FilGAP,a Rho/Rho-associated protein kinase–regulated GTPase-activating protein for Rac,controls tumor cell migration

Tumor cells exhibit two interconvertible modes of cell motility referred to as mesenchymal and amoeboid migration. Mesenchymal mode is characterized by elongated morphology that requires high GTPase Rac activation, whereas amoeboid mode is dependent on actomyosin contractility induced by Rho/Rho-associated protein kinase (ROCK) signaling. While elongated morphology is driven by Rac-induced protrusion at the leading edge, how Rho/ROCK signaling controls amoeboid movement is not well understood. We identified FilGAP, a Rac GTPase-activating protein (GAP), as a mediator of Rho/ROCK-dependent amoeboid movement of carcinoma cells. We show that depletion of endogenous FilGAP in carcinoma cells induced highly elongated mesenchymal morphology. Conversely, forced expression of FilGAP induced a round/amoeboid morphology that requires Rho/ROCK-dependent phosphorylation of FilGAP. Moreover, depletion of FilGAP impaired breast cancer cell invasion through extracellular matrices and reduced tumor cell extravasation in vivo. Thus phosphorylation of FilGAP by ROCK appears to promote amoeboid morphology of carcinoma cells, and FilGAP contributes to tumor invasion.     

Hic-5-Reduced Cell Spreading on Fibronectin: Competitive Effects between Paxillin and Hic-5 through Interaction with Focal Adhesion Kinase

Hic-5 is a paxillin homologue that is localized to focal adhesion complexes. Hic-5 and paxillin share structural homology and interacting factors such as focal adhesion kinase (FAK), Pyk2/CAKbeta/RAFTK, and PTP-PEST. Here, we showed that Hic-5 inhibits integrin-mediated cell spreading on fibronectin in a competitive manner with paxillin in NIH 3T3 cells. The overexpression of Hic-5 sequestered FAK from paxillin, reduced tyrosine phosphorylation of paxillin and FAK, and prevented paxillin-Crk complex formation. In addition, Hic-5-mediated inhibition of spreading was not observed in mouse embryo fibroblasts (MEFs) derived from FAK(-/-) mice. The activity of c-Src following fibronectin stimulation was decreased by about 30% in Hic-5-expressing cells, and the effect of Hic-5 was restored by the overexpression of FAK and the constitutively active forms of Rho-family GTPases, Rac1 V12 and Cdc42 V12, but not RhoA V14. These observations suggested that Hic-5 inhibits cell spreading through competition with paxillin for FAK and subsequent prevention of downstream signal transduction. Moreover, expression of antisense Hic-5 increased spreading in primary MEFs. These results suggested that the counterbalance of paxillin and Hic-5 expression may be a novel mechanism regulating integrin-mediated signal transduction.     

Hic-5 promotes endothelial cell migration to lysophosphatidic acid

Endothelial cell migration is critical for proper blood vessel development. Signals from growth factors and matrix proteins are integrated through focal adhesion proteins to alter cell migration. Hydrogen peroxide-inducible clone 5 (Hic-5), a paxillin family member, is enriched in the focal adhesions in bovine pulmonary artery endothelial (BPAE) cells, which migrate to lysophosphatidic acid (LPA) on denatured collagen. In this study, we investigate the role of Hic-5 in LPA-stimulated endothelial cell migration. LPA recruits Hic-5 to the focal adhesions and to the pseudopodia in BPAE cells plated on collagen, suggesting that recruitment of Hic-5 to focal adhesions is associated with endothelial cell migration. Knockdown of endogenous Hic-5 significantly decreases migration toward LPA, confirming involvement of Hic-5 in migration. To address the role of Hic-5 in endothelial cell migration, we exogenously expressed wild-type (WT) Hic-5 and green fluorescent protein Hic-5 C369A/C372A (LIM3 mutant) constructs in BPAE cells. WT Hic-5 expression increases chemotaxis of BPAE cells to LPA, whereas migration toward LPA of the green fluorescent protein Hic-5 C369A/C372A-expressing cells is similar to that shown in vector control cells. