Integrins Uncouple Src-induced Morphological and Oncogenic Transformation*

Expression of activated mutants of c-Src in epithelial cells can induce tumorigenicity. In addition to such oncogenic transformation, the cells undergo a dramatic morphological transformation: cell-cell contacts are disrupted, spreading on extracellular matrix proteins is suppressed, actin stress fibers and focal contacts are lost, and podosomes are formed. We have previously shown that integrin αvβ3 strongly supports Src-mediated oncogenic transformation through an interaction at the β3 cytoplasmic tail. Our current findings demonstrate that this interaction does not affect Src-mediated morphological alterations, thus separating oncogenic from morphological transformation. Moreover, β1 and β3 integrins differently affect the various aspects of Src-induced morphological transformation. High levels of β3, but not β1, integrins can prevent Src-induced cell rounding although stress fiber disassembly and podosome formation still occur. Studies using chimeric integrin subunits demonstrate that this protection requires the β3 extracellular domain. Finally, like tumor formation, podosome assembly occurs independent of β3 phosphorylation. Instead, phosphorylation of β1 is required to suppress Rho-mediated contractility in order to assemble podosomes. Thus, integrins regulate Src-mediated oncogenic transformation and various aspects of morphological transformation through dissociable pathways.

The ubiquitously expressed Src family kinase c-Src is involved in pro-survival and mitogenic signaling cascades (1). Activated mutants of Src, including the oncogenic product of Rous sarcoma virus (v-Src), can induce anchorage-and growth factor-independent growth of cell lines in vitro and tumor formation in vivo (2)(3)(4). c-Src has been found to play a critical role in the development of cancer in mice (5,6), and expression and/or activity of c-Src is frequently increased in human melanoma and carcinomas of the breast, colon, and other epithelia (4,7,8). Activation of Ras, phosphatidylinositol 3-kinase, and Stat3 has been implicated in Src-mediated oncogenic transformation (3).
In addition to its role in mitogenic signaling, c-Src is a critical regulator of both cadherin-and integrin-mediated adhesion structures (9,10). While low levels of c-Src kinase activity or kinase-independent functions of c-Src can support the formation of cell-cell or cell-matrix adhesions (11)(12)(13), c-Src kinase activation typically stimulates the disassembly of these structures (14,15). Indeed, expression of activated mutants of Src in epithelial cells induces scattering, loss of cytoskeletal contractility, weak adhesion, cell rounding, and the formation of highly dynamic cell-matrix adhesions termed podosomes that are considered to be hotspots for invasion and matrix remodeling (9, 16 -18).
It is not clear to what extent the signaling pathways activated by Src that are involved in oncogenic transformation overlap with those involved in the morphological transformation. Moreover, the different aspects of Src-induced morphological transformation may be connected (e.g. they may all be explained to some extent by loss of actomyosin contractility) or may involve activation of distinct signaling processes (e.g. separable alterations at cell-cell junctions, within the cytoskeletal contractility machinery, and at cell-matrix adhesions). In cellmatrix adhesions, integrins can serve as direct phosphorylation substrates of v-Src, which suppresses integrin function and weakens cell-matrix adhesion. Phosphorylation of the cytoplasmic domain of ␤1 integrins was shown to be critical for v-Src-mediated morphological transformation (19). Others have found that v-Src phosphorylates and reduces the affinity of ␤3, but not of ␤1, integrins, and instead an indirect mechanism that disrupts ␤1 integrin-mediated cell adhesion was proposed (20,21).
