Formation of E-Cadherin-Mediated Cell-Cell Adhesion Activates Akt and Mitogen Activated Protein Kinase via Phosphatidylinositol 3 Kinase and Ligand-Independent Activation of Epidermal Growth Factor Receptor in Ovarian Cancer Cells

Pradeep Reddy1, Lian Liu1, Chong Ren1, Peter Lindgren, Karin Boman, Yan Shen, Eva Lundin, Ulrika Ottander, Miia Rytinki and Kui Liu

Departments of Medical Biochemistry and Biophysics (P.R., L.L., C.R., Y.S., M.R., K.L.), Clinical Science/Obstetrics and Gynecology (P.L., U.O.), Radiation Sciences (K.B.), and Medical Biosciences (E.L.), Umeå University, SE-901 87, Umeå, Sweden

Address all correspondence and requests for reprints to: Dr. Kui Liu, Department of Medical Biochemistry and Biophysics, Umeå University, SE-901 87, Umeå, Sweden. E-mail: kui.liu{at}medchem.umu.se.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
E-cadherin is a well characterized adhesion molecule that plays a major role in epithelial cell adhesion. Based on findings that expression of E-cadherin is frequently lost in human epithelial cancers, it has been implicated as a tumor suppressor in carcinogenesis of most human epithelial cancers. However, in ovarian cancer development, our data from the current study showed that E-cadherin expression is uniquely elevated in 86.5% of benign, borderline, and malignant ovarian carcinomas irrespective of the degree of differentiation, whereas normal ovarian samples do not express E-cadherin. Thus, we hypothesize that E-cadherin may play a distinct role in the development of ovarian epithelial cancers. Using an E-cadherin-expressing ovarian cancer cell line OVCAR-3, we have demonstrated for the first time that the establishment of E-cadherin mediated cell-cell adhesions leads to the activation of Akt and MAPK. Akt activation is mediated through the activation of phosphatidylinositol 3 kinase, and both Akt and MAPK activation are mediated by an E-cadherin adhesion-induced ligand-independent activation of epidermal growth factor receptor. We have also demonstrated that suppression of E-cadherin function leads to retarded cell proliferation and reduced viability. We therefore suggest that the concurrent formation of E-cadherin adhesion and activation of downstream proliferation signals may enhance the proliferation and survival of ovarian cancer cells. Our data partly explain why E-cadherin is always expressed during ovarian tumor development and progression.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
OVARIAN CANCER IS a common fatal cancer and a major cause of death from gynecological malignancies (1, 2, 3). In contrast to other types of human epithelial cancers, this disease is characterized by more local growth of tumor mass without typical symptoms, and when it is diagnosed, it has usually spread into the peritoneal cavity (1, 3, 4). More than 90% of malignant ovarian cancers are epithelial in type and originate from ovarian surface epithelial (OSE) cells. OSE cells covering deep clefts, invaginations, and inclusion cysts of the ovaries have long been considered as precursors of ovarian carcinomas (for papers and reviews see Refs.1, 2, 3 and Refs.5, 6, 7).

E-cadherin is a 120-kDa transmembrane molecule that plays a major role in epithelial cell adhesion. E-cadherin molecules can establish calcium-dependent homophilic interactions through their extracellular domain, and these bindings are stabilized by the association of the intracellular domain with {alpha}-, ß-, and {gamma}-catenins, which are linked to F actin to form functional adherens junctions (8, 9). Due to the fact that expression of E-cadherin is frequently lost in human epithelial cancers such as bladder, lung, and pancreatic carcinomas, this molecule has been implicated as a tumor suppressor in carcinogenesis (10, 11, 12, 13, 14).

E-cadherin exhibits a distinct expression pattern in the development of ovarian carcinomas, however. Although healthy OSE cells covering the ovarian surface do not express E-cadherin, E-cadherin is expressed in OSE cells covering deep clefts, invaginations, and inclusion cysts of the ovaries (4, 15, 16), where early malignant changes are believed to take place (1, 2, 3, 5, 6, 7). E-cadherin is detected more frequently in OSE cells from patients with a family history of ovarian cancers (17). By mostly immunohistochemical analyses of ovarian tumor samples, E-cadherin has not been found in normal ovaries, but has been found constantly in benign, borderline, and malignant ovarian tumors of all stages, as well as in metastases from such ovarian tumors (4, 18, 19, 20). In vitro experiments have shown that introduction of E-cadherin expression into human OSE cells induces epithelial-differentiation markers associated with preneoplastic, metaplastic OSE, and OSE-derived primary carcinomas (16) and enhances tumorigenesis of OSE cells that lead to the development of invasive adenocarcinomas (2). Moreover, in immortalized mouse OSE cells transfected with E-cadherin, the cells exhibited higher proliferation rate and gained anchorage-independent growth ability, although they were not found to be tumorigenic in xenograft models with nude mice (Dr. Barbara Vanderhyden, personal communication).

Whether or not the expression of E-cadherin in OSE cells is functionally significant in the neoplastic transformation of the cells is still unknown. However, it would be intriguing to study whether the formation of intercellular E-cadherin homophilic binding may trigger proliferation signals in OSE and ovarian cancer cells. In the present study using the E-cadherin-expressing ovarian cancer cell line OVCAR-3, we have studied the activation of phosphatidylinositol 3-kinase (PI3 K) and MAPK pathways upon establishment of E-cadherin-mediated cellular contacts. The possible mechanisms by which Akt and MAPK are activated by E-cadherin were also investigated.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
E-Cadherin Is Not Expressed in Normal Ovaries but in Benign, Borderline, and Malignant Ovarian Epithelial Tumors
To systematically reveal the expression pattern of E-cadherin in normal ovaries and in ovarian carcinomas, we have measured E-cadherin levels in a number of human ovaries and also in ovarian tumor samples. As shown in Fig. 1Go, normal ovaries express very little or no E-cadherin (Fig. 1AGo). However, as shown in Fig. 1Go, B–H, and also as summarized in Table 1Go, most of the ovarian epithelial cancers express high levels of E-cadherin regardless of tumor type, stage of malignancy, or stage of differentiation (Table 1Go). A major band of 120 kDa and a minor band of 80 kDa, representing a breakdown product of E-cadherin (21), were observed. Among them, all mucinous benign tumors tested (seven of seven), all mucinous borderline tumors tested (seven of seven), all mucinous cancers (four of four), 50% of serous benign tumors (three of six), 75% of serous borderline tumors tested (three of four), 80% of serous cancers (four of five), and all endometrioid cancers tested (four of four) expressed high levels of E-cadherin (Fig. 1Go, B–H; and Table 1Go). In total, 86.5% of all ovarian tumors tested expressed high levels of E-cadherin (Table 1Go).



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Fig. 1. E-Cadherin Expression in Normal Human Ovaries and Human Ovarian Epithelial Tumors

Seven normal ovaries (A) and 37 human ovarian epithelial tumors (B–H), as indicated in the figure and as described in Table 1Go, were homogenized as described in Materials and Methods. Protein concentrations were measured, and equal amounts of total proteins (50 µg) were analyzed by SDS-PAGE and blotted for detection of E-cadherin and ß-actin.

