Src-induced de-regulation of E-cadherin in colon cancer cells requires integrin signalling

Although Src expression and activity are often elevated in colon cancer, the precise consequences of overexpression of the non-catalytic Src homology (SH) domains, or enhanced catalytic activity, are unknown. We show that, in KM12C colon cancer cells, elevated Src activity causes the components of adherens junctions, including vinculin, to be redistributed to Src-induced integrin adhesion complexes. Specifically, elevated Src activity blocks proper assembly of cell cell contacts after cells are switched from media containing a low level of calcium to media containing a high level of calcium, and E-cadherin remains internalized. In contrast, although elevated expression of the non-catalytic domains of Src is sufficient to induce assembly of integrin adhesion complexes, it does not induce disorganization of E-cadherin-associated intercellular contacts. Surprisingly, Src-induced disruption of E-cadherin localization requires specific integrin signalling, because E-cadherin redistribution is blocked by loss of cell-matrix interaction, or by inhibitory antibodies to alpha(v) or beta(1) integrin subunits. Furthermore, phosphorylation of the integrin-regulated focal adhesion kinase (FAK) on Src-specific sites is required for Src-induced de-regulation of E-cadherin, demonstrating interdependence between integrin-induced signals and cadherin-associated adhesion changes induced by Src.     

Involvement of cell-cell interactions in the rapid stimulation of Cas tyrosine phosphorylation and Src kinase activity by transforming growth factor-beta 1

Transforming growth factor-beta (TGF-beta) regulates a wide range of physiological and pathological cellular processes, including cell migration, mesenchymal transition, extracellular matrix synthesis, and cell death. Cas (Crk-associated substrate, 130 kDa), an adaptor protein localized at focal adhesions and stress fibers, is also known to have important functions in cell migration and the induction of immediate-early gene expression. Here, we report that a rapid and transient tyrosine phosphorylation of Cas is induced by TGF-beta 1 and that E-cadherin-mediated cell-cell interaction and the Src kinase pathway are involved in this early TGF-beta signaling. The addition of TGF-beta 1 to epithelial cells rapidly induced tyrosine phosphorylation of Cas and promoted the formation of complexes between focal adhesion molecules. Cas phosphorylation required the integrity of the actin cytoskeleton but was not dependent on cell adhesion, implying that Cas-dependent signaling may be distinct from integrin signaling. TGF-beta 1 also stimulated Src kinase activity, and specific inhibitors of Src completely blocked the induction of Cas phosphorylation by TGF-beta 1. The Cas phosphorylation and Src kinase activation seen in our results were induced in an epithelial phenotype-specific manner. Stable transfection of E-cadherin to L929 cells and L cells as well as E-cadherin blocking assay revealed that E-cadherin-mediated cell-cell interactions were essential for both Cas phosphorylation and Src kinase activation. Taken together, our data suggest that rapid Cas phosphorylation and Src kinase activation may play a novel role in TGF-beta signal transduction.     

An active Src kinase-beta-actin association is linked to actin dynamics at the periphery of colon cancer cells

Src controls the dynamic actin cytoskeleton in fibroblasts and in cancer cells, although it is not known how direct its effects are. Using FRET/FLIM imaging, we found that wild type Src associates directly, or indirectly, with peripheral beta-actin at integrin adhesions after serum stimulation, and that an active Src kinase domain is essential. Beta-actin can be directly tyrosine-phosphorylated by Src in vitro, and in a Src-dependent manner in cells. Moreover, beta-actin dynamics are suppressed when Src is rendered kinase-inactive. Surprisingly, debilitating mutations in the Src SH2 or SH3 domains do not suppress association of Src with beta-actin. This may therefore be an example of a spatially regulated Src kinase/substrate interaction that is controlling peripheral actin dynamics. Interestingly, there is no FRET between Src and beta-actin at cadherin-mediated cell-cell contacts, despite apparent co-localization there, demonstrating precise spatial specificity of Src/beta-actin complexes.     