To clarify how different integrins regulate the various aspects of Src-mediated morphological transformation and how this relates to oncogenic transformation, we have expressed a c-Src mutant that is constitutively in an open, primed conformation (c-Src[Y530F], here referred to as Src YF ) in the context of wild type, chimeric, and mutant ␤1 and ␤3 integrin subunits in two independent ␤1-deficient cell lines. While overexpression of ␣v␤3 augments Src YF -mediated tumor growth through an interaction at the ␤3 cytoplasmic tail (22), the ␣v␤3 extracellular domain protects against Src YF -induced cell rounding. Moreover, like tumor formation Src YF -induced podosome assembly occurs independent of ␤3 phosphorylation. Instead, phosphorylation of ␤1 is required to suppress Rho-mediated contractility in order to assemble podosomes. Thus, integrins uncouple Src YF -mediated oncogenic transformation and various aspects of morphological transformation.
Short Term Adhesion Assays-Adhesion assays were performed in 96-well tissue culture plates that were coated with 5 g/ml FN in PBS overnight at 4°C, blocked with 2% heat-denatured bovine serum albumin for 2 h at 37°C, and washed once with PBS. Cells were trypsinized, collected in culture medium, washed once with PBS, resuspended in Dulbecco's modified Eagle's medium/0.5% bovine serum albumin, and added to the plate at 2 ϫ 10 4 cells/well. After 15 min of incubation at 37°C, unattached cells were removed by rinsing the wells with PBS; the remaining attached cells were lysed and stained overnight at 37°C in 3.75 mM p-nitrophenyl N-acetyl-␤-D-glucosamide/0.05 M sodium citrate/0.25% Triton X-100. Stopbuffer (50 mM glycine, pH 10.4, 5 mM EDTA) was added, and the A 405 was determined in triplicate wells and related to the A 405 measured in wells in which all 2 ϫ 10 4 cells were stained to calculate the percentage of adhered cells.
Immunofluorescence and Flow Cytometry-For immunofluorescence, cells were fixed in 4% formaldehyde, permeabilized in 0.4% Triton X-100, blocked with 2% bovine serum albumin, and incubated with anti-paxillin antibody or anti-human ␤3 (23C6), followed by Alexa-488-conjugated secondary antibody, rhodamine-phalloidin or TOPRO-3 staining (Molecular Probes). Preparations were mounted in Poly Aquamount (Polysciences, Inc.) and analyzed using a Bio-Rad Radiance 2100 confocal system. Images were obtained using a ϫ40 or ϫ60 oil objective and imported in Adobe Photoshop. For flow cytometry and cell sorting, cells were trypsinized, collected in culture medium, washed with PBS, and incubated with primary antibodies in PBS containing 2% serum for 1 h at 4°C. Cells were then washed in PBS, incubated with phycoerythrin-or allophycocyanin-conjugated secondary antibodies for 1 h at 4°C, washed in PBS, and analyzed on a FACSCalibur or sorted on a FACStar plus (BD Biosciences).
Rho Activity Assays-Cells were plated overnight to subconfluency before lysis in Nonidet P-40 lysis buffer (0.5% Nonidet P-40, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM MgCl 2 , 10% glycerol, supplemented with a protease inhibitor mix (Sigma-Aldrich)), and lysates were clarified by centrifugation at 14,000 rpm for 20 min at 4°C. A 1% aliquot was removed for determination of total quantities of RhoA. Clarified lysates were then incubated for 45 min at 4°C with a glutathione S-transferase fusion protein of the Rho-binding domain of the Rho effector protein Rhotekin. Complexes were bound to glutathione-conjugated beads and washed three times in Nonidet P-40 lysis buffer. The samples were analyzed by SDS-PAGE and Western blotting.