 

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Table 1. Patient Material and Histopathological Criteria Regarding Ovarian Tissues According to the World Health Organization Classification

 
E-Cadherin-Mediated Cell-Cell Adhesion Triggers Akt and MAPK Activation in OVCAR-3 Cells
The universally elevated E-cadherin expression in ovarian epithelial tumors relative to normal ovaries suggests that E-cadherin may play a role in the development of ovarian carcinomas. As uncontrolled cell proliferation is one of the early steps of tumorigenesis, we have studied the possible correlation between the establishment of intercellular E-cadherin homophilic ligations and the activation of intracellular proliferation and survival signals.

Using an E-cadherin-expressing ovarian cancer cell line, OVCAR-3, we adopted a well-established model for studying E-cadherin-mediated cell-cell junctions, in which the Ca2+-dependent E-cadherin-mediated adhesion was disrupted by EGTA and restored by physiological concentrations of Ca2+ contained in fresh media (22, 23). As shown in Fig. 2AGo, E-cadherin is expressed at sites of cell-cell contact in serum-starved OVCAR-3 cells before disruption by EGTA (Fig. 2AGo, E-cadherin, Control). The anti-E-cadherin antibody we used for immunofluorescence studies recognizes the intracellular tail of the E-cadherin molecule, so the fluorescence signal seen actually indicates the location of the intracellular part of the E-cadherin molecule. As a parallel marker, ß-catenin, which links E-cadherin to the cytoskeleton, was found to be expressed in both the cell membrane area as well as in the cytoplasm (Fig. 2AGo; ß-catenin, Control). The cell borders were also outlined by the tight junction protein zonula occludens-1 (ZO-1) (Fig. 2AGo; ZO-1, Control). Addition of EGTA (4 mM) for 30 min substantially disrupted E-cadherin-mediated cellular adhesions, which was evidenced also by the disturbed expression of ß-catenin and ZO-1 along the cell contacts (Fig. 2AGo, EGTA, 30 min). However, upon replenishment with fresh Ca2+-containing (1.8 mM) medium, the expression of E-cadherin was found to be relocated to sites of cell-cell adhesion, reflecting the reestablishment of homophilic binding between E-cadherin molecules (Fig. 2AGo; E-cadherin restoring calcium 5 min). Expression of ß-catenin and ZO-1 was also restored at cell borders upon the reestablishment of cell-cell adhesion (Fig. 2AGo; ß-catenin and ZO-1 restoring calcium 5 min).



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Fig. 2. E-Cadherin-Mediated Cellular Adhesion Induces Akt and MAPK Activation

A, Starved OVCAR-3 cell monolayers were left untreated (Control) or treated with 4 mM EGTA for 30 min (EGTA 30 min). The EGTA-containing medium was then replaced with serum-free, calcium-containing (1.8 mM) medium for 5 min (E-Cad Restoring Calcium 5 min). Afterward, the cells were fixed, permeabilized, and stained with anti-E-cadherin, anti-ß-catenin, and anti-ZO-1 antibodies followed by fluorescein isothiocyanate-conjugated secondary antibodies and prepared for microscopic analysis at a magnification of x63. B, OVCAR-3 cells were starved of serum overnight and treated with 4 mM EGTA for 30 min and subsequently restored with calcium-containing (1.8 mM) serum-free medium for 5–90 min, as indicated. As a control, starved OVCAR-3 cells were left untreated, treated with EGTA only, or with 30% fetal bovine serum (FBS) for 30 min. After calcium restoration, cell lysates were blotted with anti-phospho-Akt (Ser473) (P-Akt) and anti-phospho-p44/42 MAPK (Thr202/Tyr204) (P-MAPK). Amounts of similar proteins loaded were monitored using blots with anti-Akt (Total Akt) and anti-p44/42 MAPK (Total MAPK). The Western blot shows a representative result from three independent experiments, and normalized values (mean ± SD) from three independent experiments for changes in levels of P-Akt and P-MAPK were also shown. C, Phosphorylated Akt was immunoprecipitated from cell lysates and an Akt kinase assay was performed using recombinant GSK as Akt substrate, as described in Materials and Methods. Both Akt kinase activities (Akt activity) and the amount of immunoprecipitated phosphorylated Akt (IP-P-Akt) are shown. Normalized values (mean ± SD) from three independent experiments for changes in Akt kinase activities were also shown. Lowercase letters (a, b, c, and d) indicate significant differences (P < 0.05).

 
As shown in Fig. 2BGo, basal levels of active Akt and p44/42 MAPK in starved OVCAR-3 cells and OVCAR-3 cells treated with only EGTA were low, as indicted by levels of phosphorylated Akt (P-Akt) and MAPK (P-MAPK) (Fig. 2BGo, lanes 1 and 2). However, both Akt and MAPK were quickly activated (within 5 min, the shortest time studied) by the formation of E-cadherin-mediated cell-cell junctions (Fig. 2BGo, lane 3). This activation of Akt and MAPK was seen from 5–30 min after calcium restoration (Fig. 2BGo, lanes 3–6). The activation of Akt and MAPK decreased after 30 min and was at low levels till 90 min after calcium restoration, which was the longest time used (Fig. 2BGo, lanes 7–9). It is noteworthy that levels of activated Akt and MAPK upon establishment of cellular adhesion were comparable to those activated by fetal bovine serum treatment for 30 min (Fig. 2BGo, lane 10). As an internal control, P-Akt and P-MAPK levels in OVCAR-3 cells treated with only EGTA for identical lengths of time, but not given fresh calcium-containing medium, were also measured. EGTA treatment by itself did not cause any change in the activation status of Akt and MAPK (data not shown).

To study this question in greater depth, we have also evaluated the Akt kinase activities during the time course of the calcium switch in OVCAR-3 cells by measuring the enzymatic activity of immunoprecipitated P-Akt in an in vitro reaction system in which recombinant glycogen synthase kinase (GSK) was used as an Akt substrate. Thus, in accordance with the levels of activated Akt (Fig. 2BGo), calcium restoration also caused an increase in Akt kinase activity (Fig. 2CGo; Akt activity, lanes 3–7). The increased Akt activity was caused by the increased amounts of P-Akt that could be immunoprecipitated (Fig. 2CGo, IP-P-Akt).