Src and FAK signalling controls adhesion fate and the epithelial-to-mesenchymal transition

Src kinase controls cellular adhesions, including cadherin-based intercellular adhesions and integrin-mediated cell-matrix adhesions. In epithelial cells, Src activation, or increased signalling from migratory growth factor receptors via Src, induces an adhesion switch that enhances dynamic cell-matrix adhesions and migratory capacity while suppressing intercellular contact. Moreover, Src and the associated tyrosine kinase FAK are at the heart of the recently identified crosstalk between integrin- and cadherin-mediated adhesions of epithelial cells, particularly during the epithelial-to-mesenchymal transition.     

Src and FAK mediate cell–matrix adhesion‐dependent activation of met during transformation of breast epithelial cells

Cell–matrix adhesion has been shown to promote activation of the hepatocyte growth factor receptor, Met, in a ligand‐independent manner. This process has been linked to transformation and tumorigenesis in a variety of cancer types. In the present report, we describe a key role of integrin signaling via the Src/FAK axis in the activation of Met in breast epithelial and carcinoma cells. Expression of an activated Src mutant in non‐neoplastic breast epithelial cells or in carcinoma cells was found to increase phosphorylation of Met at regulatory tyrosines in the auto‐activation loop domain, correlating with increased cell spreading and filopodia extensions. Furthermore, phosphorylated Met is complexed with β1 integrins and is co‐localized with vinculin and FAK at focal adhesions in epithelial cells expressing activated Src. Conversely, genetic or pharmacological inhibition of Src abrogates constitutive Met phosphorylation in carcinoma cells or epithelial cells expressing activated Src, and inhibits filopodia formation. Interestingly, Src‐dependent phosphorylation of Met requires cell–matrix adhesion, as well as actin stress fiber assembly. Phosphorylation of FAK by Src is also required for Src‐induced Met phosphorylation, emphasizing the importance of the Src/FAK signaling pathway. However, stimulation of Met phosphorylation by addition of exogenous HGF in epithelial cells is refractory to inhibition of Src family kinases, indicating that HGF‐dependent and Src/integrin‐dependent Met activation occur via distinct mechanisms. Together these findings demonstrate a novel mechanism by which the Src/FAK axis links signals from the integrin adhesion complex to promote Met activation in breast epithelial cells. J. Cell. Biochem. 107: 1168–1181, 2009. © 2009 Wiley‐Liss, Inc.     

Cbl associates with Pyk2 and Src to regulate Src kinase activity, alpha(v)beta(3) integrin-mediated signaling, cell adhesion, and osteoclast motility

The signaling events downstream of integrins that regulate cell attachment and motility are only partially understood. Using osteoclasts and transfected 293 cells, we find that a molecular complex comprising Src, Pyk2, and Cbl functions to regulate cell adhesion and motility. The activation of integrin alpha(v)beta(3) induces the [Ca(2+)](i)-dependent phosphorylation of Pyk2 Y402, its association with Src SH2, Src activation, and the Src SH3-dependent recruitment and phosphorylation of c-Cbl. Furthermore, the PTB domain of Cbl is shown to bind to phosphorylated Tyr-416 in the activation loop of Src, the autophosphorylation site of Src, inhibiting Src kinase activity and integrin-mediated adhesion. Finally, we show that deletion of c Src or c-Cbl leads to a decrease in osteoclast migration. Thus, binding of alpha(v)beta(3) integrin induces the formation of a Pyk2/Src/Cbl complex in which Cbl is a key regulator of Src kinase activity and of cell adhesion and migration. These findings may explain the osteopetrotic phenotype in the Src(-/-) mice.     

Coordination of cell polarization and migration by the Rho family GTPases requires Src tyrosine kinase activity.

