Elevated expression or activity of the epidermal growth factor receptor (EGFR) is common in ovarian cancer and is associated with poor patient prognosis. A naturally occurring EGFR mutation termed variant III (EGFRvIII) has been detected in many human tumors, including those of the ovary. This mutant receptor does not bind EGF; however, it is constitutively active as detected by receptor dimerization, autophosphorylation, and stimulation of signal transduction cascades. To identify the consequences of EGFRvIII expression in ovarian tumor cells, we introduced EGFRvIII into the epithelial ovarian cancer cell line OVCA 433. The EGFRvIII-transfected cells displayed a motile phenotype, defects in cell spreading, and decreased integrin α2 protein expression as detected by Western blot analysis and flow cytometry. Inhibition of EGFRvIII catalytic activity using the EGFR-selective tyrphostin AG1478 restored integrin α2 expression within 4 to 8 hours after treatment. The modulation of integrin α2 expression corresponded to marked changes in the actin cytoskeleton as detected by redistribution of filamentous-actin. Furthermore, focal adhesions were evident only when EGFRvIII activity was inhibited. Together, these findings suggest that expression of the constitutively active mutant EGFRvIII promotes changes in cell shape and focal adhesion formation, mediated in part through specific modulation of integrin α2 expression and function. We conclude that EGFR-activating mutations, such as EGFRvIII, in ovarian cancer may contribute to a more aggressive disease.
- ovarian cancer
- integrin α2
- EGF receptor
Epithelial ovarian carcinoma (EOC) is the leading cause of death from gynecologic malignancy, resulting in ∼16,090 deaths in 2004 ( 1, 2). Due to the current inability to detect disease confined to the ovary (stage I), ∼75% of women are initially diagnosed with disseminated intra-abdominal disease (stages III-IV) and have a 5-year survival of <20%, whereas patients diagnosed with cancer localized to the ovary have a >90% 5-year survival ( 3, 4). Early events in the metastasis of EOC involve dissociation of cells from the primary tumor and interaction with mesothelial cells lining the inner surface of the peritoneal cavity as well as the interstitial collagen-rich submesothelial extracellular matrix to invade adjacent pelvic organs ( 5). Integrin-mediated adhesive events are involved in each step in metastatic progression ( 5– 7), and previous studies indicate that interaction of ovarian tumor cells with interstitial collagens may represent an important early event unique to EOC i.p. dissemination ( 8– 10).
EOC displays many genetic defects and altered expression of oncogenes and tumor suppressors, including elevated epidermal growth factor receptor (EGF) expression or activity ( 4). Aberrant EGFR activation may play a critical role in tumor progression and metastasis through regulation of cell-cell and cell-matrix interactions, cell migration, and induction of many matrix-degrading proteases ( 11, 12). There are multiple mechanisms of EGFR activation in the tumor microenvironment, including ligand-dependent and ligand-independent receptor activation and mutations that alter receptor activity. Many different naturally occurring EGFR mutations occur in human tumors ( 13– 15), and the best characterized is an oncogenic mutant designated de2-7 EGFR or EGFRvIII. EGFRvIII has been detected in numerous tumor types ( 16– 18), including those of the ovary ( 19, 20). This form of the EGFR displays constitutive activity and is transforming in the absence of ligand ( 21, 22). Activation of the EGFR by various mechanisms including mutation may be a factor in the progression of ovarian cancer.
Numerous studies have shown cooperation between integrin and EGF-mediated signaling pathways in the control of mitogenic, motogenic, and cell survival pathways ( 23– 26). EGFR activation can modulate integrin function by regulating the expression and/or activity of numerous integrins, leading to altered adhesion, motility, and invasive capacity ( 27– 29). Ovarian cancer cells preferentially adhere to interstitial type I collagen primarily mediated via the α2β1 and α3β1 integrins ( 8, 9), and EGFR seems to regulate various aspects of α2β1 integrin functions. The α2 integrin cytoplasmic domain is required for EGF-stimulated migration ( 30, 31), and EGF selectively increases expression of α2 but not β1 integrin in several cell types ( 32, 33). Furthermore, colocalization and direct interaction between integrin α2β1 and the EGFR has been reported in human epithelial A431 cells ( 23). These observations suggest that aberrant EGFR activity may alter integrin α2 expression and/or function.
