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Molecular Cancer Research 2:533-540 (2004)
© 2004 American Association for Cancer Research

Angiogenesis, Metastasis, and the Cellular Microenvironment

EphA2 Induction of Fibronectin Creates a Permissive Microenvironment for Malignant Cells1

Min Hu1, Kelly L. Carles-Kinch2, Daniel P. Zelinski1 and Michael S. Kinch2

1 Department of Basic Medical Sciences, Purdue University Cancer Center, West Lafayette, Indiana and 2 MedImmune, Inc., Gaithersburg, Maryland

Requests for reprints: Michael S. Kinch, MedImmune, Inc., 35 West Watkins Mill Road, Gaithersburg, MD 20878. Phone: 240-632-4639; Fax: 301-527-4200. E-mail: kinchm{at}medimmune.com


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Normal and metastatic cells continuously exchange information with the surrounding tissue environment, and this communication governs many aspects of cell behavior. In particular, the physical placement or adhesions of cells within their environment are increasingly understood to facilitate this communication. Classically, cell-cell and cell-extracellular matrix adhesions have been viewed as separable events that are independently controlled. This simple view is changing, as evidence emerges of coordinated regulation of cellular adhesions. Here, we show that the EphA2 tyrosine kinase, which is overexpressed in many aggressive cancers, regulates a fine balance of cell-cell and cell-extracellular matrix adhesions in epithelial cells. EphA2 selectively inhibits cell-cell adhesions by increasing cell attachment and up-regulating the extracellular matrix protein fibronectin. We also show that fibronectin can contribute to important aspects of malignant character. Antibody-based targeting of EphA2 inhibits malignant cell growth by decreasing fibronectin and thereby inducing apoptotic death. Our findings strengthen a concept that cancer progression is regulated by a bidirectional communication between tumor cells and their surrounding microenvironment.


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 References
 
An important concept in cancer research has arisen with the understanding that tumor cells share a complex relationship with their local surroundings (1-4). The composition and three-dimensional organization of the local microenvironment provide structural and chemical cues that govern many aspects of cancer cell behavior, including cell growth, drug sensitivity, survival, and invasiveness (5, 6). Tumor cells synthesize growth factors and proteases that work together to modify and update the composition and organization of the local microenvironment (7-9). Likewise, cancer-associated stromal elements can promote the growth and invasiveness of benign epithelial cells (10-12).

This ability to remodel the local microenvironment plays an important role in metastasis. Metastatic cells gain the ability to colonize microenvironments that are inhospitable to normal cells (2, 13-15). For example, normal mammary epithelial cells will generally not survive if transplanted into a foreign microenvironment such as the lung. Yet, lung metastases are a frequent cause of breast cancer pathology (16). A conventional explanation for these differences is that tumor cells have bypassed the need for structural or signaling cues that govern growth and survival. For example, integrins provide physical attachments and signals that regulate normal cell growth and survival (17-22). The aforementioned paradigm predicts that malignant cells circumvent the need for integrin function.

Recent studies have linked the EphA2 receptor tyrosine kinase with cancer and metastasis. EphA2 is frequently overexpressed in cancer cells and EphA2 overexpression is sufficient to confer tumorigenic and metastatic character (23-32). As an example, EphA2 has been shown to contribute to many different aspects of breast cancer, including metastasis and hormone sensitivity (27, 28, 30).

In normal cells, EphA2 is stimulated by ligands that are anchored to the membrane of adjacent cells (33). These interactions require stable intercellular adhesions and are important because ligand stimulation causes EphA2 to transmit signals that negatively regulate cell growth, migration, and invasiveness (30, 34, 35). Cancer cells often have unstable cell-cell contacts. Consequently, EphA2 ligand binding is decreased, which causes EphA2 to promote (rather than inhibit) oncogenesis (27). These changes are reversible, as restoration or mimicry of ligand binding (e.g., using monoclonal antibodies) inhibits tumor cell growth and survival (36, 37).

In the present study, we show a link between EphA2 and fibronectin that relates to important aspects of metastatic behavior. First, EphA2 overexpression increases fibronectin and thereby positively regulates tumor cell growth and survival. In the converse situation, EphA2 antibodies decrease fibronectin production and in doing so decrease certain aspects of metastatic potential. These findings further support the concept that a dynamic interplay between malignant cells and their microenvironment critically controls tumor cell behavior.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 References
 
Decreased Cell-Cell Adhesions in EphA2-Transformed Cells
EphA2 is frequently overexpressed in breast cancer, where it functions as an oncoprotein (27). To evaluate the biological consequences of EphA2 overexpression in mammary epithelial cells, bulk cultures of control (empty vector) or EphA2-transfected MCF-10A mammary epithelial cells (MCF-10AEphA2) were used as described previously (Fig. 1A; ref. 27). Microscopic analyses revealed dramatic changes in the morphology and intercellular adhesions of EphA2-transformed epithelial cells. Whereas vector-transfected control cultures of MCF-10A cells displayed a characteristic epithelial morphology, MCF-10AEphA2 cells adopted a fibroblast-like morphology, spread onto the underlying substrate, and resisted cell-cell contacts, even when cultured at high cell density (Fig. 1B). Analyses of bulk culture transfections revealed a remarkable uniformity in this mesenchymal phenotype, and comparable results were obtained using at least three different and independent transfections of MCF-10A cells.



