In parallel experiments, the effect on cell scattering was determined by microscopic evaluation and photography. Scattered colonies were scored, and the bar graph summarizes the mean percentage of scattering activity from three experiments performed in duplicate. Error bars indicate SD of the mean. Bar, 100 µm. *, P < 0.05 vs. untreated controls.

 

Figure 7
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  		FIG. 7. Effect of DN-p38 on ovarian cancer cell invasion. Cells transiently transfected with a FLAG-tagged DN-p38 were tested for their ability to invade through the Matrigel-coated filters for 24 h in the presence or absence of 25 pM EGF and 25 pM HGF. Micrographs show invaded cells on the lower side of the filters. The results shown are the mean number of invading cells per field (average of five fields per filter) from three independent experiments performed in duplicate. Error bars indicate the SD of the mean. Bar, 100 µm. *, P < 0.05 vs. untreated controls.

 
p38 MAPK is prominently activated by EGF, but not HGF
Next we examined whether the ERK1/2 and p38 MAPK pathways were differentially regulated by EGF and HGF. SKOV-3 and CaOV-3 cells were treated with 25 pM EGF or 25 pM HGF for an increasing time course of 15, 30, 60, and 180 min, and the activation of ERK1/2 and p38 MAPK was determined by immunoblot analysis using antibodies specific for the phosphorylated forms of these MAPKs. The membranes were then reprobed to evaluate total ERK1/2 or p38 MAPK expression. As shown in Fig. 8Go, whereas both EGF and HGF significantly increased the phosphorylation of ERK1/2, their effects on ERK1/2 vary in kinetics. Prominent and sustained activation (180 min) of ERK1/2 was shown in HGF-activated cells. Activation of ERK1/2 was relatively rapid and transient, reaching a maximum 15 min (30 min for CaOV-3) after EGF stimulation and decline thereafter. In contrast to ERK1/2, activation of p38 MAPK was shown only in EGF- but not HGF-activated cells.


Figure 8
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  		FIG. 8. Time course activation of ERK1/2 and p38 MAPK. SKOV-3 and CaOV-3 cells were treated with 25 pM EGF (A) or 25 pM HGF (B) for the indicated durations. Activities of ERK1/2 and p38 MAPK were determined by Western blotting using antibodies specific for phosphorylated, activated forms of ERK1/2 (p-ERK1/2), and p38 MAPK (p-p38 MAPK). The membranes were stripped and reprobed with antibodies to total ERK1/2 and p38 MAPK. β-Actin was also included to confirm equal protein loading. The relative intensity of phosphorylated forms of ERK1/2 and p38 MAPK was normalized to total values of ERK1/2 and p38 MAPK, which were determined by densitometric analysis. The bar graph summarizes the results from three independent experiments.

 
The enhanced migratory/invasive phenotype is associated with increased MMP-9 proteolytic activity
Because MMP-9 expression is correlated with the invasive behavior in advanced ovarian cancers (21), the effects of EGF and HGF on MMP-9 activation were examined by gelatin zymography assay. As shown in Fig. 9AGo, an induction (1.8-fold) of MMP-9 was detected in EGF-treated CaOV-3 cells. In contrast, the level of MMP-2 expression was refractory to EGF stimulation. Consistent with our recently published report (16), HGF also enhanced MMP-9 activation (2.5-fold). The simultaneous addition of both ligands further increased MMP-9 activation (7.3-fold). To determine whether activation of p38 MAPK plays a role in the regulation of MMP-9 activity, cells were pretreated with SB203580 or DN-p38, followed by treatment with EGF. Similar to the results obtained for invasion and motility, targeted inhibition of p38 MAPK selectively depressed MMP-9 levels without affecting MMP-2 in conditioned media, suggesting a role for this MAPK in MMP-9 production (Fig. 9BGo).


Figure 9
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  		FIG. 9. EGF induces MMP-9 proteolytic activity. A, Conditioned media from treated cells were collected and subjected to gelatin zymography. B, Cells were pretreated with 10 µM SB203580 (SB) or transiently transfected with DN-p38 or p38 MAPK (p38)-specific siRNA in the presence or absence of 25 pM EGF. Aliquots of conditioned medium normalized for same total protein concentration were collected and subjected to gelatin zymography. The signal intensity was determined by densitometry, and MMP-9 activity was calculated relative to that of MMP-2, which was arbitrarily assigned a value of ‘1.’ Migrations of the MMP-9 and MMP-2 gelatinase activities are indicated by arrows. C, Equal amount of protein (20 µg) was analyzed by Western blot using specific antibodies recognizing TIMP-1. β-Actin was included as a loading control. *, P < 0.05; **, P < 0.005 vs. untreated controls.