Activation of Akt and MAPK by Formation of Cellular Contact Requires the Engagement of E-Cadherin
To study whether E-cadherin is essential for the activation of Akt and MAPK upon the reestablishment of cell-cell contacts, or whether the kinase activation was caused by other adhesion molecules, or even by the calcium ion in the changed culture media, we performed experiments with OVCAR-3 cells that were pretreated with the E-cadherin-neutralizing antibody SHE 78–7 (5 µg /ml). As shown in Fig. 3Go, serum-starved cells without any treatment (Fig. 3Go, lane 1), or cells treated with EGTA alone (Fig. 3Go, lane 2), exhibited low basal levels of P-Akt and P-MAPK. Similar to the data shown in Fig. 2BGo, both Akt and MAPK were substantially activated by the reestablishment of cell-cell contacts (5 min after Ca2+ restoration; Fig. 3Go, lane 3, P-Akt and P-MAPK). However, in cells that had been pretreated with E-cadherin-neutralizing antibody, activation of Akt and MAPK by Ca2+ restoration was greatly blocked (Fig. 3Go, lane 4; P-Akt and P-MAPK), indicating that the activation of P-Akt and P-MAPK was substantially reduced by the blockage of E-cadherin homophilic binding. Suppression of Akt and MAPK activation was also seen with the E-cadherin-neutralizing antibody SHE 78–7 at concentrations of 10 and 20 µg/ml (data not shown). Similar effects on blockage of Akt and MAPK activation were also seen by the use of another widely used E-cadherin-neutralizing antibody HECD-1 (data not shown). Taken together, our results demonstrate that the formation of E-cadherin homophilic binding is responsible for the major part of the activation of Akt and MAPK in OVCAR-3 cells.



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Fig. 3. Activation of Akt and MAPK by Formation of Cell Adhesion Requires Engagement of E-Cadherin

OVCAR-3 cells were starved overnight and treated with 4 mM EGTA to disrupt E-cadherin-mediated cell-cell contacts as described in Materials and Methods. The cells were lysed after 5 min of calcium restoration. As controls, serum-starved cells were left untreated, treated with EGTA alone, or pretreated with anti-E-cadherin antibody (SHE 78–7, 5 µg/ml) before EGTA and calcium treatment. Cell lysates were assayed for phosphorylated Akt (P-Akt) and MAPK (P-MAPK) levels as well as total levels of Akt (Total Akt) and MAPK (Total MAPK). The Western blot shows a representative result (three independent experiments). Normalized values (mean ± SD) from three independent Western blots for P-Akt and P-MAPK were also shown. Lowercase letters (a and b) indicate significant differences (P < 0.05). E-Cad Ab, E-Cadherin antibody.

 
E-Cadherin-Mediated Activation of Akt was through PI3 K and MAPK Activation via Raf-MAPK Kinase (MEK)
To determine the pathways by which Akt and MAPK were activated by E-cadherin, OVCAR-3 cells were pretreated with the PI3 K inhibitor wortmannin (1 µM), MEK1 and MEK 2 inhibitor U0126 (10 µM), or protein kinase A (PKA) inhibitor H89 (10 µM) for 30 min. The E-cadherin-mediated cell-cell adhesions were then disrupted with EGTA and restored by addition of fresh calcium-containing medium. As shown in Fig. 4AGo, PI3 K inhibitor completely blocked the activation of Akt (Fig. 4AGo; P-Akt, lane 4 vs. lane 3), but had very little effect on MAPK activation (Fig. 4AGo, P-MAPK, lane 4 vs. lane 3) by E-cadherin-mediated cellular adhesion. The inhibitor of MEK, U0126, on the other hand, completely abolished MAPK activation (Fig. 4AGo, lane 5; P-MAPK), but had little effect on Akt activation (Fig. 4AGo, lane 5; P-Akt), indicating that MAPK was completely activated by MEK.



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Fig. 4. E-Cadherin-Mediated Activation of Akt is via PI3 K, and MAPK Activation Is via Raf-MEK

A, OVCAR-3 cells were starved of serum, left untreated, or treated with the PI3 K inhibitor wortmannin (1 µM), the MEK inhibitor U0126 (10 µM), or the PKA inhibitor H89 (10 µM) for 30 min before the disruption and reestablishment of E-cadherin-mediated adhesions by EGTA and calcium-containing medium, respectively. The cells were lysed after 5 min of calcium restoration and assayed for phosphorylated p70 S6 kinase (P-p70S6K), Akt (P-Akt), and MAPK (P-MAPK) levels. As controls, total amounts of p70 S6 kinase (Total p70S6K), Akt (Total Akt), and MAPK (Total MAPK) were also measured. The Western blot shows a representative result from three independent experiments. Normalized values (mean ± SD) from three independent Western blots for P-Akt and P-MAPK were also shown. B, OVCAR-3 cells were serum starved, left untreated, or treated with E-cadherin-blocking antibody (SHE 78–7, 5 µg/ml) before being treated with 4 mM EGTA and restored with calcium. The cells were lysed after 5 min of calcium restoration. Cellular lysates were assayed for levels of phosphorylated MEK (P-MEK), total MAPK (Total MAPK), phosphorylated Raf (Ser 338) (P-Raf), and total Raf (Total Raf). The Western blot shows a representative gel from three independent experiments. Normalized values (mean ± SD) from three independent Western blots for P-MEK and P-Raf were also shown. Lowercase letters (a, b, and c) indicate significant differences (P < 0.05). Wort, Wotmannin; E-Cad Ab, E-cadherin antibody.

 
As a control, an inhibitor of PKA, H89, showed no inhibitory effect on the E-cadherin-activated Akt and MAPK (Fig. 4AGo, lane 6). In addition, the activation of another kinase that is downstream of PI3 K, p70S6 kinase, was also studied by measuring the level of phosphorylated p70 S6 kinase (P-p70S6K). We found that although wortmannin abolished part of the PI3 K-mediated phosphorylation of p70S6 kinase (Fig. 4AGo, lane 4), p70S6K was not significantly activated by the reestablishment of E-cadherin binding in OVCAR-3 cells (Fig. 4AGo, lane 3; P-p70S6K). MEK inhibitor U0126 and PKA inhibitor H89 showed no effect on the activation state of p70S6 kinase (Fig. 4AGo, lanes 5 and 6; P-p70 S6K).

The upstream kinases that cause the activation of MAPK, MEK, and Raf were also studied. As shown in Fig. 4BGo, levels of phosphorylated MEK (P-MEK, lane 3) and Raf (P-Raf, lane 3) were elevated by Ca2+ restoration, indicating that Raf and MEK were activated by the formation of E-cadherin adhesions (Fig. 4BGo, P-MEK and P-Raf, lane 3 vs. lanes 1 and 2). However, in OVCAR-3 cells that had been pretreated with E-cadherin-neutralizing antibody SHE 78–7 (5 µg/ml), the activation of MEK and Raf was largely suppressed (Fig. 4BGo; P-MEK and P-Raf, lane 4 vs. lane 3). Similar inhibitory effects were also observed with higher concentrations of SHE 78–7 and with another E-cadherin-neutralizing antibody, HECD-1 (data not shown). Our data therefore suggest that E-cadherin activates MAPK by the classical Raf-MEK-MAPK pathway.

