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Originally published In Press as doi:10.1074/jbc.M405123200 on June 16, 2004

J. Biol. Chem., Vol. 279, Issue 35, 37191-37200, August 27, 2004
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Epidermal Growth Factor Receptor Inhibition Promotes Desmosome Assembly and Strengthens Intercellular Adhesion in Squamous Cell Carcinoma Cells*

Jochen H. Lorch{ddagger}§, Jodi Klessner{ddagger}||**, J. Ken Park{ddagger}||, Spiro Getsios{ddagger}||{ddagger}{ddagger}, Yvonne L. Wu{ddagger}||, M. Sharon Stack{ddagger}§§, and Kathleen J. Green{ddagger}||¶¶

From the ||Departments of Pathology and Dermatology, {ddagger}The Robert H. Lurie Cancer Center, the §Division of Hematology and Oncology, and the §§Department of Cell and Molecular Biology, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611

Received for publication, May 7, 2004 , and in revised form, June 8, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The epidermal growth factor receptor (EGFR) has been proposed as a key modulator of cadherin-containing intercellular junctions, particularly in tumors that overexpress this tyrosine kinase. Here the EGFR tyrosine kinase inhibitor PKI166 and EGFR blocking antibody C225, both of which are used clinically to treat head and neck cancers, were used to determine the effects of EGFR inhibition on intercellular junction assembly and adhesion in oral squamous cell carcinoma cells. EGFR inhibition resulted in a transition from a fibroblastic morphology to a more epithelial phenotype in cells grown in low calcium; under these conditions cadherin-mediated cell-cell adhesion is normally reduced, and desmosomes are absent. The accumulated levels of desmoglein 2 (Dsg2) and desmocollin 2 increased 1.7-2.0-fold, and both desmosomal cadherin and plaque components were recruited to cell-cell borders. This redistribution was paralleled by an increase in Dsg2 and desmoplakin in the Triton-insoluble cell fraction, suggesting that EGFR blockade promotes desmosome assembly. Importantly, E-cadherin expression and solubility were unchanged. Furthermore, PKI166 blocked tyrosine phosphorylation of Dsg2 and plakoglobin following epidermal growth factor stimulation, whereas no change in phosphorylation was detected for E-cadherin and {beta}-catenin. The increase in Dsg2 protein was in part due to the inhibition of matrix metalloproteinase-dependent proteolysis of this desmosomal cadherin. These morphological and biochemical changes were accompanied by an increase in intercellular adhesion based on functional assays at all calcium concentrations tested. Our results suggest that EGFR inhibition promotes desmosome assembly in oral squamous cell carcinoma cells, resulting in increased cell-cell adhesion.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell-cell adhesion plays a critical role in cancer invasion and metastasis. Loss of cell adhesion and alterations in the expression of cadherins are common features of malignant cells and markers for aggressive tumor growth and poor prognosis (1-3). Adherens junctions and desmosomes are key mediators of cell-cell adhesion in epithelial tissues (4, 5). In adherens junctions, the classic cadherins are linked indirectly to the microfilament cytoskeleton through adapter molecules including the armadillo family member {beta}-catenin, which associates with the cadherin tail, and {alpha}-catenin, which interacts with {beta}-catenin and associates directly or indirectly with actin. In desmosomes, specialized desmosomal cadherins, desmoglein(s) and desmocollin(s), are linked indirectly to the intermediate filament (IF)1 cytoskeleton through their own set of adapter proteins, the armadillo family member plakoglobin, which associates with desmosomal cadherin tails, and the IF-associated protein desmoplakin. The adapter proteins of adhesive junctions not only tether the cadherin tails to the cytoskeleton, they also regulate junction assembly state and adhesive strength and can propagate intracellular signals that control cell motility, growth, and differentiation. Although loss of adherens junctions and their components has frequently been described for various tumors, less attention has been focused on possible alterations in desmosome-dependent adhesion or signaling that might contribute to tumor progression or invasion (5, 6).

The mechanisms by which tumor cells alter intercellular adhesion during tumor invasion and metastasis are not well understood. Growth factors such as EGF, hepatocyte growth factor, and transforming growth factor-{alpha} that are motogenic as well as mitogenic play active roles in tumor progression and wound healing (7, 8). Elevated levels of EGFR and transforming growth factor-{alpha} are early markers of carcinogenesis in head and neck cancer (9), and EGFR confers greater metastatic potential to SCCs (10). Growth factor-dependent tyrosine phosphorylation of junctional components, in particular phosphorylation of the armadillo family proteins {beta}-catenin and plakoglobin by EGFR or its downstream targets, has been proposed as a critical step in modulating intercellular adhesion and association of cadherin-catenin complexes with the cytoskeleton (see Refs. 11-16 and for review see Refs. 17 and 18). However, activation of EGFR can also lead to the induction of matrix metalloproteinases (MMPs) whose targets have recently expanded to include cadherins, and fragments generated by MMP-dependent E-cadherin cleavage promote motility (19-22). These observations suggest that growth factor-dependent phosphorylation of intercellular junction components and proteolytic processing of cadherins may cooperate to reduce intercellular adhesion and consequently facilitate tumor cell migration.

In light of these observations it might be predicted that interfering with EGFR activity would strengthen intercellular adhesion; however, little attention has been paid to the effect of growth factor inhibitors on adhesive strength. EGFR small molecule receptor tyrosine kinase inhibitors such as gefitinib (Iressa) and PKI166, as well as monoclonal antibodies such as C225 (cetuximab), are currently undergoing clinical testing. Squamous cell cancer of the head and neck has become a prime target for these drugs (23, 24) because overexpression of the EGFR is present in >90% of all cases (9). Efforts to determine the mechanism through which these drugs effect tumor growth have focused primarily on cell growth and survival rather than cell adhesion (see Refs. 25-27 and for a review see Ref. 28).