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  		FIGURE 1. EphA2 transformation changes epithelial cell adhesions. A. Protein levels of EphA2 were evaluated in control and MCF-10AEphA2 using Western blot analysis as indicated in Materials and Methods. Membranes were probed with ß-catenin to confirm equal sample loading. Numbers, relative molecular mobilities of standards (in kDa). B. Gross morphology and subcellular distribution of E-cadherin (a marker of cell-cell contacts) and paxillin (a marker of cell-ECM attachments) were compared in vector-transfected (control) or MCF-10AEphA2 cells using phase-contrast or epifluorescence microscopy (400x). Note that EphA2-overexpressing cells did not organize into colonies even at high cell density and that this reflected decreased cell-cell contacts (middle) and increased focal adhesions (right). Bar, 50 µm. C. Cell-cell adhesion was evaluated in control and MCF-10AEphA2 cells using hanging-drop microscopic analyses. Columns, cell-cell adhesion assays, fraction of individual cells (which lack cell-cell contact, No) relative to the total cell number (Nt). D. Cell-substratum adhesions were evaluated in control and EphA2-overexpressing cells as indicated in Materials and Methods. Note that cycloheximide blocks the ECM attachments of EphA2-transfected cells, which indicates a need for de novo protein synthesis. *, P < 0.0003.

 
Objective measures of cell-cell adhesion revealed that >95% of control MCF-10A more readily organized into cell/cell colonies, whereas <20% of EphA2-transformed cells were found within cell colonies (P < 0.0002; Fig. 1C). Comparable results were obtained when evaluating cell-cell contacts from samples in monolayer culture and in hanging-drop analyses (data not shown).

The decreased cell-cell contacts of EphA2-overexpressing cells are related to altered subcellular distribution of E-cadherin. Whereas E-cadherin was restricted to sites of cell-cell contact between nontransformed controls, it was diffusely distributed in MCF-10AEphA2 cells (Fig. 1B, middle). These changes did not relate to changes in E-cadherin protein levels or surface expression (data not shown). Similarly, there were no differences in the expression, cadherin binding properties, or phosphotyrosine content of E-cadherin or catenin molecules in EphA2-overexpressing cells (data not shown).

High Levels of Fibronectin in EphA2-Overexpressing Cells
Cell-cell and cell-extracellular matrix (ECM) adhesions are inversely regulated in epithelial cells (38). Thus, we postulated that EphA2 might destabilize cell-cell adhesions by increasing ECM interactions. Objective assays revealed a >20-fold increase in the ability of EphA2-transformed cells to adhere to plastic supports as compared with nontransformed controls (P < 3 x 10–4; Fig. 1C). Consistent with this, immunostaining for the focal adhesion protein paxillin revealed larger and more numerous focal adhesions in MCF-10AEphA2 cells than in matched controls (Fig. 1B).

Flow cytometric analyses did not relate the increased cell-ECM attachments to changes in the surface levels of various integrins including {alpha}5, {alpha}v, ß1, or ß3. Consistent with this, control and MCF-10AEphA2 cells were comparably able to attach and spread on exogenously provided (purified) ECM proteins, including fibronectin, collagen, and Matrigel (data not shown).

Rather, the differences in cell morphology and adhesion are related to altered levels of endogenous ECM proteins. Initial support for this hypothesis was provided by evidence that the increased cell-ECM attachments of MCF-10AEphA2 cells required de novo protein synthesis (Fig. 1D). Indeed, Western blot analyses revealed 30-fold higher levels of fibronectin in MCF-10AEphA2 cells than in matched controls (Fig. 2A). The levels of secreted fibronectin were also elevated in EphA2-transformed cells (Fig. 2B). This effect was specific for fibronectin, as laminin and ß-catenin did not differ between the two cell populations.



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  		FIGURE 2. Elevated fibronectin levels in MCF-10AEphA2 cells. A. Whole cell lysates from control and MCF-10AEphA2 cells were subjected to Western blot analyses with laminin (LN)- or fibronectin (FN)-specific antibodies. Membranes were reprobed with ß-catenin antibodies to confirm equal loading. Numbers, relative mobility of molecular mass markers (in kDa). B. Fibronectin secretion by control (Ctrl) and EphA2-transformed cells was measured in cell culture supernatants. *, P < 0.004.

