A novel role of nectins in inhibition of the E-cadherin-induced activation of Rac and formation of cell-cell adherens junctions

Nectins are Ca(2+)-independent immunoglobulin (Ig)-like cell-cell adhesion molecules. The trans-interactions of nectins recruit cadherins to the nectin-based cell-cell adhesion, resulting in formation of cell-cell adherens junctions (AJs) in epithelial cells and fibroblasts. The trans-interaction of E-cadherin induces activation of Rac small G protein, whereas the trans-interactions of nectins induce activation of not only Rac but also Cdc42 small G protein. We showed by the fluorescent resonance energy transfer (FRET) imaging that the trans-interaction of E-cadherin induced dynamic activation and inactivation of Rac, which led to dynamic formation and retraction of lamellipodia. Moreover, we found here that the nectins, which did not trans-interact with other nectins (non-trans-interacting nectins), inhibited the E-cadherin-induced activation of Rac and reduced the velocity of the formation of the E-cadherin-based cell-cell AJs. The inhibitory effect of non-trans-interacting nectins was suppressed by the activation of Cdc42 induced by the trans-interactions of nectins. These results indicate a novel role of nectins in regulation of the E-cadherin-induced activation of Rac and formation of cell-cell AJs.     

E-cadherin-mediated cell-cell attachment activates Cdc42

E-cadherin is a transmembrane protein that mediates Ca(2+)-dependent cell-cell adhesion. Cdc42, a member of the Rho family of small GTPases, participates in cytoskeletal rearrangement and cell cycle progression. Recent evidence reveals that members of the Rho family modulate E-cadherin function. To further examine the role of Cdc42 in E-cadherin-mediated cell-cell adhesion, we developed an assay for active Cdc42 using the GTPase-binding domain of the Wiskott-Aldrich syndrome protein. Initiation of E-cadherin-mediated cell-cell attachment significantly increased in a time-dependent manner the amount of active Cdc42 in MCF-7 epithelial cell lysates. By contrast, Cdc42 activity was not increased under identical conditions in MCF-7 cells incubated with anti-E-cadherin antibodies nor in MDA-MB-231 (E-cadherin negative) epithelial cells. By fusing the Wiskott-Aldrich syndrome protein/GTPase-binding domain to a green fluorescent protein, activation of endogenous Cdc42 by E-cadherin was demonstrated in live cells. These data indicate that E-cadherin activates Cdc42, demonstrating bi-directional interactions between the Rho- and E-cadherin signaling pathways.     

CD151 regulates epithelial cell-cell adhesion through PKC- and Cdc42-dependent actin cytoskeletal reorganization

CD151, a member of the tetraspanin family proteins, tightly associates with integrin alpha3beta1 and localizes at basolateral surfaces of epithelial cells. We found that overexpression of CD151 in A431 cells accelerated intercellular adhesion, whereas treatment of cells with anti-CD151 mAb perturbed the integrity of cortical actin filaments and cell polarity. E-Cadherin puncta formation, indicative of filopodia-based adhesion zipper formation, as well as E-cadherin anchorage to detergent-insoluble cytoskeletal matrix, was enhanced in CD151-overexpressing cells. Levels of GTP-bound Cdc42 and Rac were also elevated in CD151-overexpressing cells, suggesting the role of CD151 in E-cadherin-mediated cell-cell adhesion as a modulator of actin cytoskeletal reorganization. Consistent with this possibility, engagement of CD151 by the substrate-adsorbed anti-CD151 mAb induced prominent Cdc42-dependent filopodial extension, which along with E-cadherin puncta formation, was strongly inhibited by calphostin C, a protein kinase C (PKC) inhibitor. Together, these results indicate that CD151 is involved in epithelial cell-cell adhesion as a modulator of PKC- and Cdc42-dependent actin cytoskeletal reorganization.     

Rap1 regulates the formation of E-cadherin-based cell-cell contacts

In epithelial tissues, cells are linked to their neighbors through specialized cell-cell adhesion proteins. E-cadherin is one of the most important membrane proteins for the establishment of intimate cell-cell contacts, but the molecular mechanism by which it is recruited to contact sites is largely unknown. We report here that the cytoplasmic domain of E-cadherin interacts with C3G, a guanine nucleotide exchange factor for Rap1. In epithelial cell cultures, ligation of the extracellular domain of E-cadherin enhances Rap1 activity, which in turn is necessary for the proper targeting of E-cadherin molecules to maturing cell-cell contacts. Furthermore, our data suggest that Cdc42 functions downstream of Rap1 in this process. We conclude that Rap1 plays a vital role in the establishment of E-cadherin-based cell-cell adhesion.     