Here we employed two EGFR inhibitors with distinct mechanisms of action, the small molecule inhibitor PKI166 and the monoclonal antibody (mAb) C225, to block activation of the EGFR in oral SCC cells. PKI166 competitively inhibits autophosphorylation of the catalytic domain of EGFR/ERB2 tyrosine kinase (23), whereas C225 competitively inhibits receptor binding of EGF and other EGFR ligands (24). Both of these agents altered cell morphology and enhanced adhesive strength in oral SCCs. These alterations were accompanied by inhibition of desmosomal protein tyrosine phosphorylation and MMP-dependent processing, as well as accumulation of desmosomal cadherins and plaque proteins at cell-cell junctions. This assembly process occurred even under low calcium conditions in which cells are able to establish E-cadherin-mediated, but not desmosomal, contacts. Importantly, however, the increases in adhesive strength occurred at all calcium concentrations tested, supporting the physiological relevance of the action of these agents. Somewhat surprisingly, EGFR inhibition preferentially affected desmosomes, as under these conditions E-cadherin expression and solubility were unchanged as was {beta}-catenin phosphorylation. Collectively, our results suggest that inhibition of EGFR may not only affect tumor growth through inhibiting EGFR-dependent mitogenic stimulation but may also inhibit invasion and metastasis by re-establishing intercellular contacts between tumor cells. These findings also raise the intriguing possibility that the observed increase in intercellular adhesive strength in SCC68 cells might be due to the preferential effect of PKI166 on desmosome, rather than adherens junction, assembly.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—SCC68 squamous cell cancer cells (a gift from James Rheinwald, Harvard University) were cultured in keratinocyte SFM (Invitrogen) supplemented with 1 ml of bovine pituitary extract and EGF (0.3 ng/ml) as provided by the supplier. SCC68 cells were grown to 80% confluence in 100-mm culture dishes before passaging. Prior to treatment, cells were trypsinized and replated into growth medium containing 0.09 mM Ca2+ for 24 h. For some immunofluorescence experiments, cells were replated into growth medium with 0.125 mM calcium added. In the case of PKI166, drug was added to final concentrations ranging from 0.1 to 10 µM in 0.07% dimethyl sulfoxide (Me2SO) for the dose-response experiment, and 7 µM for all other experiments, and cells were harvested at the indicated time points. The vehicle 0.07% Me2SO was used as a control. C225 was added to final concentrations ranging from 0.1 to 10 µg/ml for the dose-response experiment and 10 µg/ml for all other experiments. Endobulin, a human nonspecific immunoglobulin preparation, was used as the inactive control at the same concentrations. The EGFR/ERB2-specific small molecule tyrosine kinase inhibitor PKI166 was a kind gift from Novartis AG (Basel, Switzerland) and stored in Me2SO at -20 °C. The EGFR-specific inhibiting antibody Cetuximab (C225) was purchased from Merck. Endobulin was purchased from Baxter (Munich, Germany). The broad spectrum MMP inhibitor GM6001 was obtained from Chemicon (Temecula, CA), dissolved in Me2SO, and stored at -20 °C.

Antibodies—The following antibodies used in this study were described previously. Mouse monoclonal antibodies 6D8 against Dsg2, 5H10 against {beta}-catenin, and 11E4 against plakoglobin were gifts from Dr. M. Wheelock (University of Nebraska, Omaha). The monoclonal mouse antibody 3.1 directed against Dsg1/2 and the anti-E-cadherin antibody HECD-1 were purchased from Research Diagnostics (Flanders, NJ). A mouse monoclonal antibody 4B2 directed against the cytoplasmic domain of Dsg1/2 was utilized for immunoprecipitations.2 To detect desmoplakin, a rabbit polyclonal antibody NW6 was used (29). The mouse monoclonal {beta}-catenin antibody used for immunoprecipitations was purchased from Transduction Laboratories (Lexington, KY). The rabbit {beta}-catenin antibody 2206 used for immunoblotting was purchased from Sigma. The 795 rabbit polyclonal anti-E cadherin antibody was a gift from Dr. R. Marsh and Robert Brackenbury. The chicken 1407 polyclonal anti-plakoglobin antibody was described previously (16). Anti-phosphotyrosine mouse monoclonal antibody 4G10 and anti-phosphotyrosine horseradish peroxidase-conjugated PY99 were from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit and mouse IgG (clone MOPC 31C) were obtained from Sigma. EGFR monoclonal antibodies Ab12 and Ab13 were purchased from Neomarkers (Fremont, CA). For immunoblotting, the following dilutions were used: concentrated 11E4 supernatant (1:2000), NW6 (1:2500), 1407 (1:5000), Ab13 (1:500), 6D8 ascites (1:1000), 5H10 hybridoma supernatant (1:100), 4G10 (1:1000), and PY99 (1:200). Primary antibodies were diluted in 5% milk in phosphate-buffered saline (PBS). Secondary antibodies, goat anti-mouse peroxidase, goat anti-rabbit peroxidase, and goat anti-chicken peroxidase (Kirkegaard & Perry Laboratories, Gaithersburg, MD) were used at a dilution of 1:5000 in PBS, 5% milk. Antibodies were detected using enhanced chemiluminescence (Amersham Biosciences). For immunoprecipitations, 2-5 µl of concentrated 11E4, 30 µl of 4B2 supernatant, 3 µl of rabbit or mouse IgG, 2 µl of the E-cadherin 795 rabbit polyclonal antibody, 3 µg of monoclonal {beta}-catenin antibody, 5 µl of 6D8 ascites, and 5 µl of EGFR Ab12 antibody were used. For immunofluorescence, the following antibody dilutions were used: 6D8 (1:50), NW6 (1:200), HECD1 supernatant (1:2), and KSB17.2 (1:200).