 
Fibronectin Regulates the Adhesion of EphA2-Transformed Cells
We then asked if high levels of fibronectin contributed to the altered adhesive phenotype of EphA2-overexpressing cells. To test this, MCF-10AEphA2 cells were treated with monoclonal antibodies that antagonize {alpha}5ß1 integrin (the major fibronectin receptor). These antibodies decreased substrate attachments by at least 60% (P < 0.0008) yet increased cell-cell contacts at least 10-fold (Fig. 3). Thus, integrin function increases cell-ECM adhesions and decreases cell-cell contacts in EphA2-transformed cells.



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  		FIGURE 3. Integrin function regulates cell-ECM and cell-cell adhesions. A. ECM attachment assays were conducted using MCF-10AEphA2 cells, which had been cultured in the presence of adhesion-blocking monoclonal antibodies specific for {alpha}5 or ß1 integrins. B. MCFEphA2 cells were incubated with integrin antibodies for 24 hours before assessing cell-cell adhesions using monolayer scrape assays. Integrin antibodies decreased cell-ECM attachments and increased cell-cell adhesions. *, P < 0.0003; **, P < 0.0005.

 
We then asked if fibronectin could similarly alter the adhesive interactions of nontransformed epithelial cells. For this, MCF-10A cells were plated atop fibronectin-coated supports for 24 hours, with Matrigel-coated supports providing a negative control (Fig. 4). When incubated in the presence of fibronectin, the localization of E-cadherin into intercellular contacts was diminished (Fig. 4A). Similarly, cell-cell adhesions were similarly decreased in samples that had been incubated on fibronectin (relative to Matrigel-treated controls; P < 0.0007; Fig. 4B).



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  		FIGURE 4. Fibronectin binding regulates cell-cell and cell-ECM attachments. MCF-10A cells were plated atop membranes, which had been coated with either fibronectin or a control substrate (C, Matrigel), and incubated for 24 hours before assessing cell adhesion. A. Immunolocalization of E-cadherin and paxillin were used to assess cell-cell and cell-ECM attachments. B. Objective analyses of cell-cell and cell-ECM adhesions were conducted following incubation with fibronectin. *, P < 0.002; **, P < 0.0007. C. Western blot analyses of immunoprecipitated EphA2 revealed higher levels of EphA2 following incubation with fibronectin. Exposure to fibronectin decreased the phosphotyrosine content of EphA2, reflecting decreased ligand binding.

 
High levels of EphA2 autophosphorylation generally relate to decreased ECM attachments, whereas low levels of EphA2 phosphotyrosine content have been linked with unstable cell-cell adhesion (30, 35). Thus, EphA2 was immunoprecipitated and subjected to Western blot analyses with phosphotyrosine-specific antibodies (4G10). EphA2 phosphotyrosine content was reduced following exposure to fibronectin (Fig. 4C). Notably, the levels of EphA2 protein were higher following incubation with fibronectin, which is consistent with the emerging concept that phosphorylated EphA2 is fated for degradation, whereas unphosphorylated EphA2 can accumulate on tumor cells (39). Various markers of cell-ECM attachments, including the focal adhesion protein paxillin, revealed increased ECM adhesions by cells that had been exposed to fibronectin (Fig. 4A) and objective measure of ECM attachment confirmed this observation (Fig. 4B). These results indicate that increased exposure to fibronectin can recapitulate certain characteristics of tumor cells that have high levels of EphA2.

EphA2 Antibodies Decrease Fibronectin and Induce Apoptosis
The studies above link EphA2 overexpression with fibronectin. Here, we took the opposite approach by asking if targeted intervention against EphA2 would alter fibronectin levels. To test this, MDA-MB-231 breast tumor cells were treated with EphA2 antibodies for 0 to 24 hours. Certain antibodies that mimic the action of ligand (so-called agonist antibodies) inhibit malignant behavior in vitro and in vivo (36, 37). These same reagents decreased the levels of fibronectin within 4 hours and low levels of fibronectin persisted for at least the following 20 hours (Fig. 5). This outcome did not reflect a general decrease in protein levels as neither paxillin nor ß-catenin changed following incubation with EphA2 antibodies. A drop in EphA2, which began within 1 hour, preceded the decrease in fibronectin. We confirmed that these outcomes were not unique to MDA-MB-231 cells, as EphA2 antibodies similarly decreased fibronectin levels in other cell systems (including MCF-10AEphA2 cells; data not shown).



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  		FIGURE 5. EphA2 antibodies induce fibronectin degradation. MDA-MB-231 cells were incubated in the presence or absence of agonistic EphA2 antibodies (EA2) for the times indicated. Samples were then extracted and subjected to Western blot analyses with antibodies specific for EphA2 (top). Parallel samples were evaluated using antibodies specific for fibronectin (middle). Paxillin-specific antibodies confirmed equal sample loading (bottom).