FN Matrix Assembly Assays-To visualize FN matrix assembly, cells were plated on FN-coated coverslips for 4 h and subsequently incubated for an additional 20 h in medium containing 10% FN-depleted serum supplemented with 10 g/ml biotinylated FN. Cells were fixed in 4% formaldehyde, blocked with 2% bovine serum albumin, and stained with streptavidin-Texas Red. Subsequently, coverslips were permeabilized in 0.4% Triton X-100 and stained with TOPRO-3. For biochemical analysis of FN matrix assembly cells were labeled with biotinylated FN as described above and lysed in DOC buffer (1% sodium deoxycholate, 20 mM Tris-HCl, pH 8.5, 2 mM N-ethylmaleimide, 2 mM iodoacetic acid, 2 mM EDTA, and 2 mM phenylmethylsulfonyl fluoride). Lysates were passed through a 23-gauge needle, and deoxycholate-insoluble material was collected by centrifugation at 14,000 rpm for 20 min at 4°C. The pellet was washed once with DOC buffer, resolved in reduced sample buffer, and analyzed by SDS-PAGE and Western blotting.
Integrin Immunoprecipitations-Prior to immunoprecipitation some cells were stimulated with 3 mM H 2 O 2 and 1 mM sodium orthovanadate for 20 min to maximize phosphorylation. Cells were lysed for 15 min at 4°C in lysis buffer (1% Nonidet P-40, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM sodium vanadate, 0.5 mM sodium fluoride, and protease inhibitor mixture (Sigma-Aldrich)). Lysates were clarified by centrifugation at 14,000 rpm for 15 min at 4°C and precleared with protein A-Sepharose (Amersham Biosciences) for 2 h at 4°C. Proteins were immunoprecipitated overnight at 4°C with antibodies to ␤1 (K20) or ␤3 (SSA6) coupled to protein A-Sepharose. The beads were resolved in reduced sample buffer and analyzed by SDS-PAGE and Western blotting.

Morphological Transformation by Src YF Does Not Require ␤1
Integrins-Src activation causes a dramatic change in cellular morphology by interfering with adhesion and cytoskeletal organization, processes in which integrin signaling plays a critical role. To investigate the role of ␤1 integrins in Src-mediated morphological transformation, we expressed Src YF in two independent ␤1-deficient cell lines. As described for Src activation in other cell types (9,16), expression of Src YF in GE11 and GD25 cells caused disruption of cell-cell contacts and cell scattering (Fig. 1A).
Expression of Src YF also caused a dramatic reorganization of the actin cytoskeleton: F-actin bundles and ruffles disappeared and, instead, actin clusters were formed that resemble podosomes (Fig. 1B). Initial adhesion (e.g. 15 min) of GE11 and GD25 cells to FN is weak ( Fig. 2A), but at later time points (1 h) they do fully adhere and spread (Fig. 2B). Expression of Src YF interfered with this spreading, causing a rounded or fusiform phenotype, which was maintained after overnight culture (Figs. 1B and 2B). These experiments show that all aspects of Src YF -induced morphological transformation can occur in ␤1 null cells, arguing against a requirement for ␤1 integrins per se.
Different Aspects of Src YF -mediated Morphological Transformation Can Be Separated; Distinct Roles for ␤1 and ␤3 Integrins-Expression of ␤1 in GE11 and GD25 cells led to a strong increase in cell adhesion to FN (ϳ70% of the cells attached at 15 min after plating) that was suppressed by Src YF (Fig. 2A). At later times (e.g. 1 h after plating), GE␤1 and GD␤1 cells had all adhered regardless of the absence or presence of Src YF , but in the presence of Src YF cells remained rounded (Fig.  2B). In complete contrast, overexpression of ␤3 in the ␤1 null cells led to a similar increase in adhesion and spreading to FN as expression of ␤1 but this was only minimally affected by Src YF  ( Fig. 2, A and B). Notably, after overnight culture Src YF ␤1-expressing cells retained a fusiform or even rounded shape whereas Src YF ␤3-expressing cells remained well spread (Fig.  4A). This indicates that Src YF did not simply delay ␤1-integrinmediated spreading but caused a permanent morphological alteration that was not seen in the context of ␣v␤3. Finally, expression of ␤1 in GESrc YF ␤3 cells did not alter the well spread morphology of these cells, indicating that ␣v␤3-mediated protection against Src YF -induced cell rounding was dominant (supplemental Fig. S1, A and B).