Src SH3/2 domain-mediated peripheral accumulation of Src and phospho-myosin is linked to deregulation of E-cadherin and the epithelial-mesenchymal transition

Elevated Src kinase in epithelial cancer cells induces adhesion changes that are associated with a mesenchymal-like state. We recently showed that Src induces dynamic integrin adhesions in KM12C colon cancer cells, whereas E-cadherin-dependent cell-cell contacts become disorganized. This promotes a fibroblastic-like morphology and expression of the mesenchymal marker vimentin. Furthermore, Src-induced deregulation of E-cadherin, and the associated mesenchymal transition, is dependent on integrin signaling (Avizienyte et al., Nat. Cell Biol. 2002, 4, 632-638), although the nature of downstream signals that mediate these Src- and integrin-dependent effects are unknown. Here we show that the SH2 and SH3 domains of Src mediate peripheral accumulation of phospho-myosin, leading to integrin adhesion complex assembly, whereas loss of SH2 or SH3 function restores normal regulation of E-cadherin and inhibits vimentin expression. Inhibitors of MEK, ROCK, or MLCK also suppress peripheral accumulation of phospho-myosin and Src-induced formation of integrin-dependent adhesions, whereas at the same time restoring E-cadherin redistribution to regions of cell-cell contact. Our data therefore implicate peripheral phospho-myosin activity as a point of convergence for upstream signals that regulate integrin- and E-cadherin-mediated adhesions. This further implicates spatially regulated contractile force as a determinant of epithelial cell plasticity, particularly in cancer cells that can switch between epithelial and mesenchymal-like states.     

Discoidin domain receptor 1 activation suppresses alpha2beta1 integrin-dependent cell spreading through inhibition of Cdc42 activity

Upregulation and overexpression of discoidin domain receptor 1 (DDR1) have been implied in the regulation of kidney development and progression of cancers. Our previous studies with Mardin-Darby canine kidney (MDCK) cells showed that overexpression of DDR1 inhibited cell spreading, whereas dominant negative DDR1 promoted cell spreading on collagen-coated dish. Cell spreading is an important characteristic for cell differentiation and survival. However, little is known about the molecular mechanisms underlying the role of DDR1 in cell spreading. We have found here a novel signaling pathway of DDR1 consisting of Cdc42 that regulates the assembly and disassembly of cytoskeleton and cell spreading in MDCK cells. Cell spreading involves the organization of cytoskeleton that is mainly regulated by Rho-family GTPases. We assessed the activity of Rho-family GTPases and transfected MDCK cells with constitutively active or dominant negative GTPases, and quantified the extent of cell spreading. These results showed that DDR1 decreased the filamentous actin ratio and Rac1/Cdc42 activities, but had no effects on RhoA activity. Neither constitutively active nor dominant negative Rac1 altered DDR1-inhibited cell spreading. Constitutively active Cdc42 could rescue the DDR1-inhibited cell spreading, whereas dominant negative Cdc42 inhibited cell spreading, indicating that DDR1-inhibited cell spreading is Cdc42 dependent. With the use of alpha(2)beta(1) integrin blocking antibody, we showed that collagen-induced Cdc42 activation was mediated by alpha(2)beta(1) integrin. Moreover, ectopic FAK expression enhanced the Cdc42 activity. Reducing FAK activity by dominant negative FAK (FRNK) markedly abolished the Cdc42 activity. These findings show that DDR1a/b activation inhibits cell spreading through suppressing alpha(2)beta(1) integrin-mediated Cdc42 activation.     

Trans-interactions of nectins induce formation of filopodia and Lamellipodia through the respective activation of Cdc42 and Rac small G proteins

Nectins and afadin constitute a novel cell-cell adhesion system that plays a cooperative role with cadherins in the organization of adherens junctions (AJs). Nectins are Ca(2+)-independent immunoglobulin-like cell-cell adhesion molecules, and afadin is a nectin- and actin filament-binding protein that connects nectins to the actin cytoskeleton. Rac and Cdc42 small G proteins have been implicated in the organization of AJs, but their modes of action remain unknown. The trans-interaction of E-cadherin has recently been shown to induce the activation of Rac, but not that of Cdc42. We show here that the trans-interactions of nectins induce the formation of filopodia and lamellipodia through the respective activation of Cdc42 and Rac. The Cdc42 activation is necessary, but not sufficient, for the Rac-induced formation of lamellipodia, whereas the Rac activation is not necessary for the Cdc42-induced formation of filopodia. These effects of nectins require their cytoplasmic tail but not their association with afadin. We propose here the functional relationship between nectins and the small G proteins in the organization of AJs.     

