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[Cancer Research 65, 9280-9286, October 15, 2005]
© 2005 American Association for Cancer Research

Cell and Tumor Biology

Down-regulation of Integrin {alpha}2 Surface Expression by Mutant Epidermal Growth Factor Receptor (EGFRvIII) Induces Aberrant Cell Spreading and Focal Adhesion Formation

Yan Ning1, Reema Zeineldin2, Yueying Liu3, Martina Rosenberg1, M. Sharon Stack3 and Laurie G. Hudson1

1 Health Sciences Center, College of Pharmacy and 2 Chemical and Nuclear Engineering Department, University of New Mexico, Albuquerque, New Mexico; and 3 Department of Cell and Molecular Biology, Northwestern University Feinberg Medical School, Chicago, Illinois

Requests for reprints: Laurie G. Hudson, College of Pharmacy, MSC09 5360, University of New Mexico, Albuquerque, NM 87131-0001. Phone: 505-272-2482; Fax: 505-272-6749; E-mail: lghudson{at}unm.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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 {alpha}2 protein expression as detected by Western blot analysis and flow cytometry. Inhibition of EGFRvIII catalytic activity using the EGFR-selective tyrphostin AG1478 restored integrin {alpha}2 expression within 4 to 8 hours after treatment. The modulation of integrin {alpha}2 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 {alpha}2 expression and function. We conclude that EGFR-activating mutations, such as EGFRvIII, in ovarian cancer may contribute to a more aggressive disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
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 {alpha}2ß1 and {alpha}3ß1 integrins (8, 9), and EGFR seems to regulate various aspects of {alpha}2ß1 integrin functions. The {alpha}2 integrin cytoplasmic domain is required for EGF-stimulated migration (30, 31), and EGF selectively increases expression of {alpha}2 but not ß1 integrin in several cell types (32, 33). Furthermore, colocalization and direct interaction between integrin {alpha}2ß1 and the EGFR has been reported in human epithelial A431 cells (23). These observations suggest that aberrant EGFR activity may alter integrin {alpha}2 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 {alpha}2, but not integrin {alpha}3, in parental OVCA 433– and EGFRvIII-expressing cells. Furthermore, integrin {alpha}2, but not integrin {alpha}3 or integrin ß1, cell surface localization and protein expression are dynamically regulated by EGFRvIII. Based on these findings, we propose that regulation of integrin {alpha}2 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
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
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-{alpha}2 integrin antibodies (1:100 dilution, Chemicon clones P1E6 and CLB150, Temecula, CA), anti-{alpha}3 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 x 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 {alpha}2 (1:100 dilution, clone P1E6, Chemicon) and integrin {alpha}3 (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 x 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 {alpha}2 (1:100, BD PharMingen, San Diego, CA), mouse anti-integrin {alpha}3 (1:100, Chemicon), and mouse anti-integrin ß1 (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 {alpha}2 (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 {alpha}2 (1:200, BD PharMingen), mouse anti-integrin ß1 (1:200, Chemicon), or mouse anti-integrin {alpha}3 (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 {alpha}2 (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 ß-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 ß1 (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 2x 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 ß1 (1:200, Chemicon).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
EGFRvIII alters the morphology and spreading of ovarian tumor cells. Ovarian cancer cells adhere preferentially to interstitial type I collagen mediated by {alpha}2ß1 and {alpha}3ß1 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 {alpha}2ß1 and/or {alpha}3ß1 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 {alpha}2–blocking antibodies significantly reduced cell spreading, whereas integrin {alpha}3–blocking antibodies had no effect (Fig. 2B), illustrating a key role of integrin {alpha}2 in mediating OVCA 433 cell spreading on type I collagen.



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  		Figure 1. Detection of EGFRvIII in stably transfected cells and morphology of OVCA 433 cells and EGFRvIII-expressing clones on type I collagen. A, detection by immunofluorescence microscopy and Western blot analysis. Cells were fixed and stained with chicken anti-EGFRvIII Ab1825 antibody as described in Materials and Methods. 433, parental OVCA 433 cell line; A1, EGFRvIII clone A1; B6, EGFRvIII clone B6. B, for Western blot, 35 µg of total cell lysate were loaded per well. An antibody to the intracellular domain of EGFR and EGFRvIII was used to detect both wild type and EGFRvIII expression. Note that EGFRvIII was detected in EGFRvIII-expressing cells only. C, OVCA 433 cells were plated on type I collagen (5 µg/cm2) for 24 hours and treated without or with 25 nmol/L EGF for an additional 24 hours. EGFRvIII-expressing clones A1 and B6 were plated under identical conditions in the absence of EGF. Representative of a minimum of three independent experiments.