We have reported that expression of ␤1 integrins in GE11 and GD25 cells stimulates Rho-mediated cytoskeletal contrac-tility and FN matrix assembly, whereas overexpression of ␤3 in ␤1 null cells is unable to do so (24). We wondered whether higher levels of Rho-mediated cytoskeletal contractility could also explain the inhibition of cell spreading in the Src YF -transformed cells expressing ␤1 integrins. However, in the presence of Src YF , RhoA-GTP levels in ␤1-expressing cells were dramatically suppressed to levels that were comparable with those in cells lacking ␤1 (Fig. 3A). Moreover, FN matrix assembly, a process that requires Rho-mediated contractility, was strongly reduced upon introduction of Src YF (Fig. 3, B and C).
Subsequently, we investigated whether ␤1 and ␤3 integrins affected Src YF -mediated podosome assembly. Despite the markedly different sensitivities of ␤1and ␤3-mediated adhesion and spreading to suppression by Src YF (Fig. 2), loss of F-actin stress fibers and conversion of focal adhesions into podosomes was seen in each case (Fig. 4A). Podosomes of Src YF ␤1 cells often consisted of F-actin dots that were tightly sealed  together, whereas more dispersed, individual, small F-actin dots were present in Src YF ␤3 cells, which may be explained by increased cell spreading (see Fig. 4A, insets). The podosomes that were formed in each of the Src YF -transformed cell types were dependent on Src YF kinase activity, because treatment with the Src-selective kinase inhibitor PP2 led to their disassembly (Fig. 4, B and C).
Taken together, these results demonstrate that (i) high levels of ␤3, but not ␤1, integrins protect Src YF -transformed cells from rounding up and (ii) two typical aspects of Src YF -induced morphological transformation, cell rounding and podosome formation, are distinct processes and are differently affected by the integrin expression profile.
Src YF -induced Podosomes Are Proteolytically Active Irrespective of the Integrin Type-Formation of podosomes is a morphological hallmark of Src transformation, and these adhesions are thought to be hotspots for invasion and proteolytic remodeling of the extracellular matrix (17,18). We next tested whether the integrin expression profile affected the proteolytic activity of these podosomes. No matrix degradation was observed to be associated with focal contacts in GE␤1 and GE␤3 cells in the absence of Src YF when plated on immobilized FITC-labeled FN (Fig. 5). By contrast, podosomes formed in GESrc YF ␤1 and GESrc YF ␤3 cells were both able to degrade FITC-FN. Proteolytic activity was often evident at sites outside cell borders, indicating that cells had moved along these sites (Fig. 5, arrowheads). Thus, podosomes in Src YF -transformed cells are proteolytically active, irrespective of the integrin composition.

Oncogenic and Morphological Transformations Are Separated by Distinct Integrin Domains-We
have previously shown that ␣v␤3 strongly supports Src YF -mediated tumorigenesis through an interaction between the ␤3 cytoplasmic domain and the Src homology 3 domain (22). We examined whether this was related to the capacity of ␣v␤3 to protect cells against Src YFinduced rounding (Figs. 2 and 4). Therefore, we expressed a chimeric ␤1 ex 3 in subunit, consisting of a ␤1 extracellular and transmembrane region fused to the cytoplasmic tail of ␤3, or an inverse ␤3 ex 1 in integrin in GESrc YF cells (supplemental Fig.  S1C). Using these chimeric integrins we demonstrated that the ␤3 cytoplasmic domain was required and sufficient for the stimulation of Src YF -mediated tumor growth (Fig.  6C, left graph, and Ref. 22). In complete contrast, ␣v␤3-mediated protection against Src YF -induced cell rounding required the ␤3 extracellular domain: ␤3 ex 1 in failed to support tumor growth but effectively rescued short term cell adhesion and subsequent spreading, whereas the opposite was the case for a ␤1 ex 3 in chimera (Fig. 6, A and C). Like adhesion and spreading, the appearance of podosomes was unaffected by the integrin cytoplasmic tail swap: podosomes in the presence of ␤1 ex 3 in resembled those of ␤1-expressing cells and were often sealed together, whereas podosomes of ␤3 ex 1 in -expressing cells were comparable with those expressing ␤3, consisting mainly of dispersed small F-actin dots (Fig. 6B). These results demonstrate that (i) high levels of ␣v␤3 support Src YF -mediated tumor formation and protect against Src YF -induced loss of adhesion and spreading through distinct mechanisms and (ii) Src YF -mediated oncogenic and morphological transformation can be separated.