Ligand-Independent Activation of Epidermal Growth Factor (EGF) Receptor Is Necessary for E-Cadherin-Mediated Akt and MAPK Activation
E-cadherin is an adhesion molecule with no reported enzymatic activities (8, 9), and Akt and MAPKs are known to be activated by various types of receptor protein tyrosine kinases (RPTKs) or protein tyrosine kinases (PTKs) (24). We therefore hypothesized that the E-cadherin activation of Akt and MAPK may be achieved by activation of certain PTKs or RPTKs. To address this question, we pretreated OVCAR-3 cells with a set of inhibitors that specifically inhibit various PTKs of interest, including Genistein (a general PTK inhibitor), PP1 (Src inhibitor), Tyrphostin AG 1478 (EGF receptor inhibitor), Tyrphostin AG 825 (ErbB-2 inhibitor), GTP-14564 (Kit, c-fms, and platelet-derived growth factor receptor ß inhibitor), and AGL 2263 (insulin receptor and IGF receptor inhibitor). Although most of the inhibitors did not appear to have any inhibitory effect on E-cadherin-mediated activation of Akt and MAPK, the general PTK inhibitor Genistein and the EGF receptor inhibitor Tyrphostin AG 1478 significantly suppressed the E-cadherin activation of Akt (Fig. 5AGo; P-Akt; lanes 4 and 7 vs. lane 2) and MAPK (Fig. 5AGo; P-MAPK; lanes 4 and 7 vs. lane 2).



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Fig. 5. Activation of EGF Receptor Is Necessary for E-Cadherin-Mediated Activation of Akt and MAPK

A, OVCAR-3 cells were serum starved overnight and treated for 1 h with the Src inhibitor PP1 (500 nM); a general tyrosine kinase inhibitor Genistein (50 µM); c-fms, c-kit, wt-FLT3, and platelet-derived growth factor receptor ß-inhibitor GTP-14564 (1 µM); IGF-I receptor and insulin receptor inhibitor AGL 2263 (1 µM); EGF receptor inhibitor Tyrphostin AG 1478 (100 nM); or ErbB-2 inhibitor Tyrphostin AG 825 (1 µM). After disruption of E-cadherin-mediated cell-cell contacts with 4 mM EGTA for 30 min and reestablishment of E-cadherin adhesion with fresh calcium for 5 min, the cells were lysed. As controls, serum-starved cells were treated with EGTA but not with calcium. Cellular lysates were assayed for phosphorylated Akt (P-Akt), total Akt (Total Akt), phosphorylated MAPK (P-MAPK), and total MAPK levels (P-MAPK). The Western blot shows a representative result from three independent experiments). Normalized values (mean ± SD) from three independent Western blots for P-Akt and P-MAPK were also shown. B, OVCAR-3 cells were serum starved overnight and treated for 1 h with PI3 K inhibitor LY294002 (50 µM), serial dilutions of EGF receptor inhibitor AG 1478 (10 nM, 100 nM, and 1 µM), and ErbB-2 inhibitor AG 825 (10 µM). After E-cadherin binding was disrupted by EGTA and restored by calcium, the cells were lysed, and levels of phosphorylated Akt (P-Akt), total Akt (Total Akt), phosphorylated MAPK (P-MAPK), total MAPK (P-MAPK), phosphorylated MEK (P-MEK), and total MEK (Total MEK) were measured. The Western blot shows a representative gel from three independent experiments. Normalized values (mean ± SD) from three independent Western blots for P-Akt, P-MEK, and P-MAPK were also shown. Lowercase letters (a, b, c, d, and e) indicate significant differences (P < 0.05).

 
The EGF receptor inhibitor AG 1478 showed a substantial suppression of E-cadherin-activated Akt and MAPK (Fig. 5AGo). Moreover, as shown in Fig. 5BGo, increasing the concentrations of AG 1478 from 10 nM to 1 µM showed an increasing ability to inhibit Akt, MAPK, and MEK activation (Fig. 5BGo; P-Akt, P-MAPK, and P-MEK; lanes 5–7). The highest AG 1478 concentration (1 µM) was able to block Akt, MAPK, and MEK activation back to basal levels (Fig. 5BGo, lane 7 vs. lanes 1 and 2). This indicates that the activation of EGF receptor is indispensable for the E-cadherin-mediated activation of Akt, MAPK, and MEK. Another PI3 K inhibitor, LY294002, also completely inhibited the activation of Akt, confirming that E-cadherin activated Akt completely through the PI3 K pathway. As a negative control, an inhibitor of ErbB-2, AG 825, did not show any inhibitory effects on Akt, MAPK, and MEK activation, even at a concentration as high as 10 µM (Fig. 5BGo, lane 8; P-Akt, P-MAPK, and P-MEK), indicating that the kinase activation in our system is not due to the activation of ErbB-2.

To prove the EGF receptor activation in OVCAR-3 cells before and after the reestablishment of E-cadherin homophilic binding, levels of phosphorylated tyrosine in immunoprecipitated EGF receptor were also measured. As shown in Fig. 6Go, phosphorylated tyrosine levels in the EGF receptor were elevated upon the establishment of E-cadherin homophilic binding in OVCAR-3 cells (Fig. 6Go, lanes 3–7). However, the activation of EGF receptor by E-cadherin was temporary, which was seen from 1–30 min after calcium restoration (Fig. 6Go, lanes 3–7). The activated EGF receptor level had decreased to basal levels at 60 min after calcium restoration, which was the longest time studied (Fig. 6Go, lane 8). Nevertheless, no physical interaction between E-cadherin and EGF receptor was observed in our experimental system by performing immunoprecipitation and Western blot under different conditions (data not shown).



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Fig. 6. Activation of EGF Receptor upon Reestablishment of E-Cadherin-Mediated Cellular Adhesion

OVCAR-3 cells were serum starved overnight, and E-cadherin-mediated adhesions were disrupted with EGTA (4 mM) for 30 min. After calcium restoration for 1, 2, 5, 15, 30, and 60 min, the cells were lysed and 500 µg of lysate were used for immunoprecipitation of EGF receptor and blot for phosphorylated tyrosine of the precipitated EGF receptor with antityrosine antibody pY99 (P-tyrosine). Levels of immunoprecipitated EGF receptor are also shown (EGF receptor) as internal controls. The Western blot shows a representative gel from three independent experiments. Normalized values (mean ± SD) from three independent Western blots for P-tyrosine of EGF receptor was also shown. Lowercase letters (a, b, and c) indicate significant differences (P < 0.05). EGFR, EGF receptor; IP, immunoprecipitation.

 
Conceivably, OVCAR-3 cells may produce either membrane-associated or soluble ligands, including EGF or TGF-{alpha}, which could bind to the extracellular ligand binding domain of EGF receptor and activate the receptor. As tested in Fig. 7AGo, TGF-{alpha} (10 ng/ml) can indeed rapidly stimulate the activation of Akt and MAPK in starved OVCAR-3 cells over a period of 15 min (Fig. 7AGo; P-Akt and P-MAPK; lanes 2–4). However, if the cells were pretreated for 24 h with a EGF receptor antibody (LA1, 10 µg/ml) that specifically blocks the ligand-binding domain of the receptor, the TGF-{alpha}-activated P-Akt and P-MAPK levels were greatly reduced (Fig. 7AGo; P-Akt, P-MAPK; lanes 6–8). This indicates that TGF-{alpha} indeed activates Akt and MAPK through binding to the EGF receptor, and moreover, the EGF receptor-neutralizing antibody (LA1) we used is effective in blocking the ligand-receptor interaction.