BACKGROUND: The ability of a cell to polarize and move is governed by remodeling of the cellular adhesion/cytoskeletal network that is in turn controlled by the Rho family of small GTPases. However, it is not known what signals lie downstream of Rac1 and Cdc42 during peripheral actin and adhesion remodeling that is required for directional migration. RESULTS: We show here that individual members of the Rho family, RhoA, Rac1, and Cdc42, direct the specific intracellular targeting of c-Src tyrosine kinase to focal adhesions, lamellipodia, or filopodia, respectively, and that the adaptor function of c-Src (the combined SH3/SH2 domains coupled to green fluorescent protein) is sufficient for targeting. Furthermore, Src's catalytic activity is absolutely required at these peripheral cell-matrix attachment sites for remodeling that converts RhoA-dependent focal adhesions into smaller focal complexes along Rac1-induced lamellipodia (or Cdc42-induced filopodia). Consequently, cells in which kinase-deficient c-Src occupies peripheral adhesion sites exhibit impaired polarization toward migratory stimuli and reduced motility. Furthermore, phosphorylation of FAK, an Src adhesion substrate, is suppressed under these conditions. CONCLUSIONS: Our findings demonstrate that individual Rho GTPases specify Src's exact peripheral localization and that Rac1- and Cdc42-induced adhesion remodeling and directed cell migration require Src activity at peripheral adhesion sites.     

Phospholipase Cgamma2 modulates integrin signaling in the osteoclast by affecting the localization and activation of Src kinase

Integrin engagement induces a cascade of signaling pathways that include tyrosine phosphorylation of numerous proteins that lead to modulation of the actin cytoskeleton. Src is a major intracellular mediator of integrin-dependent functions, but the mechanism(s) by which Src is regulated in response to integrin signals is not fully understood. Here, we demonstrate an important role for phospholipase C gamma 2 (PLCgamma2) in Src activation in the osteoclast. Through analysis of primary cells from PLCgamma2(-/-) mice, PLCgamma2 was found to be an important regulator of alpha(v)beta(3) integrin-mediated bone osteoclast cell adhesion, migration, and bone resorption. Adhesion-induced PYK2 and Src phosphorylation is decreased in the absence of PLCgamma2, and the interaction of Src with beta(3) integrin and PYK2 is dramatically reduced. Importantly, PLCgamma2 was found to be required for proper localization of Src to the sealing actin ring, and this function required both its catalytic activity and adapter domains. Based on these results, we propose that PLCgamma2 influences Src activation by mediating the localization of Src to the integrin complex and thereby regulating integrin-mediated functions in the osteoclast.     

Formation of E-cadherin-mediated cell-cell adhesion activates AKT and mitogen activated protein kinase via phosphatidylinositol 3 kinase and ligand-independent activation of epidermal growth factor receptor in ovarian cancer cells

E-cadherin is a well characterized adhesion molecule that plays a major role in epithelial cell adhesion. Based on findings that expression of E-cadherin is frequently lost in human epithelial cancers, it has been implicated as a tumor suppressor in carcinogenesis of most human epithelial cancers. However, in ovarian cancer development, our data from the current study showed that E-cadherin expression is uniquely elevated in 86.5% of benign, borderline, and malignant ovarian carcinomas irrespective of the degree of differentiation, whereas normal ovarian samples do not express E-cadherin. Thus, we hypothesize that E-cadherin may play a distinct role in the development of ovarian epithelial cancers. Using an E-cadherin-expressing ovarian cancer cell line OVCAR-3, we have demonstrated for the first time that the establishment of E-cadherin mediated cell-cell adhesions leads to the activation of Akt and MAPK. Akt activation is mediated through the activation of phosphatidylinositol 3 kinase, and both Akt and MAPK activation are mediated by an E-cadherin adhesion-induced ligand-independent activation of epidermal growth factor receptor. We have also demonstrated that suppression of E-cadherin function leads to retarded cell proliferation and reduced viability. We therefore suggest that the concurrent formation of E-cadherin adhesion and activation of downstream proliferation signals may enhance the proliferation and survival of ovarian cancer cells. Our data partly explain why E-cadherin is always expressed during ovarian tumor development and progression.     