In this study, we report that cell spreading on type I collagen is impaired in ovarian tumor cell lines expressing EGFRvIII when compared with the parental or vector control cells. Cell morphology, spreading, and assembly of focal adhesions are restored upon inhibition of EGFRvIII catalytic activity. The observed modulation of cell shape was selectively mediated by integrin α2, but not integrin α3, in parental OVCA 433– and EGFRvIII-expressing cells. Furthermore, integrin α2, but not integrin α3 or integrin β1, cell surface localization and protein expression are dynamically regulated by EGFRvIII. Based on these findings, we propose that regulation of integrin α2 by EGFRvIII is a key determinant in the altered morphology and impaired spreading observed in cells expressing this constitutively active form of the EGFR.
Materials and Methods
Cell culture and treatment. Ovarian carcinoma cell line OVCA 433 was generously provided by Dr. Robert Bast Jr. (M.D. Anderson Cancer Center, Houston, TX) and grown as described previously ( 34– 36). The OVCA 433 cell line was selected for transfection based on moderate expression of endogenous wild-type EGFR compared with other ovarian tumor lines, as determined by Western blot analysis and evidence of intact EGF-dependent signaling pathways (data not shown; refs. 37, 38). The EGFRvIII construct was a generous gift of Dr. David Moscatello (Thomas Jefferson University, Philadelphia, PA). OVCA 433 cells were cotransfected with a vector containing a neomycin resistance gene and either the EGFRvIII construct in PLTR2 vector or the PLTR2 vector at a 1:10 ratio by calcium phosphate method. Multiple clones were selected in presence of 300 μg/mL G418 (Life Technologies/Bethesda Research Laboratories, Gaithersburg, MD). EGFRvIII expression was verified by Western blot using an antibody directed against the intracellular domain of the EGFR (clone 1005, Santa Cruz Biotechnology, Santa Cruz, CA) to detect both wild-type EGFR and EGFRvIII and by immunofluorescence using an antibody directed against the novel epitope in EGFRvIII (Ab1825). Ab1825 was raised in chickens by Aves Laboratory (Tigard, OR) as described elsewhere ( 39) with the following modifications: the anti-peptide antibody was produced for the peptide LEEKKGNYVVTDHC after adding an amino caproic acid just upstream of the COOH-terminal cysteine, which was used for conjugation to keyhole limphet hemacyanin. Two EGFRvIII-transfected clones, designated EGFRvIIIA1 and EGFRvIIIB6, were selected for extensive characterization. For experiments involving EGF (Biomedical Technologies, Stoughton, MA), OVCA 433 cell lines were placed into MEM containing 0.1% (w/v) bovine serum albumin (BSA) for 24 hours before growth factor addition, as described previously ( 35, 36). Treatment with the EGFR catalytic inhibitor AG1478 (Calbiochem, La Jolla CA) was conducted in complete growth medium unless otherwise noted in the figure legends.
Cell spreading and spreading inhibition assay. To evaluate the integrin dependence of spreading, 24-well plates were coated by passive adsorption with type I collagen [20 μg/mL in 0.1 mol/L sodium carbonate (pH 9.0)] at 4°C overnight and blocked by incubating with 3% BSA in MEM at 37°C for 1 hour. OVCA 433 cells were trypsinized, neutralized, and resuspended in serum-free MEM at a concentration of 100,000 cells/mL before incubation with anti-α2 integrin antibodies (1:100 dilution, Chemicon clones P1E6 and CLB150, Temecula, CA), anti-α3 integrin antibodies (1:100 dilution, Chemicon clone ASC-6), or control mouse IgG for 30 minutes at room temperature with gentle rotation. Cells (100,000/mL) were added to the coated wells in a volume of 0.5 mL for the indicated time periods. Spreading was monitored by fixing and staining cells with Diff-quik. After acquisition of digital images, cell length was measured and expressed in arbitrary units.