 
We then asked whether EphA2 antibodies are directly cytotoxic to tumor cells. MDA-MB-231 breast cancer cells were seeded onto polyhydroxylethyl methacrylate–coated dishes (to prevent ECM attachments) in the presence of absence of anti-EphA2 (5 µg/mL EA5), with an isotype-matched antibody (1A7) providing a negative control. EA5 treatment induced DNA strand breaks (using FITC-dUTP and terminal deoxynucleotide transferase; Fig. 6A) and cleavage of the DNA repair enzyme poly(ADP-ribose) polymerase (Fig. 6B). Consequently, there were 70% fewer viable cells in the EA5-treated than 1A7-treated cultures within 24 hours (data not shown). Similar results were obtained using other EphA2 agonistic antibodies (e.g., B233 and EA5), thus indicating that this class of EphA2 antibodies can mediate active killing of tumor cells.



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  		FIGURE 6. EphA2 antibodies induce apoptosis of tumor cells. MDA-MB-231 cells were cultured in polyhydroxylethyl methacrylate–coated dishes in the presence of EA5 or vehicle control or a matched isotype control (1A7) for 0 to 8 hours. A. Cells were then harvested, fixed, stained with propidium iodide and FITC-dUTP/terminal deoxynucleotide transferase, and subjected to flow cytometric analyses to evaluate the fraction of cells with DNA strand breaks. Log-phase cells in monolayer culture provided a negative control for apoptosis (data not shown). B. Western blot analyses revealed poly(ADP-ribose) polymerase (PARP) cleavage within 2 hours. Membranes were stripped and reprobed with ß-catenin antibodies to verify equal sample loading.

 
Our final series of studies asked if fibronectin signaling could mimic the increased survival of EphA2-overexpressing cells. To test this, {alpha}5ß1 integrin was aggregated at the cell surface of MCF10A cells using monoclonal antibodies. This particular cell model was selected, as it requires stable ECM binding for continued survival. Whereas control cultures of MCF-10A cells were largely nonviable following suspension atop polyhydroxylethyl methacrylate, {alpha}5ß1 aggregation increased cell survival under these same conditions (Fig. 7).



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  		FIGURE 7. Integrin signaling promotes cell survival. {alpha}5ß1 integrin was aggregated on the surface of MCF-10A using monoclonal antibodies ({alpha}5ß1 XL) prior to suspending the samples atop polyhydroxylethyl methacrylate–coated tissue culture plates. After 7 days, the number of viable cells was evaluated using trypan blue exclusion and phase-contrast microscopy.

 

   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 References
 
The major finding of this study is that EphA2 overexpression causes dramatic changes in cell morphology and behavior and that these changes involve fibronectin. We also show that high levels of fibronectin contribute to aspects of tumor cell growth and survival that are unique to malignant cells. Finally, we link the selective targeting of malignant cells with EphA2 antibodies with decreased fibronectin in vitro.

The present results break new ground in linking EphA2 oncogenic activity with fibronectin. The finding that fibronectin decreases cell-cell contacts is intriguing because EphA2 function is highly sensitive to the stability of cell-cell contacts (30). In particular, ligand-bound EphA2 transmits signals that negatively regulate tumor cell growth and survival (30, 34, 35). In the absence of ligand binding, EphA2 enhances (rather than inhibits) these same behaviors (27). These results suggest that EphA2 overexpression may alter its own function by regulating ligand binding and suggest a means by which expression and function are coordinately regulated in normal and cancer cells.

Phosphorylated EphA2 is rapidly degraded, whereas unphosphorylated EphA2 accumulates at the cell surface (36, 39). The present results suggest that EphA2 overexpression and altered function may be induced other stimuli that up-regulate EphA2 or fibronectin or that decrease intercellular contacts. Changes in the expression or function of certain tumor suppressors (e.g., E-cadherin and DCC) and many oncogenes (e.g., Ras and Src) induce similar changes in cellular adhesion, and any of these could up-regulate EphA2. Such a mechanism may help to explain the prominence and frequency of EphA2 overexpression in cancer, which has been linked to many solid tumors (24, 27-29, 40-44).

Recent studies indicate that the levels of EphA2 phosphotyrosine content can be regulated by associated tyrosine phosphatases including the low molecular weight tyrosine phosphatase (45, 46). The levels of low molecular weight tyrosine phosphatase in breast epithelial cells (including MCF-10A and MCF-7) do not change following ectopic overexpression of EphA2 (M.S. Kinch, data not shown). This outcome suggests that EphA2 functions downstream of low molecular weight tyrosine phosphatase in promoting transformation. These results also indicate that the altered adhesions documented herein are regulated by EphA2 but not by low molecular weight tyrosine phosphatase.