Podosome Formation Requires Src YF -mediated Phosphorylation of the ␤1 Cytoplasmic Tail to Suppress Cytoskeletal Contractility-Integrin cytoplasmic tails serve as direct phosphorylation substrates of v-Src, which impairs their adhesive function (19,21). Analysis of immunoprecipitated integrin ␤ subunits demonstrated that ␤1 and ␤3 can both be tyrosinephosphorylated by Src YF (Fig. 7A), although phosphorylation was very low compared with maximal levels reached with pervanadate (Fig. 7B). Using single tyrosine point mutants we have found that phosphorylation of either of the two tyrosines in the ␤3 cytoplasmic tail is not required for ␣v␤3-mediated support of tumor growth (22). We observed that these mutations also did not affect morphological transformation by Src YF (data not shown). Moreover, expression of a non-phosphorylatable ␤3 Y747F,Y759F (␤3 YYFF ) subunit did not change Src YF -mediated morphological transformation when compared with wild type ␤3: actin stress fibers were absent and podosomes were formed in the presence of Src YF and ␤3 YYFF (Fig. 7C). By contrast, in cells expressing ␤1, integrin phosphorylation was crucial for Src YF -mediated morphological transformation. When a nonphosphorylatable ␤1 Y783F,Y795F (␤1 YYFF ) subunit was expressed in GESrc YF and GDSrc YF cells (supplemental Fig. S1, D and E), Src YF -induced podosome formation was completely abolished (Fig. 7, D-F). Instead of podosomes that were formed in Src YF ␤1 cells, F-actin stress fibers and focal contacts were restored in Src YF ␤1 YYFF cells and eventually these cells became considerably more spread. These results indicate that phosphorylation of ␤1, but not ␤3, cytoplasmic tails is important for Src YF -mediated morphological transformation. Most likely, phosphorylation is required to suppress Rho-mediated cytoskeletal contractility that is promoted by ␤1, but not by ␤3, integrins and would interfere with podosome formation (Fig. 3).

DISCUSSION
In summary (see Fig. 8), we show that (i) Src-mediated oncogenic and morphological transformations are distinct processes; (ii) podosome formation and cell rounding are independent aspects of Src-mediated morphological transformation (e.g. all cells expressing high levels of integrin subunits containing ␤3 extracellular domain contain podosomes but remain well spread); (iii) ␣v␤3 supports Src YF -mediated tumor formation and protects against Src YF -induced loss of adhesion and spreading through distinct mechanisms (e.g. experiments using ␤1 ex 3 in and ␤3 ex 1 in chimeras indicate that the ␤3 cytoplasmic domain supports Src-mediated tumor growth whereas the ␤3 extracellular domain protects against Src-induced cell rounding); and (iv) Src-induced podosome assembly in the presence of ␤1 requires phosphorylation of the integrin cytoplasmic domain to reduce cytoskeletal contractility (e.g. ␤1 YYFF ). In the absence of ␤1 integrins, ␤3 does not promote Rho-mediated cytoskeletal contractility (24) and podosomes can be formed without Src-mediated phosphorylation of integrin tails (e.g. ␤3 YYFF ).