For spreading inhibition experiments, cells were lifted in Dulbecco's PBS (DPBS) containing 50 mmol/L EDTA and plated in six-well plates at 1 × 104 cells/cm2 in a final volume of 1 mL per well of growth medium with or without AG1478 (2 μmol/L) or function-blocking antibodies directed against integrin α2 (1:100 dilution, clone P1E6, Chemicon) and integrin α3 (1:100 dilution, clone P1B5, Chemicon). For the indicated assays, wells were coated overnight at 4°C with 1 mL of type I collagen solution diluted at 5 μg/cm2. The cells were examined 24 hours after plating under an inverted microscope (Olympus 1 × 70, Melville, NY). Spreading was defined as flattened cells without the refractile, fibroblastic morphology determined and expressed as a percentage of the total cells ± SD. At least 150 cells per well were counted, and the data represent the mean of three independent experiments.
Immunofluorescence. EGFRvIII-expressing cells or control OVCA 433 cells (parental or vector control) were treated with 2 μmol/L AG1478 or 25 nmol/L EGF as indicated in the figure legends and fixed with freshly prepared 3.7% (w/v) formaldehyde in PBS [137 mmol/L NaCl, 2.7 mmol/L KCl, 8.1 mmol/L Na2HPO4, and 1.5 mmol/L KH2PO4 (pH 7.4)] containing 0.8 mmol/L MgCl2 and 0.18 mmol/L CaCl2 for 45 minutes at 4°C, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, and blocked with 10% nonfat milk/PBS for 1 hour at 37°C. Fixed cells were incubated with chicken anti-EGFRvIII (1:800, Aves Lab, Inc., Tigard, OR), mouse-anti-integrin α2 (1:100, BD PharMingen, San Diego, CA), mouse anti-integrin α3 (1:100, Chemicon), and mouse anti-integrin β1 (1:200, Chemicon) for 1 hour at 37°C. After washing thrice with PBS, samples were incubated with FITC-conjugated anti-chicken IgG (1:500, Chemicon) or mouse IgG (1:500, Chemicon). For double staining, cells were grown in type I collagen (5 μg/cm2, BD Biosciences, Bedford, MA)–coated slides, treated with AG 1478 and function-blocking antibodies directed against integrin α2 (1 μg/mL, clone P1E6, Chemicon). After 24 hours, cells were fixed and blocked with 10% nonfat milk/PBS for 1 hour at 37°C, and incubated with mouse anti-paxillin (1:500, Upstate Biotechnology, Lake Placid, NY) for 1 hour at 37°C, and FITC-conjugated anti-mouse IgG (1:200, Chemicon) for 1 hour at 37°C. After washing with thrice PBS, TRITC-labeled Phalloidin (0.5 μg/mL, Sigma, St. Louis, MO) was added for 30 minutes at room temperature. Cells were examined using an Olympus BH2-RFCA microscope (Melville, NY) and images were obtained using an Omegafire digital camera (Optronix, Goleta, CA).
Flow cytometry. Cell lines were washed twice with DPBS and then harvested with trypsin-EDTA. Cells (106) were incubated with primary antibody mouse anti-integrin α2 (1:200, BD PharMingen), mouse anti-integrin β1 (1:200, Chemicon), or mouse anti-integrin α3 (1:200, Chemicon) for 20 minutes at 4°C and then washed twice with DPBS. Cells were then stained with secondary antibody conjugated with FITC for 20 minutes at 4°C, washed twice with DPBS, and then resuspended in 0.5 mL DPBS. Flow cytometric analysis was done on a Becton Dickinson FACScan flow cytometer (Immunocytometry Systems, San Jose, CA). Mean fluorescence intensity for three independent experiments was shown, and error bars represent ±SD.