Recent evidence has linked fibronectin and integrins with metastasis and chemotherapy resistance (5, 6). In our present study, we show that tumor cells themselves can provide one source of fibronectin. Other cell types could also contribute to fibronectin and thereby favor malignant behavior. In this light, an elegant series of studies showed that cancer-associated fibroblasts can support malignant progression (10, 12). Fibroblasts are major sources of fibronectin, and it is tempting to speculate that cancer-associated fibroblasts may function, at least in part, to provide fibronectin for the developing tumor. Fibronectin can bind various soluble factors that promote cell growth, survival, and differentiation and thereby provide a local reservoir of growth and survival factors. Similarly, Matrigel is a complex mixture of soluble and insoluble components, and we cannot presently exclude the possibility that some or all of these factors contribute to the types of cell adhesion associated with Matrigel.

These findings have potential implications for the design of new therapeutic approaches for cancer. Recent studies suggest that EphA2 agonists (artificial ligands, antibodies, and peptides) may have efficacy against tumor cells as measured using preclinical models (36, 37, 47). EphA2 agonists negatively regulate ECM attachments and subsequently induce apoptosis. This finding is intriguing in light of studies linking cancer with resistance to anoikis, a form of apoptosis linked with decreased ECM attachments (20). Based on evidence linking EphA2 and fibronectin with metastasis and chemotherapy resistance, our present findings suggest that EphA2-based therapeutic targeting could provide a much-needed target for cancer by overriding phenomena such as anoikis resistance. Thus, future investigation could evaluate the potential application of EphA2-based targeting in combination with other therapies designed to enhance tumor apoptosis.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
References
 
Cell Culture and Antibodies
All control and EphA2-transfected cells were cultured as described previously (27, 36). The methods used to transfect EphA2 into MCF-10A and to evaluate the levels of EphA2 expression and enzymatic activity have also been described previously (27). In particular, the magnitude of EphA2 enzymatic activity in these cells is proportional to the degree of EphA2 overexpression (27).

To control for potential variability between transfections, all results were confirmed using at least three independent bulk culture transfections of EphA2 into MCF-10A cells. When indicated, tissue culture dishes were preincubated overnight at 4°C in PBS supplemented with 1% Matrigel (Collaborative, Bedford, MA), 100 µg/mL fibronectin (Sigma Chemical Co., St. Louis, MO), or vitronectin (from 5% horse serum, Life Technologies, Inc., Grand Island, NY). In suspension culture experiments, tissue culture dishes were coated with 20 mg/mL polyhydroxylethyl methacrylate (Sigma Chemical) in 95% ethanol and allowed to air dry before use to prevent MDA-MB-231 cells from attaching to the plates.

The EphA2 monoclonal antibody (D7) was used for Western blot analyses and was purchased from Upstate Biologicals, Inc. (Lake Placid, NY). Agonist antibodies specific for EphA2 (EA2 and EA5) were generated in our laboratory (see ref. 36 for a detailed analysis of these reagents). 1A7 antibodies were generated in our laboratory and provided an isotype (IgG1) control. Antibodies specific for fibronectin and ß-catenin were purchased from Transduction Laboratories (Lexington, KY). Antibodies specific for poly(ADP-ribose) polymerase were purchased from Cell Signaling Technology (Beverly, MA). Antibodies specific for pan-phosphotyrosine (4G10) were purchased from Upstate Biologicals. Antibodies specific for {alpha}5 and ß1 integrins were purchased from Developmental Studies Hybridoma Bank (Iowa City, IA) and used as recommended (48, 49). Rabbit anti-E-cadherin antibodies were generously provided by Dr. R. Brackenbury (University of Cincinnati, Cincinnati, OH). Paxillin antibodies were generously provided by Dr. K. Burridge (University of North Carolina, Chapel Hill, NC).

Western Blot Analysis and Immunoprecipitation
Western blot analyses were done as described (36). Antibody binding was detected by enhanced chemiluminescence (Pierce, Rockford, IL) and autoradiography (Kodak X-OMAT, Kodak, Rochester, NY). Band intensity was measured using the Kodak Image Station 440 (Kodak). To confirm equal sample loading, the blots were reprobed with antibodies specific for ß-catenin or paxillin. To determine statistical significance, at least three independent experiments were analyzed using a Student's t test, defining P < 0.05 as significant.

Immunofluorescence and Phase-Contrast Microscopy
Samples were grown on glass coverslips and prepared for immunofluorescence microscopy as detailed (36). After incubation with primary antibodies, the coverslips were labeled with rhodamine-conjugated donkey anti-mouse IgG (Chemicon, Temecula, CA). The samples were viewed using an Olympus BX-60 epifluorescence microscope (Eugene OR) outfitted with the appropriate filters, and images were recorded onto T-Max 400 professional film (Kodak). For phase-contrast microscopy, viable cell cultures were observed using an Olympus IX-70 microscope, and images were recorded onto 35 mm professional film as detailed above.