 
MMP expression is often coordinately regulated with production of its endogenous inhibitors, TIMPs (22). However, there was no significant change in the expression of TIMP-1, the endogenous inhibitor of MMP-9, after EGF treatment (Fig. 9CGo).

To evaluate the contribution of MMP-9 to EGF-induced invasion, the inhibitor SB-3CT (10 µM) and a neutralizing antibody against MMP-9 (10 µg/ml) were used. As shown in Fig. 10Go, EGF promoted Matrigel invasion in CaOV-3 and SKOV-3, and the addition of SB-3CT or anti-MMP-9 abrogated this effect. Inhibitor alone had no effect, indicating that the inhibition of invasion was not because of a cytotoxic effect of the inhibitor (Fig. 10Go). Moreover, the MMP-9-mediated effect was specific because addition of a potent MMP-2 inhibitor (OA-Hy) or other more general protease inhibitors (e.g. leupeptin, pepstatin A) did not inhibit EGF-induced cell motility (data not shown). These results indicate that the EGF-enhanced invasion was indeed mediated by increased MMP-9 activity in SKOV-3 and CaOV-3 cells.


Figure 10
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  		FIG. 10. Inhibition of MMP-9 suppresses EGF-stimulated ovarian cancer cell invasion. Cells were untreated or pretreated with SB-3CT (10 µM), or anti-MMP-9 neutralizing antibody (MMP-9 Ab) (10 µg/ml) for 30 min. Pretreated cells were then assayed for their ability to invade through the Matrigel-coated filters for 24 h in the presence or absence of 25 pM EGF. Micrographs show invaded cells on the lower side of the filters. The results shown are the mean number of invading cells per field (average of five fields per filter) from three independent experiments performed in duplicate. Error bars indicate SD of the mean. Bar, 100 µm. *, P < 0.05 vs. untreated controls.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Most deaths from ovarian cancer are due to metastasis characterized by widespread ip dissemination and malignant ascites. EGF and HGF have been known to play a predominant role in ovarian cancer by directly stimulating growth, invasiveness, and metastatic capability of cancer cells (23, 24). The present study is the first report to describe the cooperative effect of these two clinically important growth factors on ovarian cancer cell invasion. The combination of EGF and HGF at suboptimal equal molar concentrations (12.5 and 25 pM) showed synergistic effects. The concentrations of EGF and HGF in serum or ascites associated with ovarian malignancy have been approximately 1.1–84.9 fmol/ml (1.1–84.9 pM) and 2.3–11.2 ng/ml (24.5–130 pM), respectively (6, 12). This is consistent with the concentrations used in our experiments. Furthermore, we show that EGF and HGF may use independent MAPK pathways during ovarian tumor cell migration and invasion. Our results indicate that the p38 MAPK signaling pathway is critical for EGF-induced cell motility/invasion, whereas ERK1/2 appears to be required for HGF-stimulated cell migratory and invasive response.

Temporal differences in cellular signaling may have significant phenotypical manifestations. The duration of ERK1/2 signaling response affects differentiation in PC12 cells exposed to either EGF or NGF (25). Consistent with this hypothesis, we have shown that HGF-induced ERK1/2 activation was prolonged in ovarian cancer cells, but not in cells with EGF stimulation. In contrast to ERK1/2, which was effectively activated by both EGF and HGF, prominent activation of p38 MAPK was shown only in EGF-activated cells. This differential signaling may provide a mechanistic insight into the synergistic action between EGF- and HGF-mediated migratory and invasive behavior of ovarian cancer cells.

MAPKs are among the central elements that transduce extracellular signaling into cellular responses, and are believed to play pivotal roles in cell growth and survival (26). Involvement of MAPK members in other aspects of tumor progression, including invasion and metastasis, has been suggested recently. p38 MAPK has been demonstrated to be an important mediator of H-Ras-stimulated motility in human breast epithelial cells (27). The ERK1/2 pathway was involved in the invasive or migratory behavior of a number of malignancies, such as colon cancer, melanoma, breast cancer, and prostate cancer (20, 28, 29, 30, 31). Although ERK1/2 can be an upstream regulator of the p38 MAPK pathway (32), we excluded this possibility in ovarian cancer cells because treatment of cells with a specific inhibitor of MEK1 (PD98059) had no effect on p38 MAPK activation (data not shown).