Regulation of the assembly and adhesion activity of E-cadherin by nectin and afadin for the formation of adherens junctions in Madin-Darby canine kidney cells

The Ca2+-independent immunoglobulin-like molecule nectin first forms cell-cell adhesion and then assembles cadherin at nectin-based cell-cell adhesion sites, resulting in the formation of adherens junctions (AJs). Afadin is a nectin- and actin filament-binding protein that connects nectin to the actin cytoskeleton. Here, we studied the roles and modes of action of nectin and afadin in the formation of AJs in cultured MDCK cells. The trans-interaction of nectin assembled E-cadherin, which associated with p120(ctn), beta-catenin, and alpha-catenin, at the nectin-based cell-cell adhesion sites in an afadin-independent manner. However, the assembled E-cadherin showed weak cell-cell adhesion activity and might be the non-trans-interacting form. This assembly was mediated by the IQGAP1-dependent actin cytoskeleton, which was organized by Cdc42 and Rac small G proteins that were activated by the action of trans-interacting nectin through c-Src and Rap1 small G protein in an afadin-independent manner. However, Rap1 bound to afadin, and this Rap1-afadin complex then interacted with p120(ctn) associated with non-trans-interacting E-cadherin, thereby causing the trans-interaction of E-cadherin. Thus, nectin regulates the assembly and cell-cell adhesion activity of E-cadherin through afadin, nectin signaling, and p120(ctn) for the formation of AJs in Madin-Darby canine kidney cells.     

Polarized trafficking of E-cadherin is regulated by Rac1 and Cdc42 in Madin-Darby canine kidney cells

E-cadherin is a major cell-cell adhesion protein of epithelia that is trafficked to the basolateral cell surface in a polarized fashion. The exact post-Golgi route and regulation of E-cadherin transport have not been fully described. The Rho GTPases Cdc42 and Rac1 have been implicated in many cell functions, including the exocytic trafficking of other proteins in polarized epithelial cells. These Rho family proteins are also associated with the cadherin-catenin complexes at the cell surface. We have used functional mutants of Rac1 and Cdc42 and inactivating toxins to demonstrate specific roles for both Cdc42 and Rac1 in the post-Golgi transport of E-cadherin. Dominant-negative mutants of Cdc42 and Rac1 accumulate E-cadherin at a distinct post-Golgi step. This accumulation occurs before p120^ctn interacts with E-cadherin, because p120^ctn localization was not affected by the Cdc42 or Rac1 mutants. Moreover, the GTPase mutants had no effect on the trafficking of a targeting mutant of E-cadherin, consistent with the selective involvement of Cdc42 and Rac1 in basolateral trafficking. These results provide a new example of Rho GTPase regulation of basolateral trafficking and demonstrate novel roles for Cdc42 and Rac1 in the post-Golgi transport of E-cadherin. Rho family GTPases; catenin; polarity; sorting; actin     

IQGAP1 and calmodulin modulate E-cadherin function

Ca(2+)-dependent cell-cell adhesion is mediated by the cadherin family of transmembrane proteins. Adhesion is achieved by homophilic interaction of the extracellular domains of cadherins on adjacent cells, with the cytoplasmic regions serving to couple the complex to the cytoskeleton. IQGAP1, a novel RasGAP-related protein that interacts with the cytoskeleton, binds to actin, members of the Rho family, and E-cadherin. Calmodulin binds to IQGAP1 and regulates its association with Cdc42 and actin. Here we demonstrate competition between calmodulin and E-cadherin for binding to IQGAP1 both in vitro and in a normal cellular milieu. Immunocytochemical analysis in MCF-7 (E-cadherin positive) and MDA-MB-231 (E-cadherin negative) epithelial cells revealed that E-cadherin is required for accumulation of IQGAP1 at cell-cell junctions. The cell-permeable calmodulin antagonist CGS9343B significantly increased IQGAP1 at areas of MCF-7 cell-cell contact, with a concomitant decrease in the amount of E-cadherin at cell-cell junctions. Analysis of E-cadherin function revealed that CGS9343B significantly decreased homophilic E-cadherin adhesion. On the basis of these data, we propose that disruption of the binding of calmodulin to IQGAP1 enhances the association of IQGAP1 with components of the cadherin-catenin complex at cell-cell junctions, resulting in impaired E-cadherin function.     