Disruption of cytoskeletal contractility is one of the key events during Src-induced morphological transformation that enables reorganization of the actin cytoskeleton in order to assemble podosomes. Relaxation of the actin cytoskeleton requires inactivation of RhoA, and indeed expression of constitutively activated RhoA suppresses loss of stress fibers and podosome formation induced by v-Src (26). On the other hand, complete inhibition of RhoA also perturbs podosomes, indicating that local RhoA activity might still be required (27). We find that Src YF inhibits the ability of ␤1 integrins to support RhoA-mediated contractility. The kinase activity of Src YF is required for podosome formation in Src YF ␤1and Src YF ␤3-expressing cells, and Src YF phosphorylates ␤1 and ␤3 cytoplasmic domains. However, phosphorylation of ␤1, but not ␤3, is important for Src YF -mediated morphological transformation. In line with a previous report (28), mutation of the tyrosines in the ␤1 cytoplasmic tail restored focal adhesions and cell spreading. Our findings suggest that this is due to restored cytoskeletal contractility that prevents the transformation from focal contacts to podosomes in the presence of Src YF . Indeed, overexpression of ␣v␤3 fails to promote Rho-mediated cytoskeletal contractility in ␤1-null cells (24), explaining why corresponding mutations in the ␤3 subunit do not affect Src-mediated morphological transformation. Notably, in osteosarcoma cells phosphorylation of ␤3 by v-Src reduces the binding strength of ␣v␤3 to FN (21). In our studies, ␣v␤3-mediated adhesion to FN was not affected by the expression of Src YF , which may be related to differences between v-Src (which contains multiple additional mutations) and Src YF (which may closely resemble c-Src in human cancer cells where its interaction with overexpressed receptor tyrosine kinases or mutations in the C terminus can lead to enhanced priming) or to the moderate Src YF expression and integrin phosphorylation levels that we reach in GE11 and GD25 cells. Nevertheless, these levels are sufficient to cause all the aspects of morphological transformation and lead to rounding of ␤1-expressing cells. Our study dissociates Src YF -mediated oncogenic from morphological transformation and shows that different aspects of morphological transformation (e.g. podosome formation and cell rounding) involve separable, independent pathways. These findings are corroborated by studies in which mitogenic activ-ity, morphological alterations, and the anchorage independence of cells expressing mutants of v-Src were compared. It was shown that the amino-terminal domain of v-Src is important for determining cell morphology, whereas the kinase domain is essential for all three parameters (29). Also, when expressed at very low levels in Madin-Darby canine kidney cells, v-Src elicited disruption of zonula adherences, which was dissociable from oncogenic transformation, as determined by anchorageindependent growth capacity and proliferation (30). Attempts to transform c-Myc-deficient fibroblasts with v-Src resulted in morphological transformation but failed to induce DNA synthesis and proliferation (31). All together, these studies show that signaling downstream of Src can occur through multiple independent pathways. Our current work indicates that the integrin expression profile differentially modulates all these aspects of Src transformation.
In human cancer increased expression and activity of c-Src contributes to tumor development through stimulation of mitogenic signaling pathways in which c-Src normally plays a regulatory role (10,32). In addition, reorganization of the actin cytoskeleton, cell-cell, and cell-matrix adhesions upon Src activation may contribute to tumor invasion and metastasis (4,9). Interestingly, changes in the expression profile of integrins often occur with tumor formation and during later steps of tumor progression. Increased expression levels of ␣v␤3 are associated with growth and progression of various cancers (33). For example, high levels of ␣v␤3 promote the conversion from radial to vertical growth phase in human melanoma (34,35), a cancer type in which c-Src activity is frequently increased (4). Our findings suggest that such changes in integrin expression can have a dramatic impact on Src-mediated effects on growth and/or invasion of tumors. Cooperation between integrin ␣v␤3 and c-Src may be important for tumor growth, whereas shifts in the relative expression of ␤1 and ␤3 integrins might be important to control tumor cell adhesion and spreading during cancer progression.