Cell Adhesion Assays
Cells (5 x 105) were incubated overnight at 37°C for cell-cell adhesion studies (except for studies of ECM contact, where cells were incubated atop the ECM overnight). Two different types of cell-cell assays were used and yielded comparable findings. In one set of experiments, the experimental conditions (e.g., antibody treatment) were employed while the samples remained attached to the underlying substratum. The cells were then removed from tissue culture–treated plates using a cell scraper and analyzed as indicated below. To confirm these findings, additional experiments were conducted by suspending the cells using EDTA and then subjecting these samples to the experimental conditions for 30 minutes at 37°C. The results are presented as No/Nt, where No indicates the number of individual cells and Nt represents the total number of cells as determined in blinded experiments using a high-power microscope (400x).

To measure cell-substrate attachments, 5 x 105 cells were suspended in medium and plated onto 35 mm tissue culture dishes for 30 minutes at 37°C and 5% CO2. Weakly adherent cells were detached by three vigorous washes, and the remaining adherent cells were suspended with trypsin and counted using a hemacytometer. The average of at least four separate experiments is reported. For assays on dishes coated with horse serum, Matrigel, or fibronectin, cells were incubated in the presence of 25 µg/mL cycloheximide or a matched vehicle (PBS) beginning 60 minutes prior to suspension. Cells were suspended in serum-free medium prior to plating. For experiments with integrin antibodies, the culture medium was supplemented with 1% {alpha}5 or ß1 integrin ascites or 10% to 20% {alpha}5 or ß1 integrin hybridoma supernatant. Additional analysis with a nonreactive hybridoma supernatant (clone D7) provided an isotype-matched control. To determine statistical significance, all cell adhesion analyses were assessed in at least three independent experiments and were analyzed using a Student's t test, defining P < 0.05 as significant.

Soft Agar Assays
Cells (1 x 105) were seeded into each well of a six-well tissue culture–treated dish (Costar, Cambridge, MA). After 3 or 7 days of incubation at 37°C, samples were harvested in a trypsin-EDTA solution (Life Technologies) and counted using a hemacytometer (Olympus IX-70). Each experiment was repeated at least three times and representative results are reported. The statistical significance of all results was calculated using a Student's t test (Microsoft Excel, Seattle, WA), defining P < 0.05 as significant.

Apoptosis Detection and Cell Cycle Measurement
Apoptosis detection and cell cycle measurement of monolayer or suspension cells were done according to Apo-Direct kit (BD Biosciences PharMingen, Franklin Lakes, NJ). Briefly, 1 x 106 to 2 x 106 cells were harvested and washed with PBS once. Then, cells were fixed with 1% paraformaldehyde in PBS on ice for 1 hour. Cells were washed twice with PBS and stored in 70% ethanol at –20°C for at least 12 to 18 hours until ready for use. Cell suspensions were centrifuged for 5 minutes at 300 x g and the 70% ethanol was discarded. After washed twice, cells were incubated with terminal deoxynucleotide transferase enzyme and FITC-dUTP at 37°C for 2 hours. Then, cells were rinsed twice and incubated with propidium iodide/RNase staining buffer at room temperature for 30 minutes in the dark. The actively growing cells in monolayer were used as negative controls for apoptosis. The percentage of cells in each phase of cell cycle was calculated using ModFit LT program (Verity Software House, Inc., Topsham, ME). The percentage of apoptotic cells was analyzed using WinMDI 2.8 software (Scripps Research Institute, La Jolla, CA).


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
1 MedImmune, Inc., American Cancer Society, NIH/National Cancer Institute Molecular Targeting and Drug Design Program, and U.S. Army Medical Research Acquisition Activity Breast Cancer Research Program (M.S. Kinch). Back

Received February 23, 2004; revised August 30, 2004; accepted September 3, 2004.


   References
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 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