Endocytosis of E-cadherin regulated by Rac and Cdc42 small G proteins through IQGAP1 and actin filaments

E-cadherin is a key cell-cell adhesion molecule at adherens junctions (AJs) and undergoes endocytosis when AJs are disrupted by the action of extracellular signals. To elucidate the mechanism of this endocytosis, we developed here a new cell-free assay system for this reaction using the AJ-enriched fraction from rat liver. We found here that non-trans-interacting, but not trans-interacting, E-cadherin underwent endocytosis in a clathrin-dependent manner. The endocytosis of trans-interacting E-cadherin was inhibited by Rac and Cdc42 small G proteins, which were activated by trans-interacting E-cadherin or trans-interacting nectins, which are known to induce the formation of AJs in cooperation with E-cadherin. This inhibition was mediated by reorganization of the actin cytoskeleton by Rac and Cdc42 through IQGAP1, an actin filament-binding protein and a downstream target of Rac and Cdc42. These results indicate the important role of the Rac/Cdc42-IQGAP1 system in the dynamic organization and maintenance of the E-cadherin-based AJs.     

E-cadherin promotes EGFR-mediated cell differentiation and MUC5AC mucin expression in cultured human airway epithelial cells

In previous work, we showed that epidermal growth factor receptor (EGFR) activation causes mucin expression in airway epithelium in vivo and in human NCI-H292 airway epithelial cells and normal human bronchial epithelial (NHBE) cells in vitro. Here we show that the cell surface adhesion molecule, E-cadherin, promotes EGFR-mediated mucin production in NCI-H292 cells in a cell density- and cell cycle-dependent fashion. The addition of the EGFR ligand, transforming growth factor (TGF)-alpha, increased MUC5AC protein expression markedly in dense, but not in sparse, cultures. MUC5AC-positive cells in dense cultures contained 2 N DNA content and did not incorporate bromodeoxyuridine, suggesting that they develop via cell differentiation and that a surface molecule involved in cell-cell contact is important for EGFR-mediated mucin production. In support of this hypothesis, in dense cultures of NCI-H292 cells and in NHBE cells at air-liquid interface, blockade of E-cadherin-mediated cell-cell contacts decreased EGFR-dependent mucin production. E-cadherin blockade also increased EGFR-dependent cell proliferation and TGF-alpha-induced EGFR tyrosine phosphorylation in dense cultures of NCI-H292 cells, suggesting that E-cadherin promotes EGFR-dependent mucin production and inhibits EGFR-dependent cell proliferation via modulation of EGFR phosphotyrosine levels. Furthermore, in dense cultures, E-cadherin blockade decreased the rate of EGFR tyrosine dephosphorylation, implicating an E-cadherin-dependent protein tyrosine phosphatase in EGFR dephosphorylation. Thus E-cadherin promotes EGFR-mediated cell differentiation and MUC5AC production, and our results suggest that this occurs via a pathway involving protein tyrosine phosphatase-dependent EGFR dephosphorylation.     

Activation of Cdc42 by trans interactions of the cell adhesion molecules nectins through c-Src and Cdc42-GEF FRG

Nectins, Ca2+ -independent immunoglobulin-like cell-cell adhesion molecules, initiate cell-cell adhesion by their trans interactions and recruit cadherins to cooperatively form adherens junctions (AJs). In addition, the trans interactions of nectins induce the activation of Cdc42 and Rac small G proteins, which increases the velocity of the formation of AJs. We examined here how nectins induce the activation of Cdc42 in MDCK epithelial cells and L fibroblasts. Nectins recruited and activated c-Src at the nectin-based cell-cell adhesion sites. FRG, a GDP/GTP exchange factor specific for Cdc42, was then recruited there, tyrosine phosphorylated by c-Src, and activated, causing an increase in the GTP-bound active form of Cdc42. Inhibition of the nectin-induced activation of c-Src suppressed the nectin-induced activation of FRG and Cdc42. Inhibition of the nectin-induced activation of FRG or depletion of FRG by RNA interference suppressed the nectin-induced activation of Cdc42. These results indicate that nectins induce the activation of Cdc42 through c-Src and FRG locally at the nectin-based cell-cell adhesion sites.     