Immunoblotting, Cell Fractionation, Immunoprecipitation, and Concentration of Conditioned Media—For immunoblot analysis of whole cell lysates, cells were harvested in urea sample buffer, and immunoblotting of protein gels on nitrocellulose membranes was performed as described previously (30). For solubility experiments, following treatment with PKI166, Me2SO, endobulin, or C225 cells were rinsed in complete PBS and subjected to cell fractionation into Triton-soluble and Triton-insoluble pools using coimmunoprecipitation buffer (1% Triton X-100, 145 mM NaCl, 10 mM Tris-HCl, pH 7.4, 5 mM EDTA, 2 mM EGTA, 1 mM phenylmethylsulfonyl fluoride) as described previously (31). For immunoprecipitation, 1 ml of RIPA buffer (1% Triton X-100, 0.1% SDS, 10 mM Tris, 140 mM NaCl, 0.5% deoxycholate, 5 mM EDTA, 2 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate) per 100-mm cell culture dish was used for extraction, and lysates were centrifuged at 14,000 x g for 30 min at 4 °C prior to immunoprecipitation.

Immunoprecipitations were conducted using the appropriate antibody as described previously (16). For immune complex retrieval, 40 µl of Gamma Bind Plus Sepharose beads (Amersham Biosciences) were used per reaction. Immune complexes were released by incubation in reducing Laemmli sample buffer at 95 °C and were subjected to SDS-PAGE on 7.5% gels and subsequent immunoblot analysis.

Conditioned media were concentrated using Microcon Y-10 filter devices (Millipore, Bedford, MA) according to the manufacturer's instructions. The retentate was diluted in 1x urea sample buffer (32) and analyzed using 7.5% denaturing gels and subsequent immunoblotting. Scanning densitometry of immunoblots was carried out using Molecular Analyst software (Bio-Rad).

Immunofluorescence and Phase Contrast Microscopy—Cells were plated on glass coverslips for 24 h in regular growth medium. PKI166, Me2SO, endobulin, or C225 were added as indicated above. For immunofluorescence, coverslips were rinsed in PBS and fixed in methanol for 2 min at -20 °C. Alexa Fluor-conjugated secondary antibodies (Molecular Probes, Eugene, OR) were used (1:200). Processed coverslips were examined with a Leitz DMR microscope, and images were captured using a Hamamatsu Orca digital camera and Improvision Openlab software. To quantify border staining, 50 borders from three random fields were examined, and the length of the border occupied by each marker was scored as follows: one-third (black), two-thirds (gray), or the entire (white) border (see Fig. 2C). Statistical analyses were performed by using a two-tailed Student's t test. For phase contrast pictures, a Nikon Diaphot inverted microscope with a Nikon N 2000 camera was used.



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  		FIG. 2.
PKI166 treatment results in specific accumulation of desmosomal cadherins and recruitment of desmosomal cadherins and desmoplakin to cell-cell borders. A, Western blot analysis of whole cell lysates prepared from cells treated with PKI166 for 20 h were immunoblotted for Dsg2, Dsc2, desmoplakin (DP), plakoglobin (PG), E-cadherin (Ecad), and {beta}-catenin ({beta}cat). Both Dsg2 and Dsc2 increased 2-2.5-fold, whereas desmoplakin and plakoglobin as well as the adherens junction proteins E-cadherin and {beta}-catenin were not detectably changed. B, fluorescence analysis of desmosome (a-d) and adherens junction (e and f) markers after a 20-h treatment with 0.07% Me2SO4 (DMSO) only (a, c, and e) or 7 µM PKI166 (b, d and f) in medium containing 0.09 mM CaCl2. a and c represent dual label images of control cells stained with Dsg2 (antibody 6D8) and desmoplakin (antibody NW6), respectively; b and d represent corresponding dual label images of PKI166-treated cells. e and f are Me2SO- and PKI-treated single label images stained for E-cadherin (HECD-1). Dsg2 and desmoplakin (DP) were detected in a punctate desmosomal distribution at cell-cell borders in the cells treated with PKI (b and d) but exhibited a cytoplasmic distribution in Me2SO controls (a and c). E-cadherin staining was present at the cell borders in the controls and exhibited a somewhat more organized appearance following treatment with the EGFR inhibitor (e and f). Bar, 20 µm. C, borders from the immunofluorescence experiments were counted and quantified by determining the extent of staining along the length of the cell border as follows: one-third (black), two-thirds (gray), or entire (white) border. A significant increase in the total amount of Dsg2 (p < 0.01) and desmoplakin (p < 0.001) recruited to cell borders was observed in the presence of PKI166 when compared with the Me2SO controls. No statistically significant difference in the number of borders exhibiting E-cadherin staining was detected.

 
Dispase Assay—Confluent SCC68 monolayers that were seeded in triplicate onto 60-mm dishes were treated with Me2SO, PKI, endobulin, or C225 in the presence of 0.09, 0.25, 0.50, 0.75, and 1.0 mM calcium. After 20 h, cultures were washed twice in PBS and then incubated in 2 ml of dispase (2.4 units/ml; Roche Diagnostics) for 30 min. Released monolayer fragments were quantified based on size and total number. Large fragments were defined as fragments more than 5 mm in size; medium fragments were between 1 and 5 mm, and small fragments were defined as less than 1 mm in size. Under experimental conditions where fragmentation was excessive, a maximum of 400 fragments was counted. Statistical significance was determined for three independent experiments using the Student's t test.