  1. Hansen RK, Bissell MJ. Tissue architecture and breast cancer: the role of extracellular matrix and steroid hormones. Endocr Relat Cancer 2000;7:95–113.[Abstract]
  2. Onn A, Fidler IJ. Metastatic potential of human neoplasms. In Vivo 2002;16:423–9.[Medline]
  3. Weaver VM, Bissell MJ. Functional culture models to study mechanisms governing apoptosis in normal and malignant mammary epithelial cells. J Mammary Gland Biol Neoplasia 1999;4:193–201.[CrossRef][Medline]
  4. Hayward SW, Grossfeld GD, Tlsty TD, Cunha GR. Genetic and epigenetic influences in prostatic carcinogenesis. Int J Oncol 1998;13:35–47.[Medline]
  5. Hazlehurst LA, Dalton WS. Mechanisms associated with cell adhesion mediated drug resistance (CAM-DR) in hematopoietic malignancies. Cancer Metastasis Rev 2001;20:43–50.[CrossRef][Medline]
  6. Yu JL, Coomber BL, Kerbel RS. A paradigm for therapy-induced microenvironmental changes in solid tumors leading to drug resistance. Differentiation 2002;70:599–609.[CrossRef][Medline]
  7. Lynch CC, Matrisian LM. Matrix metalloproteinases in tumor-host cell communication. Differentiation 2002;70:561–73.[CrossRef][Medline]
  8. McCawley LJ, Matrisian LM. Matrix metalloproteinases: multifunctional contributors to tumor progression. Mol Med Today 2000;6:149–56.[CrossRef][Medline]
  9. Fidler IJ. Modulation of the organ microenvironment for treatment of cancer metastasis. J Natl Cancer Inst 1995;87:1588–92.[Free Full Text]
  10. Cunha GR, Hayward SW, Wang YZ, Ricke WA. Role of the stromal microenvironment in carcinogenesis of the prostate. Int J Cancer 2003;107:1–10.[CrossRef][Medline]
  11. Olumi AF, Grossfeld GD, Hayward SW, Carroll PR, Tlsty TD, Cunha GR. Carcinoma-associated fibroblasts direct tumor progression of initiated human prostatic epithelium. Cancer Res 1999;59:5002–11.[Abstract/Free Full Text]
  12. Tlsty TD, Hein P. Know thy neighbor: stromal cell can contribute oncogenic signals. Curr Opin Genet Dev 2001;11:54–9.[CrossRef][Medline]
  13. Steeg PS. Metastasis suppressors alter the signal transduction of cancer cells. Nat Rev Cancer 2003;3:55–63.[CrossRef][Medline]
  14. Radinsky R. Molecular mechanisms for organ-specific colon carcinoma metastasis. Eur J Cancer 1995;31A:1091–5.
  15. Li L, Price JE, Fan D, Zhang RD, Bucana CD, Fidler IJ. Correlation of growth capacity of human tumor cells in hard agarose with their in vivo proliferative capacity at specific metastatic sites. J Natl Cancer Inst 1989;81:1406–12.[Abstract/Free Full Text]
  16. Welch DR, Steeg PS, Rinker-Schaeffer CW. Molecular biology of breast cancer metastasis. Genetic regulation of human breast carcinoma metastasis. Breast Cancer Res 2000;2:408–16.[CrossRef][Medline]
  17. Park CC, Bissell MJ, Barcellos-Hoff MH. The influence of the microenvironment on the malignant phenotype. Mol Med Today 2000;6:324–9.[CrossRef][Medline]
  18. Ruoslahti E. Fibronectin and its integrin receptors in cancer. Adv Cancer Res 1999;76:1–20.[Medline]
  19. Keely P, Parise L, Juliano R. Integrins and GTPases in tumor cell growth, motility and invasion. Trends Cell Biol 1998;8:101–6.[CrossRef][Medline]
  20. Frisch SM, Ruoslahti E. Integrins and anoikis. Curr Opin Cell Biol 1997;9:701–6.[CrossRef][Medline]
  21. Juliano RL. Integrin signals and tumor growth control. Princess Takamatsu Symp 1994;24:118–24.[Medline]
  22. Hunter T. Oncoprotein networks. Cell 1997;88:333–46.[CrossRef][Medline]
  23. Hendrix MJ, Seftor EA, Hess AR, Seftor RE. Molecular plasticity of human melanoma cells. Oncogene 2003;22:3070–5.[CrossRef][Medline]
  24. Miyazaki T, Kato H, Fukuchi M, Nakajima M, Kuwano H. EphA2 overexpression correlates with poor prognosis in esophageal squamous cell carcinoma. Int J Cancer 2003;103:657–63.[CrossRef][Medline]
  25. Walker-Daniels J, Hess AR, Hendrix MJ, Kinch MS. Differential regulation of EphA2 in normal and malignant cells. Am J Pathol 2003;162:1037–42.[Free Full Text]
  26. Kinch MS, Moore MB, Harpole DH Jr. Predictive value of the EphA2 receptor tyrosine kinase in lung cancer recurrence and survival. Clin Cancer Res 2003;9:613–8.[Abstract/Free Full Text]
  27. Zelinski DP, Zantek ND, Stewart JC, Irizarry AR, Kinch MS. EphA2 overexpression causes tumorigenesis of mammary epithelial cells. Cancer Res 2001;61:2301–6.[Abstract/Free Full Text]