RhoA, Rac1, and Cdc42 exert distinct effects on epithelial barrier via selective structural and biochemical modulation of junctional proteins and F-actin

Epithelial intercellular junctions regulate cell-cell contact and mucosal barrier function. Both tight junctions (TJs) and adherens junctions (AJs) are regulated in part by their affiliation with the F-actin cytoskeleton. The cytoskeleton in turn is influenced by Rho family small GTPases such as RhoA, Rac1, and Cdc42, all of which constitute eukaryotic targets for several pathogenic organisms. With a tetracycline-repressible system to achieve regulated expression in Madin-Darby canine kidney (MDCK) epithelial cells, we used dominant-negative (DN) and constitutively active (CA) forms of RhoA, Rac1, and Cdc42 as tools to evaluate the precise contribution of each GTPase to epithelial structure and barrier function. All mutant GTPases induced time-dependent disruptions in epithelial gate function and distinct morphological alterations in apical and basal F-actin pools. TJ proteins occludin, ZO-1, claudin-1, claudin-2, and junctional adhesion molecule (JAM)-1 were dramatically redistributed in the presence of CA RhoA or CA Cdc42, whereas only claudins-1 and -2 were redistributed in response to CA Rac1. DN Rac1 expression also induced selective redistribution of claudins-1 and -2 in addition to JAM-1, whereas DN Cdc42 influenced only claudin-2 and DN RhoA had no effect. AJ protein localization was unaffected by any mutant GTPase, but DN Rac1 induced a reduction in E-cadherin detergent solubility. All CA GTPases increased the detergent solubility of claudins-1 and -2, but CA RhoA alone reduced claudin-2 and ZO-1 partitioning to detergent-insoluble membrane rafts. We conclude that Rho family GTPases regulate epithelial intercellular junctions via distinct morphological and biochemical mechanisms and that perturbations in barrier function reflect any imbalance in active/resting GTPase levels rather than simply loss or gain of GTPase activity. epithelium; tight junctions; paracellular permeability; Madin-Darby canine kidney cells     

Positive role of IQGAP1, an effector of Rac1, in actin-meshwork formation at sites of cell-cell contact

The small guanosine triphosphatase Rac1 is activated by E-cadherin-mediated cell-cell adhesion and is required for the accumulation of actin filaments, E-cadherin, and β-catenin at sites of cell-cell contact. However, the modes of activation and action of Rac1 remain to be clarified. We here found that suppression of IQGAP1, an actin-binding protein and an effector of Rac1, by small interfering RNA apparently reduced the accumulation of actin filaments, E-cadherin, and β-catenin at sites of cell-cell contact in Madin-Darby canine kidney II epithelial cells under the conditions in which knockdown of Rac1 reduced them. Knockdown of Rac1 did not affect the localization of these junctional components in cells expressing a constitutively active IQGAP1 mutant defective in Rac1/Cdc42 binding. Knockdown of either Rac1 or IQGAP1 accelerated the 12-O-tetradecanoylphorbol-13-acetate-induced cell-cell dissociation. The basal Rac1 activity, which was maintained by E-cadherin-mediated cell-cell adhesion, was inhibited in the IQGAP1-knocked down cells, whereas the Rac1 activity was increased in the cells overexpressing IQGAP1. Together, these results indicate that Rac1 enhances the accumulation of actin filaments, E-cadherin, and β-catenin by acting on IQGAP1 and suggest that there exists a positive feedback loop comprised of “E-cadherin-mediated cell-cell adhesion→Rac1 activation→actin-meshwork formation by IQGAP1→increasing E-cadherin-mediated cell-cell adhesion.”     

Cadherin engagement regulates Rho family GTPases.

The formation of cell-cell adherens junctions is a cadherin-mediated process associated with reorganization of the actin cytoskeleton. Because Rho family GTPases regulate actin dynamics, we investigated whether cadherin-mediated adhesion regulates the activity of RhoA, Rac1, and Cdc42. Confluent epithelial cells were found to have elevated Rac1 and Cdc42 activity but decreased RhoA activity when compared with low density cultures. Using a calcium switch method to manipulate junction assembly, we found that induction of cell-cell junctions increased Rac1 activity, and this was inhibited by E-cadherin function-blocking antibodies. Using the same calcium switch procedure, we found little effect on RhoA activity during the first hour of junction assembly. However, over several hours, RhoA activity significantly decreased. To determine whether these effects are mediated directly through cadherins or indirectly through engagement of other surface proteins downstream from junction assembly, we used a model system in which cadherin engagement is induced without cell-cell contact. For these experiments, Chinese hamster ovary cells expressing C-cadherin were plated on the extracellular domain of C-cadherin immobilized on tissue culture plates. Whereas direct cadherin engagement did not stimulate Cdc42 activity, it strongly inhibited RhoA activity but increased Rac1 activity. Deletion of the C-cadherin cytoplasmic domain abolished these effects.     