Integrin-dependent actomyosin contraction regulates epithelial cell scattering

The scattering of Madin-Darby canine kidney cells in vitro mimics key aspects of epithelial-mesenchymal transitions during development, carcinoma cell invasion, and metastasis. Scattering is induced by hepatocyte growth factor (HGF) and is thought to involve disruption of cadherin-dependent cell-cell junctions. Scattering is enhanced on collagen and fibronectin, as compared with laminin1, suggesting possible cross talk between integrins and cell-cell junctions. We show that HGF does not trigger any detectable decrease in E-cadherin function, but increases integrin-mediated adhesion. Time-lapse imaging suggests that tension on cell-cell junctions may disrupt cell-cell adhesion. Varying the density and type of extracellular matrix proteins shows that scattering correlates with stronger integrin adhesion and increased phosphorylation of the myosin regulatory light chain. To directly test the role of integrin-dependent traction forces, substrate compliance was varied. Rigid substrates that produce high traction forces promoted scattering, in comparison to more compliant substrates. We conclude that integrin-dependent actomyosin traction force mediates the disruption of cell-cell adhesion during epithelial cell scattering.     

Distinct ligand binding sites in integrin alpha3beta1 regulate matrix adhesion and cell-cell contact

The integrin alpha3beta1 mediates cellular adhesion to the matrix ligand laminin-5. A second integrin ligand, the urokinase receptor (uPAR), associates with alpha3beta1 via a surface loop within the alpha3 beta-propeller (residues 242-246) but outside the laminin binding region, suggesting that uPAR-integrin interactions could signal differently from matrix engagement. To explore this, alpha3-/- epithelial cells were reconstituted with wild-type (wt) alpha3 or alpha3 with Ala mutations within the uPAR-interacting loop (H245A or R244A). Wt or mutant-bearing cells showed comparable expression and adhesion to laminin-5. Cells expressing wt alpha3 and uPAR dissociated in culture, with increased Src activity, up-regulation of SLUG, and down-regulation of E-cadherin and gamma-catenin. Src kinase inhibition or expression of Src 1-251 restored the epithelial phenotype. The H245A and R244A mutants were unaffected by coexpression of uPAR. We conclude that alpha3beta1 regulates both cell-cell contact and matrix adhesion, but through distinct protein interaction sites within its beta-propeller. These studies reveal an integrin- and Src-dependent pathway for SLUG expression and mesenchymal transition.     

Autophagic targeting of Src promotes cancer cell survival following reduced FAK signalling

Here we describe a mechanism that cancer cells use to survive when flux through the Src/FAK pathway is severely perturbed. Depletion of FAK, detachment of FAK-proficient cells or expression of non-phosphorylatable FAK proteins causes sequestration of active Src away from focal adhesions into intracellular puncta that co-stain with several autophagy regulators. Inhibition of autophagy results in restoration of active Src at peripheral adhesions, and this leads to cancer cell death. Autophagic targeting of active Src is associated with a Src-LC3B complex, and is mediated by c-Cbl. However, this is independent of c-Cbl E3 ligase activity, but is mediated by an LC3-interacting region. Thus, c-Cbl-mediated autophagic targeting of active Src can occur in cancer cells to maintain viability when flux through the integrin/Src/FAK pathway is disrupted. This exposes a previously unrecognized cancer cell vulnerability that may provide a new therapeutic opportunity.     

Introduction of p130cas signaling complex formation upon integrin-mediated cell adhesion: a role for Src family kinases.