Western blot analysis. Cells were grown to subconfluence and solubilized in cell lysis buffer [1% SDS, 5 mmol/L EDTA, 2 mmol/L EGTA, 10 mmol/L Tris (pH 7.5)] containing a cocktail of protease inhibitor mixture (Roche Molecular Biochemicals, Indianapolis, IN). Total cellular proteins were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and blocked with 5% nonfat milk in TBS [10 mmol/L Tris, 150 mmol/L NaCl (pH 8.0)] and 0.05% Tween 20 for 1 hour at room temperature. Cellular proteins were then probed with rabbit anti-EGFR (1:1,000, Santa Cruz Biotechnology), rabbit anti-integrin α2 (1:200, Chemicon), rabbit anti-phospho-extracellular signal-regulated kinase (ERK, 1:1,000, Cell Signaling Technology, Beverly, MA), or rabbit anti-tubulin (1:500, Santa Cruz Biotechnology); washed thrice with TBS and 0.05% Tween 20 (TBST); and incubated with horseradish peroxidase–conjugated anti-rabbit IgG secondary antibody. For immunoprecipitation, cells were lysed in ice-cold lysis buffer [20 mmol/L HEPES (pH 7.4), 2 mmol/L EGTA, 50 mmol/L β-glycerophosphate, 1 mmol/L Na3VO4, 5 mmol/L NaF, 1% Triton X-100, 10% glycerol, 1 mmol/L DTT] containing a cocktail of protease inhibitor mixture (Roche Molecular Biochemicals) for 30 minutes at 4°C and clarified at 14,000 rpm at 4°C for 20 minutes. The supernatants of cell lysates were precleared with Protein A-Sepharose 4B and incubated with mouse anti-integrin β1 (2 μg, Chemicon) for 2 hours at 4°C followed by with protein A-Sepharose 4B beads overnight at 4°C. The Sepharose beads were washed thrice with 15 mmol/L CHAPS and once with deionized water and suspended in 50 μL of 2× loading buffer [62.5 mmol/L Tris-HCl (pH 6.8), 2% SDS, 25% glycerol, and 0.1% bromophenol blue]. Western blotting was done on samples, and membranes were probed with mouse anti-integrin β1 (1:200, Chemicon).
EGFRvIII alters the morphology and spreading of ovarian tumor cells. Ovarian cancer cells adhere preferentially to interstitial type I collagen mediated by α2β1 and α3β1 integrins ( 8– 10, 40) likely reflecting the phenotypic plasticity of the mesodermally derived ovarian surface epithelium as well as the interstitial collagen-rich composition of the mesothelial matrix to which ovarian cancer cells adhere and subsequently invade ( 3). Stable clonal lines of OVCA 433 cells expressing EGFRvIII were generated ( Fig. 1A-B ). EGFRvIII-expressing ovarian cancer cells are morphologically distinct from parental or control-transfected cells ( Fig. 1C). Parental OVCA 433 cells are epithelioid and adopt a spindle morphology following EGF treatment for 24 hours ( Fig. 1C). EGFRvIII-expressing clones A1 and B6 display an elongated and dispersed phenotype even in the absence of exogenous EGF ( Fig. 1C, bottom). The altered morphology associated with EGFRvIII expression has no effect on the magnitude of cell adhesion to type I collagen at multiple time points (30 minutes to 24 hours; data not shown) when compared with parental or vector control cells; however, cell spreading is substantially impaired ( Fig. 2 ). Control OVCA 433 cells attach, extend lamellipodia, and spread on type I collagen within 30 minutes after plating ( Fig. 2A, top). In contrast, EGFRvIII-expressing cells exhibit long filopodia, but few lamellipodia, resulting in an elongated phenotype ( Fig. 2A, bottom). To determine whether α2β1 and/or α3β1 integrin is necessary for cell spreading on collagen, OVCA 433 cells were plated on type I collagen in the absence or presence of integrin subunit-specific function-blocking antibodies ( Fig. 2B). Two distinct integrin α2–blocking antibodies significantly reduced cell spreading, whereas integrin α3–blocking antibodies had no effect ( Fig. 2B), illustrating a key role of integrin α2 in mediating OVCA 433 cell spreading on type I collagen.