Hanging Drop Adhesion Assays—The hanging drop assay was based on that described previously (33). Low density cultures of SCC68 cells were trypsinized, centrifuged, resuspended in culture medium, and passed though a 40-µm nylon cell strainer (BD Biosciences). Twenty-µl drops of cell suspensions incubated in the presence of either Me2SO or 7 µM PKI166 were seeded onto the inner surface of a 35-mm culture dish lid and cultured in this hanging drop for an additional 20 h. To limit evaporation over this period, 2 ml of PBS was added to the bottom of these tissue culture dishes. The total number of cells that were present in both monolayer and hanging drop cultures following 20 h of culture in the presence of PKI166 was markedly reduced compared with Me2SO controls, consistent with the negative effects on proliferation reported previously (34, 35) for this small molecule inhibitor in a variety of carcinoma cell lines. To compare similar densities of cells grown in a hanging drop after 20 h, 4 x 103 and 8 x 103 cells/drop were examined in cultures treated with Me2SO and PKI166, respectively. Similar results were obtained when identical numbers of cells (8 x 103 cells/drop) were seeded in a hanging drop and cultured for 20 h (data not shown). At the indicated time points, the culture dish lids were inverted, and the hanging drops were flattened with a glass coverslip for subsequent image analysis. To examine the adhesive strength of these cellular aggregates, parallel cultures were triturated 10 times through a 20-µl pipette tip, which had been pre-rinsed with 0.1% Triton X-100/PBS-, followed by three rinses in PBS-, prior to imaging. Three random fields of phase-contrast images from each hanging drop were acquired using a Nikon Diaphot inverted microscope with a Hamamatsu Orca digital camera and Improvision Openlab software. The total number of cells in clusters of 0-20, 21-100, or >100 cells was counted from triplicate hanging drops, and the percentage of cells in these different sized clusters was determined. Statistical differences were examined from three independent experiments using a Student's t test (p < 0.05).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Inhibition of the EGF Receptor Results in a Morphological Transition from a Mesenchymal to Epithelial Phenotype in Squamous Cell Carcinoma Cells—Treatment of epithelial cells such as A431, HT29, and squamous cell carcinoma cells with EGF results in a transition to a fibroblastic morphology, accompanied by loss of intercellular contacts and cell scattering (12). To investigate the effects of EGFR inhibition on cell-cell adhesion, we used the small molecule tyrosine kinase inhibitor PKI166 (24) and the mAb C225 (cetuximab) (23). PKI166 is a competitive inhibitor of the ATP-binding site of the cytoplasmic portion of the EGFR and ERB2, whereas C225 competitively inhibits binding of EGF and other EGFR ligands. The oral squamous cell carcinoma cell line SCC68, which is derived from an EGFR-overexpressing cancer of the tongue, was selected for these studies based on its highly invasive characteristics (36, 37).

To address the efficacy of PKI166 and C225 in SCC68 cells, EGFR was immunoprecipitated from RIPA lysates of cells treated for 20 h, and its tyrosine phosphorylation status was analyzed by immunoblot. Both agents effectively inhibited EGFR phosphorylation in SCC68 cells (Fig. 1A). Dose-response analysis showed alterations in morphology and protein expression of adhesion molecules within a clinically relevant range for PKI166 and C225 (38, 39) (Fig. 1, B and C, and data not shown), and 7 µM (PKI166) and 10 µg/ml (C225) were selected for use throughout the remainder of the study.



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  		FIG. 1.
PKI166 and C225 block EGFR activation and alter SCC68 colony morphology. A, PKI166 and C225 block EGFR phosphorylation. RIPA lysates prepared from SCC68 cells cultured under normal growth conditions with 0.09 mM calcium with Me2SO (0.07%), PKI166 (7 µM), endobulin (10 µg/ml), or C225 (10 µg/ml) for 20 h were incubated with the EGFR Ab13, and immune complexes were precipitated, as described, and blotted for phosphotyrosine with antibody PY99 (PY) and EGFR with Ab12. Whole blot staining for EGFR (Ab12) showed increased amounts of protein possibly due to up-regulation of the receptor and effective inhibition of EGFR tyrosine phosphorylation following treatment with PKI166 or C225. B, PKI166; C, C225 dose-response analyses. Western blot analysis of total cell lysates (30 µg) prepared from SCC68 cells cultured in the presence of 0, 0.1, 0.5, 1, 5, or 10 µM PKI166 or 0, 0.1, 0.5, 1, 5, or 10 µg/ml endobulin, or C225 for 20 h. The immunoblots were probed by using a mouse monoclonal antibody directed against Dsg2 (6D8), Dsc2 (7G6), or a rabbit polyclonal antibody directed against E-cadherin (Ecad) (795). Dsg2 and Dsc2 expression levels increased in a dose-dependent manner (to 1.7-2-fold) with both treatments. In contrast, E-cadherin expression levels remained relatively constant at all concentrations. D, cells were treated with PKI166 at a final concentration of 7 µM (b) or with an equivalent amount of the vehicle, Me2SO (a), for 4 h. SCC68 control cells remain loosely grouped together in control medium containing 0.09 mM CaCl2 and 0.3 ng/ml of EGF as shown by phase contrast microscopy. Cells treated with PKI166 form tightly packed cell colonies and assume a more epithelial cell morphology compared with the more fibroblastic appearance of control cells. Bar = 100 µm.

 
Cells grown for 24 h in low calcium-containing media (0.09 mM CaCl2) under which conditions intercellular adhesion is reduced and desmosomes do not form, were treated with vehicle only or PKI166. Untreated cells retained a fibroblast-like, mesenchymal appearance and were loosely grouped together (Fig. 1D, a), whereas PKI166-treated cells formed colonies that assumed a more tightly packed, cobblestone morphology with prominent cell-cell borders (Fig. 1D, b). These changes became visible after 4 h of treatment and increased during the following 20 h. Similar alterations in cell shape and colony morphology were observed using the mAb C225 (not shown).

EGF Receptor Inhibition Results in Accumulation of Desmosomal Cadherins and Promotes Their Recruitment to Cell-Cell Borders—The dose-response analysis for PKI and C225 indicated that the apparent increase in cell-cell contact was accompanied by an increase in accumulated desmosomal cadherin proteins. Both full-length forms of Dsg2 and Dsc2 increased from 1.7- to 2-fold based on densitometric analysis, although the classic cadherin E-cadherin remained constant (Fig. 1, B and C). To examine whether protein levels of other desmosomal or adherens junction components were altered by EGFR inhibition, immunoblot analysis of whole cell lysates from PKI166-treated cultures was performed for the desmosomal plaque components desmoplakin and plakoglobin and the adherens junction plaque component {beta}-catenin. The results confirmed that the levels of desmosomal cadherin proteins Dsg2 and Dsc2 were elevated 2-2.5-fold, whereas E-cadherin was unchanged (Fig. 2A). However, neither accumulation of the IF-linking protein desmoplakin nor plakoglobin nor {beta}-catenin was altered (Fig. 2A).