 


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  		Figure 2. Cell-spreading kinetics and requirement for integrin {alpha}2 on type I collagen. A, vector control OVCA 433 cells (Vec5, top) or EGFRvIII-expressing cells (A1, bottom) were plated on type I collagen–coated wells (50,000 cells per well) in a volume of 0.5 mL for the indicated times. After fixation and staining with Diff-quik, digital images were acquired for each time point. Representative images are shown. B, vector control OVCA 433 (Vec5) cells were suspended in serum-free medium (100,000 cells/mL) before incubation with the indicated antibodies for 30 minutes at room temperature. Cells were then plated on type I collagen–coated wells for the indicated times, fixed, and stained. Digital images were acquired and cell lengths were measured for cells in a minimum of three independent fields. Columns, means (arbitrary units, a.u.); bars, ±SD. +, P < 0.05; #, P < 0.005; *, P < 0.0005.

 
Integrin {alpha}2 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 {alpha}2 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 {alpha}3 expression and localization was not substantially altered as a consequence of EGFRvIII expression (Fig. 3A, middle, and B). Integrin ß1 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 {alpha}2 was decreased by 70% to 75% in EGFRvIII-expressing cells as determined by flow cytometry (Fig. 3B), and total integrin {alpha}2 protein levels were reduced (Fig. 4A, top, compare lanes 1, 3, and 5). In contrast, a modest increase in cell surface integrin {alpha}2 (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 {alpha}2 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 {alpha}2, as cell surface and total integrin ß1 expression were not substantially altered (Fig. 3B and Fig. 4A, bottom).



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  		Figure 3. Effect of EGFRvIII expression on integrin {alpha}2, {alpha}3, and ß1 levels and localization. A, parental OVCA 433 cells or EGFRvIII-expressing clones (A1 and B6) were fixed and the indicated integrins were detected by fluorescence microscopy as described in Materials and Methods. B, the surface expression of integrins {alpha}2, {alpha}3, and ß1 were compared in vector control OVCA 433 cells (Vec5) and two EGFRvIII-expressing clones (A1 and B6) using flow cytometry as described in Materials and Methods. Columns, mean fluorescence intensities for three independent experiments; bars, ±SD. *, P < 0.0005.

 


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  		Figure 4. Integrin {alpha}2 is dynamically modulated by EGFRvIII catalytic activity. A, OVCA 433 cells were treated without or with EGF (25 nmol/L) for 24 hours (lanes 1-2). EGFRvIII-expressing clones (A1 and B6) cells were treated without or with AG1478 (2 µmol/L) in growth medium for 3 days (lanes 3-6). Cell lysates were collected and total protein fractionated by polyacrylamide gel electrophoresis followed by immunoblotting for integrin {alpha}2 (top), or tubulin as a loading control (middle). Immunoprecipitation was done on cell lysates followed by immunoblotting to detect for integrin ß1 (bottom). B, integrin {alpha}2 was detected in EGFRvIII-expressing clone A1 by immunofluorescence microscopy at the indicated time points following addition (A1 + AG, top) or removal (A1 -AG, middle) of the EGFR kinase inhibitor AG1478 (2 µmol/L) from the growth medium. Parental OVCA 433 cells (bottom) were treated with EGF (25 nmol/L) for the indicated times. C, EGFRvIII-expressing clone A1 was incubated with AG1478 (2 µmol/L) for 3 days. At that time, the medium was removed and replaced with serum-free medium and cell lysates were collected at the indicated times. Total cell protein was fractionated by polyacrylamide gel electrophoresis and immunoblot analysis was done to detect integrin {alpha}2, phospho (active) ERK (P-ERK) or tubulin as a loading control.

 
To determine whether the loss of surface integrin {alpha}2 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 {alpha}2 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 {alpha}2 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 {alpha}2 from the cell surface (Fig. 4B, middle, A1-AG). Similarly, a decrease in integrin {alpha}2 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 {alpha}2 expression and cell surface localization are dynamically and reversibly modulated as a function of EGFRvIII catalytic activity and indicate that down-regulation of integrin {alpha}2 is dependent on the tyrosine kinase activity of EGFRvIII.