Integrin α2 is dynamically modulated by EGFRvIII catalytic activity. The impaired cell spreading of EGFRvIII-expressing cells on type I collagen suggested modulation of the expression and/or function of collagen-binding integrins. The effect of EGFRvIII expression on integrin levels and localization was first evaluated by immunofluorescent detection ( Fig. 3A ). Integrin α2 was localized to cell-cell borders in OVCA 433 cells, but staining was diffusely distributed and diminished in intensity in two independent EGFRvIII-expressing clonal lines ( Fig. 3A, top). In contrast, integrin α3 expression and localization was not substantially altered as a consequence of EGFRvIII expression ( Fig. 3A, middle, and B). Integrin β1 was readily detected in parental and EGFRvIII-expressing cells; however, constitutive EGFR activation led to more diffuse localization of this integrin ( Fig. 3A, bottom). This result is supported by flow cytometry to detect surface integrin levels ( Fig. 3B) and Western blot analysis of whole cell lysates ( Fig. 4A ). Cell surface integrin α2 was decreased by 70% to 75% in EGFRvIII-expressing cells as determined by flow cytometry ( Fig. 3B), and total integrin α2 protein levels were reduced ( Fig. 4A, top, compare lanes 1, 3, and 5). In contrast, a modest increase in cell surface integrin α2 (27%) was observed in control cells treated with EGF as has been reported in other cell types ( 27, 32, 33, 41). No marked changes in total integrin α2 protein ( Fig. 4A, top, compare lanes 1 and 2) or localization ( Fig. 4C, bottom) were detected following 24 hours treatment with EGF, which may reflect differences in signaling persistence between wild-type EGFR and EGFRvIII. The observed loss in EGFRvIII expressing cells was selective for integrin α2, as cell surface and total integrin β1 expression were not substantially altered ( Fig. 3B and Fig. 4A, bottom).
To determine whether the loss of surface integrin α2 was a consequence of EGFRvIII catalytic activity, cells were treated with the tyrphostin AG1478. AG1478 has been shown to effectively inhibit tyrosine phosphorylation by both the wild-type EGFR and EGFRvIII ( 14, 42). Total integrin α2 expression was increased when EGFRvIII-expressing cells were treated with AG1478 ( Fig. 4A, compare lanes 3, 4-5, and 6). In addition, restoration of cell surface integrin α2 localization occurs within 2 to 4 hours after treatment with AG1478 and is maximally increased by 8 hours ( Fig. 4B, top, A1 + AG). In the converse experiment, activation of EGFRvIII by removal of AG1478 from the culture medium is evident within 15 to 30 minutes and precedes loss of integrin α2 from the cell surface ( Fig. 4B, middle, A1-AG). Similarly, a decrease in integrin α2 protein in response to EGFRvIII activation was evident within 2 to 4 hours following withdrawal of AG1478 from the culture medium ( Fig. 4C). This response corresponds to the time course of EGFRvIII signaling under these conditions as detected by ERK and AKT phosphorylation ( Fig. 4C; data not shown). These findings show that integrin α2 expression and cell surface localization are dynamically and reversibly modulated as a function of EGFRvIII catalytic activity and indicate that down-regulation of integrin α2 is dependent on the tyrosine kinase activity of EGFRvIII.
Functional effect of integrin α2 down-regulation by EGFRvIII. Integrin α2 was found to be essential in the cell spreading of OVCA 433 cells ( Fig. 2B); therefore, experiments were done to determine whether the observed down-regulation of integrin α2 by EGFRvIII was responsible for the observed spreading defect. Inhibition of EGFRvIII catalytic activity with AG1478 for 24 hours resulted in cell spreading comparable with that observed in the parental OVCA 433 cells ( Fig. 5A ). Function-blocking integrin α2 antibodies disrupted spreading, showing the requirement for integrin α2 in this restored spreading response ( Fig. 5A-B). As with parental OVCA 433 cells, the response was selective for integrin α2, because blocking antibodies to integrin α3 did not interfere with cell spreading (data not shown). These data support a role for integrin α2 in the observed EGFRvIII-dependent change in ovarian cancer cell morphology.