To determine whether these changes in protein levels were accompanied by alterations in the assembly state of desmosomes or adherens junctions, immunofluorescence analysis was carried out using markers of plaque and transmembrane components. Under growth conditions of 0.09 (Me2SO) or 0.125 mM (endobulin) calcium, the desmosomal cadherin, Dsg2, was primarily diffuse or dotty in the cytoplasm without detectable cell-cell border localization in Me2SO and endobulin controls (Fig. 2B and Fig. 3A, a). Similar results were obtained for Dsc2. Furthermore, the plaque protein, desmoplakin, appeared as diffuse or cytoplasmic dots, without substantial intercellular staining (Figs. 2B and 3A, c). It had been reported previously (40) that treatment with an EGFR-blocking antibody was associated with a shift of E-cadherin from a cytoplasmic to a predominantly membrane-bound distribution. Under our experimental conditions E-cadherin was prominent at cell-cell borders even in low calcium (Fig. 2B and Fig. 3A, e and f).



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  		FIG. 3.
mAb C225 treatment results in recruitment of desmosomal cadherins and desmoplakin to cell-cell borders. A, fluorescence analysis of desmosome (a-d) and adherens junction (e and f) markers after endobulin control (a, c, and e) or C225 (b, d, and f) treatment. SCC68 cells treated with endobulin or C225 in the presence of 0.125 mM Ca2+ for 20 h were single-labeled for E-cadherin or double-labeled for Dsg2 and desmoplakin (DP). In the presence of endobulin, Dsg2 and desmoplakin staining was primarily diffuse and cytoplasmic (a and c). In contrast, intercellular staining was noted at many cell-cell borders in cells treated with C225 (b and d). E-cadherin staining was already evident at borders in the control cells, although staining appeared more organized following treatment with C225 (e and f). Bar, 20 µm. B, quantification of cell-cell borders. Border formation was scored by determining the amount of staining along the border as follows: one-third (black), two-thirds (gray), or entire (white) border. The amount of Dsg2 and DP at cell-cell borders was significantly increased following treatment with C225 when compared with the endobulin controls (p < 0.01). There was no significant difference in E-cadherin staining between the endobulin- and C225-treated cells.

 
Following treatment with the EGFR inhibitors, both desmoglein and desmoplakin were recruited to cell-cell borders (Fig. 2B and Fig. 3A, b and d). Both markers were organized in a discontinuous dotty pattern reminiscent of desmosomes, albeit less regularly and linearly organized than junctions observed in normal calcium. PKI166 treatment appeared somewhat more effective at inducing this recruitment than C225. In the latter case, a Ca2+ concentration of 0.125 mM was used to "prime" cell responsiveness to C225 treatment. At this concentration Dsg2 and desmoplakin were still largely absent at control borders, but recruitment to C225-treated cell borders was more prominent than in 0.09 mM Ca2+. Quantification of cell-cell borders exhibiting desmosome marker staining revealed a dramatic increase in the absolute number as well as extent of border involvement in both PKI166 and C225-treated cells (Fig. 2C and Fig. 3B). No significant difference in the number of borders was observed for E-cadherin (Fig. 2C and Fig. 3B), although borders appeared more organized in treated cell populations (Fig. 2B and Fig. 3A, e).

EGFR Inhibition Was Accompanied by a Decrease in the Solubility of Desmosomal Markers and Recruitment of Keratin IF to Desmosomes—Intercellular junction assembly and attachment to the underlying cytoskeleton is typically accompanied by a decrease in the detergent solubility of transmembrane cadherin and plaque components. As another indicator of junction assembly state, we compared the Triton solubility of adherens junction versus desmosome components in PKI166- and C225-treated cultures, using Me2SO and endobulin as controls, respectively. Following inhibition of the EGFR, the level of Dsg2 and desmoplakin in the detergent-insoluble pool increased in both cases (Fig. 4A). There was no detectable change in the solubility of E-cadherin under these culture conditions (Fig. 4A, bottom panels).



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  		FIG. 4.
EGFR inhibition specifically increases the insolubility of desmosomal components and leads to association of the IF network to cell-cell attachment sites. A, subcellular fractionation of junction components. SCC68 cells treated with Me2SO (DMSO), PKI166, endobulin, or C225 were fractionated into Triton-soluble and -insoluble pools and immunoblotted for junctional components. The level of Dsg2 (Dg3.1 antibody) and desmoplakin (DP) (NW6 antibody) in the insoluble (insol) pool increased in cells treated with either EGFR inhibitor, consistent with the formation of desmosomal complexes. E-cadherin (E-cad) solubility appeared unchanged. B, double label immunofluorescence analysis. Localization of desmoplakin (NW6) and cytokeratin (KSB 17.2) revealed little staining for desmoplakin (DP) at the cell-cell borders (top left) and perinuclear keratin IF localization (top right) in the Me2SO (DMSO) controls. Following PKI166 treatment an increase in staining for desmoplakin was observed at the cell-cell borders (middle left) frequently associated with keratin IF bundles (middle right). The boxed-in areas in are shown at a higher magnification in the bottom panels. Bar, 20 µm.

 
To address whether the increase in desmoplakin in the Triton-insoluble pool was associated with a reorganization of keratin IF in PKI166-treated cells, double staining using antibodies against desmoplakin and cytokeratin was performed. In untreated cells, very few desmosomes were present at cell-cell borders, and IF exhibited a perinuclear distribution (Fig. 4B, top). In response to PKI166 treatment keratin IF became redistributed, in many cases appearing to associate with desmoplakin-rich structures at cell-cell borders (Fig. 4B, middle and bottom). Together the results suggest that even under low calcium conditions, EGFR inhibition promotes recruitment of desmosomal proteins to cell-cell borders where they assemble into Triton-insoluble, IF-associated desmosomal structures.

PKI166 Inhibits EGF-dependent Phosphorylation of Plakoglobin and Dsg2—Studies examining the effects of tyrosine phosphorylation on the cadherin-catenin complex have largely focused on the adherens junction. To compare the effects of EGFR inhibition on adherens junction and desmosome components in SCC68 cells, we examined the tyrosine phosphorylation status of E-cadherin and {beta}-catenin as well as the desmosomal components Dsg2 and plakoglobin in EGF-stimulated cells in the absence and presence of PKI166. Surprisingly, we found that EGF stimulated the tyrosine phosphorylation of both Dsg2 and plakoglobin but not {beta}-catenin and E-cadherin in SCC68 cells (Fig. 5). PKI166 inhibited phosphorylation of the desmosomal components but had no apparent effect on the status of {beta}-catenin or E-cadherin (Fig. 5).