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Fig. 7. Activation of EGF Receptor by E-Cadherin-Mediated Cellular Adhesion Is Ligand Independent

A, Validation of EGF receptor-neutralizing antibody LA1. Starved OVCAR-3 cells, with or without EGF receptor antibody LA1 (10 µg/ml) pretreatment for 24 h, were stimulated with TGF-{alpha} (10 ng/ml) for 5, 10, and 15 min, and levels of phosphorylated Akt (P-Akt), total Akt (Total Akt), phosphorylated MAPK (P-MAPK), and total MAPK levels (P-MAPK) were measured. B, Starved OVCAR-3 cells, with or without EGF receptor antibody LA1 (10 µg/ml) pretreatment for 24 h, were subjected to calcium switch for 5, 10, and 15 min. Levels of phosphorylated Akt (P-Akt), total Akt (Total Akt), phosphorylated MAPK (P-MAPK), and total MAPK (P-MAPK) were measured. The Western blots show representative results from three independent experiments. Normalized values (mean ± SD) from three independent Western blots for P-Akt and P-MAPK were also shown. Lowercase letters (a, b, c, and d) indicate significant differences (P < 0.05). EGFR Ab, EGF receptor antibody.

 
We then used this EGF receptor-neutralizing antibody (LA1) to block the extracellular ligand-binding domain of EGF receptor in OVCAR-3 cells. We found that when OVCAR-3 cells were pretreated with LA1 (10 µg/ml) for 24 h before the calcium switch procedure, the activation of Akt and MAPK was not obviously suppressed upon addition of fresh calcium (Fig. 7BGo; P-Akt and P-MAPK; lanes 8–10 vs. lanes 3–5). Our data therefore directly indicate that the activation of Akt and MAPK by E-cadherin-mediated cell-cell adhesion is achieved through a ligand-independent activation of EGF receptor.

Determination of Downstream Effectors that Are Responsible for the E-Cadherin-EGF Receptor Activation of Akt and MAPK
Signals of the Rho family GTPases have been reported to be activated as epithelial cells reestablish contacts after chelation of extracellular calcium (22, 25, 26). As formation of E-cadherin homophilic ligation is known to regulate adhesive contacts through the activation of Rac signaling (25), by measuring levels of the active form of Rac (GTP.Rac) in OVCAR-3 cells, we have tried to determine whether E-cadherin activation of Akt and MAPK may be downstream of Rac activation. However, our data indicate that the E-cadherin activation of Akt and MAPK are not accompanied by Rac activation (data not shown). It is therefore unlikely that the activation of Akt and MAPK by E-cadherin-mediated adhesion is mediated by Rac activation.

We have also investigated molecules that function downstream of the EGF receptor, which may have different roles in the activation of Akt and MAPK upon the establishment of E-cadherin homophilic binding. Ras is a well-studied molecule that interacts with and activates Raf, which subsequently activates MEK, leading to MAPK activation in many cell types including tumor cells (24). By performing glutathione S transferase (GST)-Raf pull-down assay, we found that the reestablishment of E-cadherin binding did not lead to any change in Ras activation (data not shown). Thus, Ras is unlikely to be involved in the activation of Akt and MAPK in our system.

In addition, we have studied the activation of the adaptor protein SHC, which is known to act downstream of the EGF receptor, and can relay G protein-coupled receptor- and RPTK-induced signals (27, 28). However, our experiments showed that activation of Akt/MAPK and EGF receptor by E-cadherin-mediated cellular adhesion was not accompanied by further activated SHC (data not shown), indicating that the classic EGF receptor-SHC-MAPK pathway (27, 29) was not active in our cellular system. Nor did we see any inhibitory effects of two specific protein kinase C inhibitors, bisindolylmaleimide I and XI, on the E-cadherin-activated MAPKs (data not shown). This indicates that MAPK activation by E-cadherin-activated EGF receptor was not achieved via the known pathway of EGF receptor-phospholipase C{gamma}-protein kinase C-MAPK (29).

Among the many downstream effectors of EGF receptor, we found that GRB-associated binder 1 (Gab1) was activated concurrently with the establishment of E-cadherin-mediated cellular adhesion. As shown in Fig. 8AGo, in a similar experimental setup, as indicated by the phosphorylated tyrosine levels of immunoprecipitated Gab1, Gab1 was rapidly activated by 1 min after the replenishment of fresh calcium, and this activation reached its peak level at 2–15 min and then decreased gradually over a period of 90 min (Fig. 8AGo; P-tyrosine; lanes 3–9). As a control, the activation of Gab1 was comparable to its activation by treatment with 30% fetal bovine serum for 30 min (Fig. 8AGo, lane 10). The total Gab1 levels are also shown in Fig. 8AGo as internal controls (Fig. 8AGo; Gab 1).



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Fig. 8. Gab 1 Is Activated by EGF Receptor upon Reestablishment of Cellular Adhesion

A, OVCAR-3 cells were serum starved overnight, and E-cadherin-mediated adhesions were disrupted by EGTA (4 mM) for 30 min. After calcium restoration for 1, 2, 5, 15, 30, and 60 min, the cells were lysed and 500 µg lysate was used for immunoprecipitation of Gab 1 and blot for phosphorylated tyrosine of the precipitated Gab 1 with antityrosine antibody pY99 (P-tyrosine of Gab 1). Levels of immunoprecipitated Gab 1 are also shown (Gab 1). B, OVCAR-3 cells were serum starved overnight and treated for 1 h with serial dilutions of EGF receptor inhibitor AG 1478 (10 nM, 100 nM, 1 µM, and 10 µM). After E-cadherin binding was disrupted by EGTA and restored by calcium for 5 min, the cells were lysed for Gab 1 immunoprecipitation, which was followed by Western blot for pY99 (P-tyrosine of Gab 1). As a control, levels of immunoprecipitated Gab 1 are also shown (Gab 1). The Western blots show representative results from three independent immunoprecipitation-Western blots. Normalized values (mean ± SD) from three independent Western blots for P-tyrosine of immunoprecipitated Gab 1 were also shown. Lowercase letters (a, b, c, and d) indicate significant differences (P < 0.05). IP, Immunoprecipitation; FBS, fetal bovine serum.

 
To demonstrate that the tyrosine phosphorylation of Gab 1 is directly caused by EGF receptor, we have pretreated the cells with AG 1478 with a series of concentrations ranging from 10 nM to 10 µM, before the calcium switch procedure. Gab 1 was immunoprecipitated from cell lysates, and levels of phosphorylated tyrosine were measured. As shown in Fig. 8BGo, treatment with the EGF receptor inhibitor AG 1478 almost completely blocked the elevation in tyrosine phosphorylation of Gab 1 caused by calcium switch (Fig. 8BGo, lanes 4–7 vs. lane 3), indicating that the activation of Gab 1 during E-cadherin-mediated cell-cell adhesion formation is achieved by the tyrosine kinase activity of EGF receptor.