Using a chemical inhibitor, dominant negative mutant of p38 MAPK, and p38 MAPK-specific siRNA, we showed that the p38 MAPK signaling pathway is required for the induction of MMP-9 in EGF-stimulated ovarian cancer cells. Induction of MMP-9 expression has been found to be mediated by ERK1/2 and PI3K/Akt pathways, in which the transcription factors nuclear factor {kappa}B and activating protein-1 are involved (33). A role for p38 MAPK in regulating MMP-9 expression has not been fully elucidated. The p38 MAPK pathway targets multiple transcription factors, including c-Jun (34), c-fos (35), and ATF2 (36), and putative binding sites for these DNA-binding proteins are present in the MMP-9 promoter (37, 38). Whether these putative regulatory elements participate in the EGF-mediated activation of MMP-9 gene remains to be determined.

An essential part of invasion and metastasis includes degradation of the basement membrane by members of the MMP family. MMP-2 and MMP-9, which can degrade collagen IV, the major structural collagen of the basement membrane, have been suggested to be critical in the metastatic process of ovarian cancer (39). The present study indicates that EGF- and HGF-enhanced cell migration and invasion are through increased expression and proteolytic activity of MMP-9. The coincident induction of MMP-9 appears to be functionally significant because inclusion of MMP-9 inhibitor or an anti-MMP-9 neutralizing antibody interferes with growth factor-induced cellular invasion. In contrast to MMP-2, which is constitutively expressed, MMP-9 levels are usually low, and enzyme expression is known to be induced by extracellular factors, including EGF, HGF, and inflammatory cytokines such as TNF{alpha} (40). The MMP-9 to MMP-2 ratio is high in ovarian cancer tissue (41), and elevated levels of MMP-9 protein have been detected in invasive epithelial ovarian carcinoma patient specimens. Moreover, experimental metastasis is suppressed by a synthetic MMP inhibitor (21, 42, 43). The naturally occurring inhibitor TIMP-1 is an important controlling factor in the regulation of the activities of MMPs (22). It appears that activation of MMP-9 was not related to a decrease in TIMP-1 in the EGF- and/or HGF-induced ovarian cancer cell invasion and motility. However, there was a net increase in the MMP-9 to TIMP-1 ratio, which favors extracellular remodeling and thereby facilitates tumor progression.

More recently, we have found that HGF-induced cell invasion and MMP production are significantly dependent on PI3K/Akt signaling (16). PI3K was also required for HGF-stimulated disruption of adherens junctions in Madin-Darby canine kidney cells (44). In contrast to HGF, although involvement of PI3K in both EGF-stimulated invasion and MMP-9 production of ovarian cancer cells was seen, PI3K activity was not required for disruption of cell-cell junctions (10) and cell scattering (Fig. 4Go). Thus, the signaling requirements for junctional dissociation may be cell type and growth factor specific. In ovarian tumors, aberrant activity of the PI3K/Akt pathway is frequently detected, and Akt activity has been associated with high-grade tumor and aggressive clinical behavior (45, 46).

Together, the evidence suggests that EGF and HGF may use unique and overlapping signaling cascades leading to the invasive phenotype. We reveal that HGF-mediated cell migration and invasion require the coordinate activation of the PI3K/Akt and ERK1/2, whereas EGF-dependent invasive phenotype is regulated predominantly by p38 MAPK signaling. PI3K activation may be necessary for EGF-mediated tumor cell invasion and MMP-9 induction (10), but not cell scattering. The ability of EGF and HGF to act synergistically to promote gelatinase-mediated cellular invasion represents an important method for enhancing the invasiveness of ovarian cancer cells. These results provide valuable insights into the mechanisms of EGF- and HGF-induced signaling networks in the malignant progression of ovarian cancer, and may contribute to improved and more specific therapies.


   Acknowledgments
 
We thank Drs. J. Han (The Scripps Institute, La Jolla, CA) and N. Auersperg (University of British Columbia, Vancouver, British Columbia, Canada) for kindly providing us with the dominant negative p38 MAPK construct and ovarian cancer cell lines, respectively. We also thank Ms. A. Mak for excellent technical assistance.


   Footnotes
 
This work was supported by the Committee on Research and Conference Council Grant HKU 10205788/22351/25700/301/01 (to A.S.T.W.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online August 2, 2007

1 H.Y.Z. and Y.L.P. contributed equally to this manuscript. Back

Abbreviations: DN-p38, Dominant-negative p38 MAPK; EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; HGF, hepatocyte growth factor; MMP, matrix metalloproteinase; PI3K, phosphatidylinositol 3-kinase; siRNA, small interfering RNA; TIMP, tissue inhibitor of metalloproteinase.

Received March 16, 2007.

Accepted for publication July 20, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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