Regulation of Rho family GTPases by cell-cell and cell-matrix adhesion

Integrins and cadherins are transmembrane adhesion receptors that are necessary for cells to interact with the extracellular matrix or adjacent cells, respectively. Integrins and cadherins initiate signaling pathways that modulate the activity of Rho family GTPases. The Rho proteins Cdc42, Rac1, and RhoA regulate the actin cytoskeleton. Cdc42 and Rac1 are primarily involved in the formation of protrusive structures, while RhoA generates myosin-based contractility. Here we examine the differential regulation of RhoA, Cdc42, and Rac1 by integrin and cadherin signaling. Integrin and cadherin signaling leads to a decrease in RhoA activity and activation of Cdc42 and Rac1. When the normal RhoA suppression is antagonized or RhoA signaling is increased, cells exhibited impaired spreading on the matrix protein fibronectin and decreased cell-cell adhesion. Spreading on fibronectin and the formation of cell-cell adhesions is decreased in cells expressing dominant negative forms of Cdc42 or Rac1. These data demonstrate that integrins and cadherins regulate Rho proteins in a comparable manner and lead us to speculate that these changes in Rho protein activity participate in a feedback mechanism that promotes further cell-matrix or cell-cell interaction, respectively.     

Tumor progression: Small GTPases and loss of cell-cell adhesion

Tumor progression involves the transition from normal to malignant cells, through a series of cumulative alterations. During this process, invasive and migratory properties are acquired, enabling cells to metastasize (reach and grow in tissues far from their origin). Numerous cellular changes take place during epithelial malignancy, and disruption of E-cadherin based cell-cell adhesion is a major event. The small Rho GTPases (Rho, Rac and Cdc42) have been implicated in multiple steps during cellular transformation, including alterations on the adhesion status of the tumor cells. This review focuses on recent in vivo evidence that implicates RhoGTPases in epithelial tumor progression. In addition, we discuss different hypotheses to explain disruption of cadherin-mediated cell-cell adhesion, directly or indirectly, through activation of Rho GTPases. Understanding the molecular mechanism of how cadherin adhesion and RhoGTPases interplay in normal cells and how this balance is altered during cellular transformation will provide clues as to how to interfere with tumor progression.     

Cofilin-1 signaling mediates epithelial-mesenchymal transition by promoting actin cytoskeleton reorganization and cell-cell adhesion regulation in colorectal cancer cells

Colorectal cancer (CRC) is frequently a lethal disease because of metastasis. Actin cytoskeletal rearrangement is an essential step in cell migration during activation of the epithelial-mesenchymal transition (EMT) program, which is associated with metastatic properties of cancer cells. Cofilin-1 protein modulates actin dynamics by promoting actin treadmilling, thereby driving membrane protrusion and cell migration and invasion. However, the role of cofilin-1 during EMT in CRC is unknown. Here, we show that cofilin-1 and p-cofilin-1 have distinct subcellular distribution in EMT cells, as determined by super-resolution microscopy images, indicating distinct roles in different areas of cells. Silenced cofilin-1 cells treated with TGF-β (siCofilin-1/TGF-β) evaded p-LIMK2-p-cofilin-1 status, leading to recovery of E-cadherin and claudin-3 at the cell-cell contact and their respective protein levels, actin reorganization, and decreased mesenchymal protein level. Furthermore, siCofilin-1/TGF-β cells exhibited decreased migration and invasion rates as well as MMP-2 and -9 activity and augmented focal adhesion size. The expression of an inactive phospho-cofilin-1 mimetic (S3E) reduced E-cadherin and claudin-3 in cell-cell contacts, reduced their protein levels, and increased vimentin protein. Based on our findings, we suggest that cofilin-1 is crucial to switching from epithelial to mesenchymal-like morphology and cell migration and invasion by regulating actin cytoskeleton organization through activation of RhoA-LIMK2-cofilin-1 signaling, impacting the cell-cell adhesion organization of colon cancer cells in EMT.