Suppression of E-Cadherin Function Leads to Reduced Cell Proliferation and Viability
To study the physiological functions of E-cadherin-mediated cell-cell adhesion and the E-cadherin activation of PI3 K and MAPK pathways, we performed the MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl tetrazolium bromide) cell viability and proliferation assay. As shown in Fig. 9Go, compared with the control group of cells, incubation of cells with the E-cadherin-neutralizing antibody SHE 78–7 (5 µg/ml) stopped the cells from proliferating after 24 h of incubation, and there was a markedly reduced cell viability during an incubation period of 48–72 h (Fig. 9Go; E-cad Ab). Similarly, the PI3 K inhibitor LY294002 (50 µM) was found to dramatically decrease the viability of the cells (Fig. 9Go; LY294002), and the MEK 1 inhibitor PD98059 (50 µM) was found to stop the cells from proliferation (Fig. 9Go; PD98059). Thus, our data indicate further that E-cadherin adhesion as well as E-cadherin-mediated activation of PI3 K and MAPK pathways may be important for proliferation and survival of ovarian cancer cells.



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Fig. 9. Suppression of E-Cadherin, PI3 K, and MAPK Functions Reduces Cell Proliferation and Viability

OVCAR-3 cells (2 x 105) were seeded in 24-well plates in DMEM/F12 culture medium supplemented with 10% fetal calf serum. The cells were incubated with or without E-cadherin-neutralizing antibody SHE 78–7 (5 µg /ml), LY294002 (50 µM), or PD98059 (50 µM), for 24, 48, and 72 h. MTT assay was performed as described in Materials and Methods. Lowercase letters (a, b, c, d, e, f, and g) indicate significant differences (P < 0.05). E-cad Ab, E-cadherin antibody.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
E-cadherin is a major type of adhesion molecule, which forms Ca2+-dependent homophilic ligations to facilitate cell-cell contact in epithelial cells. In cancer development, E-cadherin has been generally implicated as a tumor suppressor in several types of human epithelial tumors, including bladder, lung, and pancreatic tumors, based on findings that the expression of E-cadherin is frequently lost in human epithelial cancers, and the establishment of functional cadherin complex in tumor cell lines leads to a reversion from invasive to benign epithelial phenotype (10, 11, 12, 13, 14). However, in ovarian cancers, E-cadherin has been related to tumor development based on the findings that 1) it is not expressed in normal ovaries, but in ovarian cancer precursor OSE cells and also in ovarian cancers at various stages of malignancy (our current study and Refs.4 and 18, 19, 20), and 2) exogenously introduced E-cadherin can drive normal human and mouse OSE cells toward tumorigenesis (Refs.2 and 16 ; and Dr. Barbara Vanderhyden, personal communication).

Because uncontrolled cellular proliferation is known as a prerequisite for tumor development (24), and to investigate whether the conversion between disruption and reestablishment of E-cadherin-mediated cell-cell adhesion may trigger intracellular activation of proliferation and survival signals, we have adopted a well-established calcium-restoration model (22, 23) and have demonstrated a rapid activation of Akt and MAPK upon reestablishment of E-cadherin-mediated adhesion in ovarian cancer cells. Our data show that it is not only the establishment of cell-cell adhesions but also the E-cadherin molecules that have an indispensable role in this process, as antibodies that can neutralize E-cadherin function abolished the activation of Akt and MAPK to a large extent. Our results therefore suggest that in ovarian cancer cells, E-cadherin may serve not only as an intercellular adhesion molecule, but also as an upstream regulator that triggers downstream kinase activation.

By performing signal transduction studies, we have investigated the pathways by which the establishment of E-cadherin homophilic binding activates Akt and MAPK. We have shown that E-cadherin-activated Akt can be blocked completely by PI3 K inhibitors, indicating that Akt is activated by PI3 K. We have also shown that activation of MAPK by E-cadherin-mediated cell-cell adhesion is mediated through the Raf-MEK-MAPK cascade. However, Ras, the molecule that interacts with and phosphorylates Raf in the classic MAPK pathway in many other cell types (24), was not activated by E-cadherin homophilic binding. Thus, we assume that there are other signal transduction pathways upstream of the Raf-MEK-MAPK pathway that participate in the activation of these molecules.

Because E-cadherin is known to activate the Rho family of GTPases for the regulation of adhesion properties (22, 25, 26), we have also investigated whether the key molecule for such activation, activation of Rac, is activated concurrently with activation of Akt and MAPK when E-cadherin homophilic binding was established in OVCAR-3 cells. We found that Rac is not activated when Akt and MAPK are activated by the establishment of E-cadherin-mediated cell-cell adhesion, suggesting that the activation of Rac and Akt/MAPK by E-cadherin are achieved through different pathways.

E-cadherin is a well-known adhesion molecule, which does not exhibit any enzymatic activity in its short intracytoplasmic tail (8, 9). Thus, the activation of Akt and MAPK by E-cadherin-mediated cell-cell adhesion is most likely mediated by activation of other signal transduction systems, especially the PTKs and RPTKs. We have demonstrated in the current study that E-cadherin-mediated cell-cell adhesion can trigger a ligand-independent activation of EGF receptor, which may subsequently lead to activation of Akt and MAPK. However, using the immunoprecipitation-western blot approach, we failed to detect any interaction between E-cadherin and EGF receptor, suggesting the existence of intermediate molecules between E-cadherin and EGF receptor, which relay signals initiated by intercellular adhesion to intracellular proliferation signal activation. We have also investigated more closely the signaling molecules that are downstream of the E-cadherin-mediated EGF receptor activation, which is responsible for the activation of Akt and MAPK. Our data indicate that Gab 1 was activated during the calcium switch procedure in OVCAR-3 cells, and it is probably important for activation of the downstream Akt and MAPK by EGF receptor.

Our results are supported by a previously published report that establishment of E-cadherin-mediated adhesion can activate Akt in dog kidney epithelial cells (MDCK) (30). While this paper was being revised, a study from Shen and Kramer (31) reported that E-cadherin can physically interact with and activate the EGF receptor in squamous cell carcinoma, which in turn leads to the activation of MAPK as well as elevation of the level of the antiapoptotic molecule Bcl-2. Although these results were found in cell lines other than ovarian cancer cells, they do indicate, together with our work, that E-cadherin-mediated cellular adhesion is capable of activating cellular survival pathways.

Compared with other types of tumors, ovarian epithelial cancers are characterized by greater local tumor growth initially, which is followed by spreading into the peritoneal cavity only at later stages of tumor progression (3, 4). E-cadherin may therefore play an important adhesive role in maintaining tissue integrity in this process (4, 15). From the results described in the current study, we propose that the concurrent establishment of E-cadherin-mediated cellular adhesion and activation of proliferation signals for the cells may enhance the transformation from a single layer of normal epithelial cells to neoplastic cells, as well as the development of a tumor tissue mass. Thus, distinct from its role as a tumor suppressor in other types of human epithelial cancers, E-cadherin may function as a tumor enhancer in the development of ovarian epithelial cancers.