Functional effect of integrin {alpha}2 down-regulation by EGFRvIII. Integrin {alpha}2 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 {alpha}2 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 {alpha}2 antibodies disrupted spreading, showing the requirement for integrin {alpha}2 in this restored spreading response (Fig. 5A-B). As with parental OVCA 433 cells, the response was selective for integrin {alpha}2, because blocking antibodies to integrin {alpha}3 did not interfere with cell spreading (data not shown). These data support a role for integrin {alpha}2 in the observed EGFRvIII-dependent change in ovarian cancer cell morphology.



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  		Figure 5. Integrin {alpha}2 is involved in cell spreading on collagen type I. A, EGFRvIII-expressing clones A1 and B6 were pretreated without or with AG1478 (2 µmol/L) for 24 hours, released from the plates by EDTA in PBS, and seeded in collagen type I–coated wells without or with the integrin {alpha}2 function-blocking antibody P1E6. Digital images were collected after 24 hours. Left, representative images of cells that did not receive AG1478. Left middle, cells treated with AG1478 (+AG). Right middle, cells treated with AG1478 but plated with integrin {alpha}2 function-blocking antibody P1E6. Note that cell spreading is enhanced when EGFRvIII catalytic activity is inhibited by AG1478, and this response is impaired in the presence of integrin {alpha}2–blocking antibody. Parental OVCA 433 cells and vector control cells are included for comparison (right). B, cell spreading under the indicated conditions was quantified from digital images obtained using an Olympus inverted microscope. Columns, average percentages of spreading cells from three independent experiments; bars, ±SD. Increased cell spreading in response to AG1478 and the decrease in cell spreading in AG1478-treated cells as a consequence of integrin {alpha}2–blocking antibody were statistically significant. *, P < 0.0005. The values obtained comparing cells in the absence of AG1478 with those receiving AG1478 and integrin {alpha}2–blocking antibody PIE6 statistically significant and may reflect more complete ablation of integrin {alpha}2 function than that observed with EGFRvIII activity alone. #, P < 0.05.

 
To further characterize the functional effect of integrin {alpha}2 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 {alpha}2 (PIE6, middle), but not integrin {alpha}3 (data not shown), disrupted this response. Together these data show that key functional activities of integrin {alpha}2, cell spreading and focal adhesion formation in response to matrix contact, are disrupted in EGFRvIII-expressing cells.



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  		Figure 6. Functional effect of integrin {alpha}2 down-regulation by EGFRvIII on cytoskeletal organization and focal adhesion formation. EGFRvIII-expressing cells (clone A1) or vector control OVCA 433 cells (Vec5) were seeded on collagen type I–coated slides and treated as indicated (+AG = AG1478, 2 µmol/L; P1E6 = integrin {alpha}2 function-blocking monoclonal antibody P1E6, 1 µg/mL; EGF = 25 nmol/L) for 24 hours. Filamentous-actin (F-actin) and paxillin were visualized by fluorescence microscopy as described in Materials and Methods.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
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 {alpha}2 protein and is accompanied by appearance of a fibroblastic morphology and aberrant spreading on type I collagen. Importantly, blockade of integrin {alpha}2, but not integrin {alpha}3, disrupted spreading of vector control and parental OVCA 433 cells, implicating integrin {alpha}2 in regulation of the cell shape. Inhibition of EGFRvIII catalytic activity restored integrin {alpha}2 expression, cell spreading, and assembly of focal adhesions and these restored functions were reversed by function-blocking antibodies directed against integrin {alpha}2. These findings suggest that expression of the constitutively active EGFRvIII promotes a fibroblastic phenotype through modulation of integrin {alpha}2 expression and function.

Integrin {alpha}2ß1 predominantly serves as a collagen type I receptor and integrin {alpha}2 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 {alpha}2 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 {alpha}2 did not alter adhesion to collagen type I (45) but affected cell shape. In RD cells, integrin {alpha}2 cytoplasmic domain was essential for an observed {alpha}2ß1-mediated conversion from a cell rounding to a cell-spreading phenotype (46) indicating that modulation of integrin {alpha}2 can have an effect on cell morphology in various cell types.

In ovarian carcinoma, modulation of integrin {alpha}2ß1 function could play multiple roles in tumor cell behavior. Integrin ß1 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.


   Acknowledgments
 
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 2/ 7/05. Revised 8/ 9/05. Accepted 8/16/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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