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  		FIG. 5.
EGFR inhibition blocks tyrosine phosphorylation of Dsg2 and plakoglobin. SCC68 cells were treated with PKI166 as in previous experiments. One hour prior to harvesting, all cells were treated with 10 ng/ml EGF. Cells were lysed in RIPA buffer and subjected to immunoprecipitation (IP) with subsequent immunoblotting (WB) using antibodies directed against E-cadherin (HECD1 and 795), {beta}-catenin (5H10 and C2206), Dsg2 (4B2 and Dg3.1), and plakoglobin (11E4 and 1407). Equal amounts of protein in each corresponding lane are shown in the upper blots in a-d with and without EGFR inhibitor. Blots were stripped and reprobed with PY99 antibody directed against phosphotyrosine which showed no significant amounts of tyrosine-phosphorylated E-cadherin and {beta}-catenin (a and b) in controls or PKI-treated cells. In contrast, Dsg2 and the predominantly desmosomal component plakoglobin showed tyrosine phosphorylation in the controls which decreased significantly in the cells treated with the EGFR inhibitor (c and d).

 
PKI166 Inhibits MMP-dependent Breakdown of Dsg2—Previous studies (41, 42) demonstrated that EGFR stimulation leads to an increase in MMP-9 activation. As cadherins have been recently identified as targets of MMP-dependent proteolysis, we hypothesized that proteolytic cleavage of Dsg2 may be in part responsible for the observed reduced protein levels in 0.09 mM calcium. In the absence of PKI166, immunoblotting for Dsg2 revealed the presence of both the full-length (160 kDa) product as well as a 100-kDa fragment. The amount of this fragment decreased following the inhibition of EGFR by PKI166 (Fig. 6A), which might in part contribute to the net increase of Dsg2 expression levels. Dsg2 breakdown was abrogated in the presence of the broad spectrum matrix metalloproteinase (MMP) inhibitor GM6001 with a complete disappearance of the 100-kDa fragment (Fig. 6B). A 60-kDa fragment generated from the Dsg2 extracellular domain was detected in the supernatant (Fig. 6B, lower panel). In addition, a fragment of the same size could be demonstrated in whole cell lysates despite rigorous washing prior to lysis (Fig. 6A), raising the possibility that the extracellular portion of Dsg2 remains bound to the cell surface despite being cleaved by MMP-9. These results suggest that SCC68 cells use MMP activity to modulate Dsg2 levels and that the EGFR inhibitors are able to interfere with this process.



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  		FIG. 6.
EGFR inhibition down-regulates MMP-dependent breakdown of Dsg2. SCC cells were grown and treated as described previously. A, immunoblotting of whole cell lysates with the extracellular Dsg2 domain antibody 6D8 showed that Dsg2 was broken down into a 100-kDa fragment, and this process was partly inhibited by PKI166 resulting in a net increase in unprocessed Dsg2. A smaller 60-kDa fragment may correspond to the extracellular portion of Dsg2 bound to the cell surface. B, addition of the broad spectrum MMP inhibitor GM6001 resulted in a disappearance of the 100-kDa fragment and a reduction of the 60-kDa extracellular fragment present in the conditioned media.

 
EGFR Inhibition Increases Adhesive Strength between SCC68 Cells—Because desmosomes are key providers of adhesion between keratinocytes, we tested whether the formation of desmosomes following treatment with the EGFR resulted in increased adhesive strength between cells by using a modification of a dispase-based assay described previously (43). The proteinase dispase, which cleaves extracellular matrix proteins while preserving intercellular adhesion molecules, was used to release a confluent monolayer of cells from the substrate. Typically, when this assay is performed on cells maintained in normal levels of extracellular calcium, the entire monolayer is released as a single sheet and is then subjected to defined mechanical stress. In this case, cell monolayers maintained in 0.09 mM calcium were released as fragments of varying sizes. Significantly, more large fragments were generated from PKI-treated monolayers compared with Me2SO controls (Fig. 7) while the number of small fragments decreased, suggesting that drug treatment augmented intercellular adhesiveness in these monolayers. These results indicate that EGFR blockade results in increased adhesive strength even in a low calcium environment.



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  		FIG. 7.
PKI166 induces increased cell-cell adhesion in substrate-released SCC68 cell sheets. Confluent monolayers of SCC68 cells maintained in 0.09 mM calcium, removed from the bottom of a 60-mm Petri dish with the proteinase dispase, were released as fragments of varying sizes. Fragments were counted using a semiquantitative scale of large (more than 5 mm in greatest diameter), medium (between 1 and 5 mm in greatest diameter), and small (up to 1 mm in greatest diameter) sized fragments. Following EGFR inhibition, there was a significantly greater number of large fragments compared with the controls (p < 0.05 on a two-tailed t test, n = 5), although the number of medium (p < 0.05) and small fragments decreased, indicating greater strength of cell-cell adhesion. DMSO, Me2SO.

 
To confirm further the effect of PKI166 on SCC68 cell-cell adhesion in a manner that was independent of cell attachment to a matrix, we performed adhesion assays on cells cultured in suspension for 20 h. This hanging drop assay has been employed previously to quantify changes in adhesive strength for a variety of epithelial cells, including A431 and Madin-Darby canine kidney cells (33, 44). Although we did not observe any significant differences in the size of cellular aggregates that formed following 20 h of culture in a hanging drop in the presence of either Me2SO or PKI166, the cellular aggregates present in the PKI166-treated cultures had a more compacted morphology compared with the Me2SO control cultures (data not shown). These morphological differences correlated with significant differences in the adhesive strength of the SCC68 hanging drop cultures, as assessed by subjecting cells to a shearing force. The percentage of cells present in clusters of >100 cells was significantly greater in PKI166- versus Me2SO-treated cultures following trituration (Fig. 8). In contrast, the number of cells present in smaller clusters (0-20 and 21-100) was increased in the Me2SO cultures, indicating that PKI166 enhanced the resistance of SCC68 cells to trituration under low calcium conditions independent of interactions with the extracellular matrix.