  28. Ogawa K, Pasqualini R, Lindberg RA, Kain R, Freeman AL, Pasquale EB. The ephrin-A1 ligand and its receptor, EphA2, are expressed during tumor neovascularization. Oncogene 2000;19:6043–52.[CrossRef][Medline]
  29. Easty DJ, Hill SP, Hsu MY, et al. Up-regulation of ephrin-A1 during melanoma progression. Int J Cancer 1999;84:494–501.[CrossRef][Medline]
  30. Zantek ND, Azimi M, Fedor-Chaiken M, Wang B, Brackenbury R, Kinch MS. E-cadherin regulates the function of the EphA2 receptor tyrosine kinase. Cell Growth Differ 1999;10:629–38.[Abstract/Free Full Text]
  31. Rosenberg IM, Goke M, Kanai M, Reinecker HC, Podolsky DK. Epithelial cell kinase-B61: an autocrine loop modulating intestinal epithelial migration and barrier function. Am J Physiol 1997;273:G824–32.
  32. Kinch MS, Carles-Kinch K. Overexpression and functional alterations of the EphA2 tyrosine kinase in cancer. Clin Exp Metastasis 2003;20:59–68.[CrossRef][Medline]
  33. Bartley TD, Hunt RW, Welcher AA, et al. B61 is a ligand for the ECK receptor protein-tyrosine kinase. Nature 1994;368:558–60.[CrossRef][Medline]
  34. Miao H, Wei BR, Peehl DM, et al. Activation of EphA receptor tyrosine kinase inhibits the Ras/MAPK pathway. Nat Cell Biol 2001;3:527–30.[CrossRef][Medline]
  35. Miao H, Burnett E, Kinch M, Simon E, Wang B. Activation of EphA2 kinase suppresses integrin function and causes focal-adhesion-kinase dephosphorylation. Nat Cell Biol 2000;2:62–9.[CrossRef][Medline]
  36. Carles-Kinch K, Kilpatrick KE, Stewart JC, Kinch MS. Antibody targeting of the EphA2 tyrosine kinase inhibits malignant cell behavior. Cancer Res 2002;62:2840–7.[Abstract/Free Full Text]
  37. Coffman KTH, Hu M, Carles-Kinch K, et al. Differential EphA2 epitope display on normal versus malignant cells. Cancer Res 2003;63:7907–12.[Abstract/Free Full Text]
  38. Kinch MS, Burridge K. Altered adhesions in ras-transformed breast epithelial cells. Biochem Soc Trans 1995;23:446–50.[Medline]
  39. Walker-Daniels J, Riese DJ, Kinch MS. c-Cbl-dependent EphA2 protein degradation is induced by ligand binding. Mol Cancer Res 2002;1:79–87.[Abstract/Free Full Text]
  40. D'Amico TA, Aloia TA, Moore MB, et al. Predicting the sites of metastases from lung cancer using molecular biologic markers. Ann Thorac Surg 2001;72:1144–8.[Abstract/Free Full Text]
  41. Hess AR, Seftor EA, Gardner LM, et al. Molecular regulation of tumor cell vasculogenic mimicry by tyrosine phosphorylation: role of epithelial cell kinase (Eck/EphA2). Cancer Res 2001;61:3250–5.[Abstract/Free Full Text]
  42. Walker-Daniels J, Coffman K, Azimi M, et al. Overexpression of the EphA2 tyrosine kinase in prostate cancer. Prostate 1999;41:275–80.[CrossRef][Medline]
  43. Zeng GH. High-Level expression of EphA2 receptor tyrosine kinase in prostatic intraepithelial neoplasia. Am J Pathol 2003;163:2271–6.[Abstract/Free Full Text]
  44. Nakamoto M, Bergemann AD. Diverse roles for the Eph family of receptor tyrosine kinases in carcinogenesis. Microsc Res Tech 2002;59:58–67.[CrossRef][Medline]
  45. Chiarugi P, Taddei ML, Schiavone N, et al. LMW-PTP is a positive regulator of tumor onset and growth. Oncogene 2004;23:3905–14.[CrossRef][Medline]
  46. Kikawa KD, Vidale DR, Van Etten RL, Kinch MS. Regulation of the EphA2 kinase by the low molecular weight tyrosine phosphatase induces transformation. J Biol Chem 2002;277:39274–9.[Abstract/Free Full Text]
  47. Koolpe M, Dail M, Pasquale EB. An ephrin mimetic peptide that selectively targets the EphA2 receptor. J Biol Chem 2002;277:46974–9.[Abstract/Free Full Text]
  48. Howlett AR, Bailey N, Damsky C, Petersen OW, Bissell MJ. Cellular growth and survival are mediated by ß1 integrins in normal human breast epithelium but not in breast carcinoma. J Cell Sci 1995;108:1945–57.[Abstract]
  49. Huhtala P, Humphries MJ, McCarthy JB, Tremble PM, Werb Z, Damsky CH. Cooperative signaling by {alpha}5ß1 and {alpha}4ß1 integrins regulates metalloproteinase gene expression in fibroblasts adhering to fibronectin. J Cell Biol 1995;129:867–79.[Abstract/Free Full Text]



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