A natural question that arises from the current study is what the difference is between ovarian epithelial cancers and other types of epithelial cancers, such as lung, bladder, and pancreatic cancers, in which E-cadherin has been well identified as a tumor suppressor. Although we do not yet understand the in-depth mechanisms behind this phenomenon, we have hypothesized that the substantial hormonal influences on the development and progression of ovarian cancers may drive E-cadherin to play an extraordinary role in the development of ovarian epithelial cancers. However, we have some preliminary data indicating that treatment of OVCAR-3 cells with hormones, including progesterone, estradiol, FSH, LH, or GnRH, does not alter the expression level of E-cadherin (data not shown). Moreover, in our screening of expression levels of E-cadherin, EGF receptor, P-Akt, P-MAPK, as well as urokinase plasminogen activator, matrix metalloproteinase 2, and matrix metalloproteinase 9 (as metastasis markers) in various types of ovarian cancer samples, we did not see any correlation in expression patterns, where, for example, activation of Akt and MAPK in tumor samples was activated in proportion to the degree of overexpression of E-cadherin. Our explanation for this result is either that Akt or MAPK activation is not proportional to the E-cadherin expression level, or the phosphorylation status of Akt and MAPK had been disturbed due to improper handling during the procedures of surgical removal and storage of patient samples. Thus, mechanisms of how E-cadherin activates Akt and MAPK in vivo remain elusive at this stage.

To our knowledge, this study is the first to report that in ovarian cancer cells, E-cadherin-mediated cellular adhesion is capable of activating proliferation and survival signals including EGF receptor, PI3 K (Akt), and MAPK pathways. We have also demonstrated that inhibition of E-cadherin function can lead to reduced cell proliferation and viability. Our results are supported by data obtained from another E-cadherin expressing ovarian cancer cell line CaOV-3, where we have seen almost identical results on the activation of Akt and MAPK via ligand-independent activation of EGF receptor by E-cadherin-mediated adhesion. This suggests that the E-cadherin-mediated activation of survival signals may be a common feature of this adhesion molecule in ovarian cancers. However, at the current stage of the study, we still do not know why E-cadherin shows a different role in ovarian cancers; nor do we know the detailed mechanisms of how E-cadherin activates downstream signals. Questions raised from this study remain to be investigated further by screening for molecules that interact with E-cadherin in ovarian cancer cells and tumor samples. Signal transduction studies, including the use of dominant negative and/or constant active DNA constructs of various kinases, and RNA interference approaches will help to determine the signaling pathways through which Akt and MAPK are activated by E-cadherin in ovarian cancer cells.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Ovarian Cancer Samples from Patients
Studies with human materials in this report were approved by the Human Ethics Committee at the Medical Faculty of Umeå University and Umeå University Hospital. Upon laparotomy at the Department of Obstetrics and Gynecology and Gynecologic Oncology, Umeå University Hospital, ovarian tumors were removed from 38 women. Microscopically and histopathologically normal ovaries, similarly removed for clinical reasons, were obtained from seven women. No patient had received any hormonal therapy during the preceding month. Their mean age (±SE) was 57.8 (1.9) yr. As judged by patient history and hormone analyses, 11 women (25%) were premenopausal and 34 women (75%) were postmenopausal at the time of surgery (Table 1Go). According to World Health Organization classification, the ovarian tumors were all classified (32) as surface epithelial-stromal (Table 1Go). Thirteen (35%) were judged to be malignant, 11 (30%) were borderline, and 13 (35%) were judged to be benign (Table 1Go). Four (31%) of the ovarian cancers were highly differentiated (grade 1), three (23%) were moderately differentiated (grade 2), and six (46%) were poorly differentiated (grade 3). Staging was performed according to International Federation of Gynecology and Obstetrics (FIGO). Seven patients (54%) were in stage I, and six (46%) were in stage III.

Immediately after surgical removal, the ovaries or ovarian tumor samples were transported to the Department of Pathology, dissected, and frozen in liquid nitrogen and kept at –80 C until the time of analysis. The samples were homogenized in a lysis buffer containing 20 mM Tris-HCl (pH 7.5), 137 mM sodium chloride, 15% glycerol (vol/vol), 1% Nonidet P-40, 20 mM sodium fluoride, 10 mM sodium phosphate (NaPPi), 25 mM ß-glycerolphosphate, 2 mM benzamidine, 2 µg/ml aprotinin, 2 µg/ml leupeptin, 1 mM sodium vanadate, and 2 mM phenylmethylsulfonylfluoride. Protein concentrations were measured and 50 µg of total proteins were loaded onto SDS-PAGE gels and blotted for determination of E-cadherin and ß-actin levels.

Cells and Culture Conditions
OVCAR-3 cells were provided by Dr. Karin Sundfeldt of Göteborg University, Sweden, and were routinely maintained at 37 C in DMEM/F12 (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% fetal calf serum and 100 U/ml penicillin and 100 µg/ml streptomycin, in a humidified atmosphere of 5% CO2 and 95% air. For all experiments, the cells were grown as a confluent monolayer to optimize cell-cell contacts and to minimize the influence of integrin-extracellular matrix interactions (33).

The OVCAR-3 cells were serum starved overnight, and E-cadherin-mediated cell-cell contacts were disrupted by the addition of EGTA to the medium to a final concentration of 4 mM, for 30 min at 37 C. Thereafter, intercellular interactions were allowed to reestablish in the presence of fresh calcium-containing medium (final concentration of CaCl2, 1.8 mM) (22, 23). At different time points (1–90 min) after calcium restoration, the cells were harvested, lysed in a lysis buffer containing 50 mM Tris-HCl (pH 8.0), 120 mM NaCl, 20 mM NaF, 20 mM ß-glycerol phosphate, 1 mM EDTA, 6 mM EGTA (pH 8.0), 1% Nonidet P-40, 1 mM dithiothreitol, 5 mM benzamidine, 1 mM phenylmethylsulfonylfluoride, 250 µM sodium vanadate, 2 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 µg/ml pepstatin, on ice for 20 min, and centrifuged in a refrigerated microcentrifuge at 14,000 rpm for 15 min. Supernatants were collected and prepared for further analysis.