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  		FIG. 8.
PKI166 increases cell-cell adhesion in suspension cultures of SCC68 cells. Hanging drop assays were performed as described under "Experimental Procedures." The stacked bar graphs represent the percentage of cells in clusters of 0-20 cells (black), 21-100 cells (gray), and >100 cells (white) under these conditions. Greater than 1 x 103 cells were counted from each hanging drop, and the average of three independent cultures are presented in the bar graphs. The distribution of SCC68 cell aggregates was similar between cultures treated with Me2SO (DMSO) and PKI166. In contrast, SCC68 cells cultured in the presence of PKI166 were more resistant to trituration compared with Me2SO control cultures.

 
To examine whether the observed increases in adhesive strength also occur under more physiologically relevant conditions, dispase assays were carried out on control cells and cells treated with PKI (Fig. 9A) and C225 (Fig. 9B) under a range of calcium concentrations. At all calcium concentrations tested, a dramatic decrease in particle number was observed in cells treated with both EGFR-blocking agents. Collectively, these data indicate that EGFR inhibition increases intercellular adhesive strength and that it does so independently of contact with the substrate.



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  		FIG. 9.
EGFR inhibition increases cell-cell adhesion in substrate-released SCC68 cell sheets regardless of calcium concentration. Confluent monolayers of SCC68 cells cultured in the presence of 0.09, 0.125, 0.25, 0.75, or 1.0 mM calcium and Me2SO (DMSO) or PKI (A) or endobulin or C225 (B) for 20 h were released using dispase. Monolayers were transferred to 15-ml conical tubes containing 5 ml of PBS and inverted five times. The amount of fragmentation was quantified by counting the number of fragments. At 0.09 mM calcium, any significant differences between the endobulin- and C225-treated SCC68 cells were beyond the resolution of this assay. However, at every other calcium concentration tested, there were significantly fewer fragments generated from the PKI- and C225-treated monolayers (p < 0.001) when compared with Me2SO and endobulin control monolayers, demonstrating that EGFR inhibition strengthens cell-cell adhesion in the SCC68 cells.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Intercellular adhesive junctions have frequently been cited as potential targets of EGFR-dependent signaling thought to promote cell motility associated with tumor progression, invasion, and metastasis. Much attention has been focused on the tyrosine phosphorylation of junctionally associated armadillo proteins by EGFR family members as well as downstream effectors such as Src. However, most of these studies have focused on the arm protein {beta}-catenin and its binding partners in adherens junctions. Surprisingly, little is known about the specific effects of EGFR activity on desmosomes. Even less is known about the influence of EGFR inhibition on intercellular adhesive strength despite the fact that many tumors and tumor-derived cells overexpress EGFR, and loss of cell adhesion is a key characteristic of cancer cells.

In this study, we demonstrate that inhibition of the EGFR, using both a competitive inhibitor of the ATP-binding site (PKI166) as well as a competitive inhibitor of ligand binding (mAb C225), in the squamous cell carcinoma cell line SCC68 induces the formation of desmosomes and augments cell-cell adhesion even in low calcium conditions under which intercellular adhesion is minimal. In a third approach, we introduced a dominant negative decoy receptor into SCC68 cells by using retroviral transduction. Although the decoy receptor dampened EGFR activation, autophosphorylation was never reduced by more than 50%, possibly due to the high receptor number in these cells, and consequently did not lead to dramatic changes in cell morphology or junction assembly. Thus, only PKI166 and C225 were used for the remainder of the analysis. Adhesive strength was increased both in cell sheets, which were generated from confluent substrate-attached cultures, as well as hanging drops cultured in the absence of cell-substrate interactions, the latter observations indicating the specificity of the effect on intercellular adhesion. These increases in intercellular adhesion were also seen at higher calcium concentrations, highlighting the potential physiological relevance of PKI and C225 in increasing adhesive strength of tumor cells in patients treated with these inhibitors.

An extensive literature documents the cell scattering effects of EGFR activation and accompanying alterations in the distribution and function of classic cadherins and their associated proteins. A more recent limited number of studies have focused on the reverse, i.e. the consequences of EGFR blockade on these junctions. The expression level of E-cadherin was shown to be up-regulated following EGFR blockade in lung cancer cell lines (45), although earlier studies (46, 47) observed no change in E-cadherin expression in EGF-stimulated epithelial cells. More recently, the assembly of adherens junction complexes was observed to occur in response to EGFR inhibition (40). In addition, inhibition of Src, a known downstream effector of EGFR that can be suppressed by treatments with Iressa (ZD1839) (48), induced cell-cell contacts and the recruitment of E-cadherin to the cell membrane (49), and inhibition of EGFR was shown to prevent caveolin-dependent endocytosis of E-cadherin in colon carcinoma cells (50). Under our experimental conditions in SCCC68 cells, we did not detect major changes in E-cadherin expression levels upon EGFR inhibition. In addition, E-cadherin was already prominent at cell borders even in the untreated cells, whereas Dsg2 staining at borders was present only in PKI166- and C225-treated cells. Based on quantification of cell borders shown in Figs. 2C and 3B, PKI166 was more efficient at inducing recruitment of desmosome molecules to cell-cell borders than was C225. Although the ERB2 receptor status of SCC68 cells is not known, it seems possible that this difference could in part be explained by the dual inhibition of EGFR (ERB1) and ERB2 by PKI166. Supporting the idea that desmosome assembly was preferentially enhanced by PKI166 and C225, both desmoplakin and Dsg2 increased in the Triton-insoluble fraction of treated cells, whereas E-cadherin solubility did not change. Most interestingly, E-cadherin staining became more organized in PKI-treated cultures, consistent with previous data suggesting that adherens junction maturation requires normal desmoplakin-containing desmosomes at cell-cell borders (51).