To further characterize the functional effect of integrin α2 down-regulation by EGFRvIII, cytoskeleton organization and focal adhesion formation were evaluated. EGFRvIII-expressing cells displayed loss of organized actin stress fibers and focal adhesions when compared with untreated control cells ( Fig. 6 , compare A1 with Vec5). Inhibition of EGFRvIII catalytic activity restored actin stress fiber formation and focal adhesions as detected by paxillin staining ( Fig. 6, A1 + AG). A function-blocking antibody directed against integrin α2 (PIE6, middle), but not integrin α3 (data not shown), disrupted this response. Together these data show that key functional activities of integrin α2, cell spreading and focal adhesion formation in response to matrix contact, are disrupted in EGFRvIII-expressing cells.
Alterations in integrin-mediated adhesion have been implicated in the shedding, migration, and invasive growth of ovarian cancer cells ( 5, 8, 43). In this study, we report that a constitutively active form of the EGFR, EGFRvIII, down-regulates integrin α2 protein and is accompanied by appearance of a fibroblastic morphology and aberrant spreading on type I collagen. Importantly, blockade of integrin α2, but not integrin α3, disrupted spreading of vector control and parental OVCA 433 cells, implicating integrin α2 in regulation of the cell shape. Inhibition of EGFRvIII catalytic activity restored integrin α2 expression, cell spreading, and assembly of focal adhesions and these restored functions were reversed by function-blocking antibodies directed against integrin α2. These findings suggest that expression of the constitutively active EGFRvIII promotes a fibroblastic phenotype through modulation of integrin α2 expression and function.
Integrin α2β1 predominantly serves as a collagen type I receptor and integrin α2 expression is selectively modulated by EGFR activation in many epithelial cell types ( 27, 32, 41). It has been reported that integrin-mediated adhesion of cells expressing high levels of EGFR was decreased by exposure to EGF; however, adhesion was enhanced in cells expressing low-to-medium levels of EGFR ( 28, 44) suggesting a complex relationship between magnitude of EGFR activation and cellular response. Interestingly, reexpression of integrin α2 into a poorly differentiated fibroblastic, motile and highly invasive mammary carcinoma cell line restored an epithelial phenotype ( 45). Similar to our findings, expression of integrin α2 did not alter adhesion to collagen type I ( 45) but affected cell shape. In RD cells, integrin α2 cytoplasmic domain was essential for an observed α2β1-mediated conversion from a cell rounding to a cell-spreading phenotype ( 46) indicating that modulation of integrin α2 can have an effect on cell morphology in various cell types.
In ovarian carcinoma, modulation of integrin α2β1 function could play multiple roles in tumor cell behavior. Integrin β1 is an important mediator of ascites ovarian tumor spheroid adhesion to mesothelial cells ( 43) and ovarian tumor cells adhere preferentially to type I collagen prevalent in the mesothelial extracellular matrix ( 8, 9). Recent studies have suggested that a key role of integrins is in bidirectional transmission of mechanical force from the extracellular matrix to the cytoskeleton thereby regulating cytoskeletal organization, gene expression, proliferation, survival, and motility ( 47). In this model, focal adhesions function as mechanosensory devices ( 48, 49), such that alterations in mechanical loading (e.g., fluid shear stress or compressive forces due to ascites) can alter the three-dimensional arrangement of cytoskeletal and signaling molecules in the adhesion plaque and thereby elicit distinct cellular responses. As cell spreading regulates focal adhesion assembly and organization ( 50), it is interesting to speculate that aberrant spreading of ovarian cancer cells due to constitutive EGFR activation leads to dysregulation of adhesion-dependent signaling pathways resulting in aggressive cellular behavior characterized by fibroblastic morphology and a migratory phenotype.
Grant support: U.S. NIH/Department of Health and Human Services grant RO1 CA90492, University of New Mexico Cancer Research and Treatment Center NIH grant P20 CA888070, and University of New Mexico National Institute of Environmental Health Sciences Center grant P30 ES-012072.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Received February 7, 2005.
- Revision received August 9, 2005.
- Accepted August 16, 2005.
- ©2005 American Association for Cancer Research.