Reagents, Antibodies, and Immunological Detection Methods
Specific kinase inhibitors Tyrphostin AG 1478, GTP-14564, AGL 2263, Tyrphostin AG 825, and Genistein were purchased from Calbiochem (San Diego, CA), and PP1 was purchased from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA). Antibodies against E-cadherins, ZO-1 and ß-catenin, were purchased from BD Transduction Laboratories (San Jose, CA). Neutralizing antibodies to E-cadherin (SHE 78–7 and HECD-1) were purchased from Takara (Shiga, Japan) and were used for antibody inhibition experiments. Rabbit polyclonal antibodies against p44/42 MAPKs (MAPK, or ERK 1 and 2), phosphorylated p44/42 MAPK (Thr202/Tyr204, p-MAPK), Akt (PKB), phosphorylated Akt (Ser473), and Gab 1 were from Cell Signaling Technologies (Beverly, MA). Rat monoclonal phospho-Raf-1 (Ser338) and EGF receptor-neutralizing antibody (LA1) were from Upstate Biotechnology, Inc. (Lake Placid, NY). Mouse monoclonal antibody against Raf-1 (E-10) and antiphosphorylated tyrosine (pY99) were obtained from Santa-Cruz Biotechnology, Inc. (Santa Cruz, CA). Antimouse and antirabbit secondary antibodies coupled to horseradish peroxidase were from Bio-Rad Laboratories, Inc. (Hercules, CA). Fluorochrome-conjugated antibodies for immunofluorescence were obtained from Zymed Laboratories, Inc. (South San Francisco, CA).

Akt Kinase Assay
Akt kinase assay was performed using the kinase assay kit from Cell Signaling Technologies (Beverly, MA). Briefly, Akt was precipitated using an immobilized monoclonal antibody that preferably recognizes phosphorylated Akt at Serine 473. After washing, ATP and recombinant GSK were added to the reaction mixture and the kinase reaction was run at 30 C for 30 min. The reaction mixture was then heated at 95 C for 5 min before being run in a SDS-PAGE gel and blotted against an antibody recognizing phosphorylated GSK (Cell Signaling Technologies).

Immunofluorescence Microscopy
OVCAR-3 cells were grown on glass coverslips until multicell clusters were formed, washed with PBS, and fixed in 4% paraformaldehyde for 20 min at room temperature. After being permeabilized with 0.5% Triton X-100 for 10 min at room temperature, the cells were washed with PBS and incubated with mouse anti-E-cadherin antibody (which recognizes the intracellular part of E-cadherin) (1:100) for 1 h at 37 C. As secondary antibody, FITC antimouse IgG (H+L) antibody was used (1:50) (Zymed Laboratories). Coverslips were examined under a Zeiss Axiophot microscope (Carl Zeiss, Thornwood, NY) at a x63 magnification using appropriate filters. Images were captured using a Leica DMLB (Leica Corp., Deerfield, IL) and processed with Adobe Photoshop (Adobe Systems, San Jose, CA).

GST-Raf Pull-Down Assay for Measurement of Active Ras
The GST-Raf-RBD construct was kindly provided by Dr. Bengt Hallberg (Umeå University, Umeå, Sweden). The construct was expressed in BL21 Escherichia coli and isolated with glutathione-agarose (Amersham Biosciences, Uppsala, Sweden) according to manufacturer’s instructions. GST-Raf-RBD fusion protein (30 µg) bound to glutathione-agarose was added to each sample of cellular lysates (500 µg) and incubated on a rotator for 1 h at 4 C. The beads were then washed three times with the lysis buffer, and associated proteins were separated by 12% SDS-PAGE and blotted with an antibody against Ras (BD Transduction Laboratories) to indicate levels of active Ras. As an internal control, levels of total Ras in the corresponding lysates were also measured.

p21-Activated Kinase (PAK) Cdc42/Rac1-interactive binding domain (CRIB) Pull-Down Assay for Measurement of Active Rac
The PAK-CRIB GST fusion protein construct (a kind gift from Dr. J. Chernoff, Fox Chase Cancer Center, Philadelphia, PA) was expressed in BL21 E. coli and isolated with glutathione-agarose (Amersham Biosciences). GST fusion protein (50 µg) bound to glutathione-agarose was added to each sample of OVCAR-3 lysate (200 µg) and incubated on a rotator for 1 h at 4 C. The beads were then washed three times with lysis buffer and separated in a 12% SDS-PAGE gel. Western blot was performed with an anti-Rac monoclonal antibody (Upstate Biotechnology, Inc.) to indicate active forms of Rac. As an internal control, levels of total Rac in the corresponding lysates were also measured.

MTT Assay
Briefly, 2 x 105 OVCAR-3 cells were seeded in 24-well plates and incubated with or without E-cadherin-neutralizing antibody SHE 78–7 or mouse IgG control (5 µg /ml), LY294002 (50 µM), or PD98059 (50 µM) for 24, 48, and 72 h at 37 C in DMEM/F12 culture media supplemented with 10% fetal calf serum and 100 U/ml penicillin and 100 µg/ml streptomycin, in a humidified atmosphere of 5% CO2 and 95% air. The standard MTT assay was performed as previously described (34). Values at 0 h was defined as 1.0, and fold inductions/reductions at different time points were used to calculate the mean values ± SD.

Data Analysis
All experiments were repeated at least three times. For Western blot figures, a representative blot from three independent experiments is depicted. In addition, normalized values of Western blot as determined by densitometric analyses showing the mean ± SD from three independent experiments were also included, where the band of the highest density in a Western blot was defined as 1.0, and from this value all other values of the same blot were calculated. Note that the relative density representing the expression level of a molecule is only comparable to other time points or treatments within the same set of experiments. The relative densities are not comparable among measurements of different molecules or among different experiments. Data were analyzed by ANOVA. Differences among groups were calculated by Tukey’s multiple-comparison test, and a difference was considered to be significant with P < 0.05.


    ACKNOWLEDGMENTS
 
Acknowledgments

We thank Professor Tor Ny (Umeå University, Umeå, Sweden) for his support on this manuscript; Dr. Karin Sundfeldt (Göteborg University, Göteborg, Sweden) for providing OVCAR-3 cells; Dr. Bengt Hallberg (Umeå University) for providing the GST-Raf-RBD construct; and Dr. J. Chernoff (Fox Chase Cancer Center) for providing the PAK-CRIB GST construct.


    FOOTNOTES
 
This work was supported by the Swedish Medical Research Council (Project no. K2005-72X-15379-01A); the Swedish Cancer Foundation (Project no. 4988-B04-01XAB); Swedish Cancer Research Foundation in Norrland (LP 03-1574; LP 04-1593; LP-04-1620); and the Medical Faculty of Umeå University, Sweden.

Present address of M.R.: Department of Medical Biochemistry, University of Kuopio, POB 1627, 70211 Kuopio, Finland.

First Published Online May 31, 2005

1 P.R., L.L., and C.R. contributed equally to this study. Back

Abbreviations: CRIB, cdc42/Rac1-interactive binding domain; EGF, epidermal growth factor; GSK, glycogen synthase kinase; GST, glutathione-S-transferase; MEK, MAPK kinase; MTT, 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl tetrazolium bromide; OSE, ovarian surface epithelial; PAK, p21-activated kinase; P-Akt, phosphorylated Akt; PI 3K, phosphatidylinositol 3-kinase; PKA, protein kinase A; P-MAPK, phosphorylated MAPK; PTK, protein tyrosine kinase; RPTK, receptor protein tyrosine kinase.

Received for publication September 1, 2004. Accepted for publication May 25, 2005.


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 ABSTRACT
 INTRODUCTION
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 DISCUSSION
 MATERIALS AND METHODS
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