{beta}-catenin phosphorylation interferes with E-cadherin and {alpha}-catenin binding and is thought to weaken interactions of the adherens junction complex with the cytoskeleton (15, 52). Evidence from our laboratory demonstrated that the desmosomal cadherin Dsg2 and the armadillo protein plakoglobin are also phosphorylated by EGFR and furthermore showed that tyrosine-phosphorylated plakoglobin does not associate with the IF linker desmoplakin (16), a finding recently confirmed by others (53). Thus, like {beta}-catenin, plakoglobin may modulate interactions with the cytoskeleton through regulation of its association with a critical IF-linking protein. Here we demonstrate that the formation of desmosomes following EGFR inhibition correlates with decreased tyrosine phosphorylation of both Dsg2 and plakoglobin. Somewhat surprisingly, in this SCC68 oral squamous carcinoma system the adherens junction components E-cadherin and {beta}-catenin were only phosphorylated to a minor degree in the controls, and there was no change in phosphorylation following treatment with PKI166. These desmosome-specific alterations in tyrosine phosphorylation were accompanied by recruitment of desmoplakin to cell-cell borders and tethering of keratin IF to the plasma membrane. In light of recent work demonstrating that IF-desmosome attachment strengthens intercellular adhesion (43), these observations are consistent with the idea that EGFR blockade enhances adhesive strength by triggering desmosome assembly and attachment to the cytoskeleton.

Recent studies have uncovered a number of cadherins and associated catenins as substrates for proteolytic modifications, which in some cases were shown to compromise their adhesive function. Dsg3, Dsc3, E-cadherin, VE-cadherin, {beta}-catenin, and plakoglobin have all been shown to be processed by a variety of proteases. Furthermore, MMP-dependent shedding of the extracellular portion of E-cadherin, Dsg3, and Dsc3 has been observed following induction of apoptosis in Madin-Darby canine kidney cells (21, 54). Here we demonstrate for the first time that Dsg2, which appears to be a key mediator of desmosomal cell adhesion in SCC68 cells, is a substrate for MMPs and results in the release of a 60-kDa extracellular fragment of Dsg2 into the supernatant. That this breakdown is regulated by EGFR activity is consistent with prior results showing that MMP-9 activity is up-regulated following stimulation with EGF (19, 42). Thus, our results suggest that MMPs may not only contribute to cancer invasion and progression through breakdown of extracellular matrix but may also further contribute to this process by specifically targeting Dsg2.

Taken together, our results put the functional interplay between desmosome formation, MMP activity, and EGFR inhibitors in a new framework which may contribute to our better understanding of how this new and promising class of drugs work. Furthermore, these results may open new avenues for the development of innovative cancer treatments designed to target cell adhesion as a therapeutic strategy.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants PO1 DE12328 (Project 4) and RO1 AR41836 (to K. J. G.) and by the J. L. Mayberry Endowment. 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. Back

Present address: Klinik fur Innere Medizin IV, Martin-Luther-University Halle, 06097 Halle, Germany. Back

** Supported by National Institutes of Health Training Grant T32 CA80621. Back

{ddagger}{ddagger} Supported by a postdoctoral fellowship from the Canadian Institutes of Health Research. Back

¶¶ To whom correspondence should be addressed: Dept. of Pathology, Northwestern University Feinberg School of Medicine, 303 East Chicago Ave., Chicago, IL 60611. Tel.: 312-503-5300; Fax: 312-503-8240; E-mail: kgreen{at}northwestern.edu.

1 The abbreviations used are: IF, intermediate filament; EGFR, epidermal growth factor receptor; Dsg2, desmoglein 2; Dsc2, desmocollin 2; EGF, epidermal growth factor; MMPs, matrix metalloproteinases; PBS, phosphate-buffered saline; SCC, squamous cell carcinoma. Back

2 T. Yin, S. Getsios, R. Caldelari, A. P. Kowalczyk, E. Müller, and K. J. Green, manuscript in preparation. Back


   ACKNOWLEDGMENTS
 
We thank all those who have generously contributed antibodies, plasmids, and other reagents, including R. Marsh, R Brackenbury, M. Wheelock, K. Johnson, J. Rheinwald, A. Wells, and Novartis AG. We thank A. Huen for advice and assistance with the dispase assays, L. Hudson for helpful discussion, and A. Kowalczyk for critical reading of the manuscript. We also thank Dr. Leo Gordon in the Department of Hematology and Oncology at Northwestern University and Dr. Mechthild Hatzfeld and Hans Joachim-Schmoll at the University of Halle for providing training and research support (to J. H. L.).



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Post-transcriptional Up-regulation of ADAM17 upon Epidermal Growth Factor Receptor Activation and in Breast Tumors
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J. H. Lorch, T. O. Thomas, and H.-J. Schmoll
Bortezomib Inhibits Cell-Cell Adhesion and Cell Migration and Enhances Epidermal Growth Factor Receptor Inhibitor-Induced Cell Death in Squamous Cell Cancer
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J. J. Bech-Serra, B. Santiago-Josefat, C. Esselens, P. Saftig, J. Baselga, J. Arribas, and F. Canals
Proteomic Identification of Desmoglein 2 and Activated Leukocyte Cell Adhesion Molecule as Substrates of ADAM17 and ADAM10 by Difference Gel Electrophoresis.
Mol. Cell. Biol., July 1, 2006; 26(13): 5086 - 5095.
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T. Yin, S. Getsios, R. Caldelari, L. M. Godsel, A. P. Kowalczyk, E. J. Muller, and K. J. Green
Mechanisms of Plakoglobin-dependent Adhesion: DESMOSOME-SPECIFIC FUNCTIONS IN ASSEMBLY AND REGULATION BY EPIDERMAL GROWTH FACTOR RECEPTOR
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J. E. Koblinski, B. R. Kaplan-Singer, S. J. VanOsdol, M. Wu, J. A. Engbring, S. Wang, C. M. Goldsmith, J. T. Piper, J. G. Vostal, J. F. Harms, et al.
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