Integrin-mediated cell adhesion triggers intracellular signaling cascades, including tyrosine phosphorylation of intracellular proteins. Among these are the focal adhesion proteins p130cas (Cas) and focal adhesion kinase (FAK). Here we identify the kinase(s) mediating integrin-induced Cas phosphorylation and characterize protein-protein interactions mediated by phosphorylated Cas. We found that expression of a constitutively active FAK in fibroblasts results in a consecutive tyrosine phosphorylation of Cas. This effect required the autophosphorylation site of FAK, which is a binding site for Src family kinases. Integrin-mediated phosphorylation of Cas was not, however, compromised in fibroblasts lacking FAK. In contrast, adhesion-induced tyrosine phosphorylation of Cas was reduced in cells lacking Src, whereas enhanced phosphorylation of Cas was observed Csk- cells, in which Src kinases are activated. These results suggest that Src kinases are responsible for the integrin-mediated tyrosine phosphorylation of Cas. FAK seems not to be necessary for phosphorylation of Cas, but when autophosphorylated, FAK may recruit Src family kinases to phosphorylate Cas. Cas was found to form complexes with Src homology 2 (SH2) domain-containing signaling molecules, such as the SH2/SH3 adapter protein Crk, following integrin-induced tyrosine phosphorylation. Guanine nucleotide exchange factors C3G and Sos were found in the Cas-Crk complex upon integrin ligand binding. These observations suggest that Cas serves as a docking protein and may transduce signals to downstream signaling pathways following integrin-mediated cell adhesion.     

Tetraspanin CO-029 inhibits colorectal cancer cell movement by deregulating cell-matrix and cell-cell adhesions

Alterations in tetraspanin CO-029 expression are associated with the progression and metastasis of cancers in the digestive system. However, how CO-029 promotes cancer metastasis is still poorly understood. To determine the mechanism, we silenced CO-029 expression in HT29 colon cancer cells and found that the CO-029 knockdown significantly reduced cell migratory ability. The diminished cell migration was accompanied by the upregulation of both integrin-dependent cell-matrix adhesion on laminin and calcium-dependent cell-cell adhesion. The cell surface levels of laminin-binding integrin α3β1 and fibronectin-integrin α5β1 were increased while the level of CD44 was decreased upon CO-029 silencing. These changes contribute to the altered cell-matrix adhesion. The deregulated cell-cell adhesion results, at least partially, from increased activity of cadherins and reduced level of MelCAM. In conclusion, CO-029 functions as a regulator of both cell-matrix and cell-cell adhesion. During colon cancer progression, CO-029 promotes cancer cell movement by deregulating cell adhesions.     

The catalytic activity of the Src family kinases is required to disrupt cadherin-dependent cell-cell contacts

Despite the importance of epithelial cell contacts in determining cell behavior, we still lack a detailed understanding of the assembly and disassembly of intercellular contacts. Here we examined the role of the catalytic activity of the Src family kinases at epithelial cell contacts in vitro. Like E- and P-cadherin, Ca(2+) treatment of normal and tumor-derived human keratinocytes resulted in c-Yes (and c-Src and Fyn), as well as their putative substrate p120(CTN), being recruited to cell-cell contacts. A tyrosine kinase inhibitor with selectivity against the Src family kinases, PD162531, and a dominant-inhibitory c-Src protein that interferes with the catalytic function of the endogenous Src kinases induced cell-cell contact and E-cadherin redistribution, even in low Ca(2+), which does not normally support stable cell-cell adhesion. Time-lapse microscopy demonstrated that Src kinase inhibition induced stabilization of transiently formed intercellular contacts in low Ca(2+). Furthermore, a combination of E- and P-cadherin-specific antibodies suppressed cell-cell contact, indicating cadherin involvement. As a consequence of contact stabilization, normal cells were unable to dissociate from an epithelial sheet formed at high density and repair a wound in vitro, although individual cells were still motile. Thus, cadherin-dependent contacts can be stabilized both by high Ca(2+) and by inhibiting Src activity in low (0.03 mM) Ca(2+) in vitro.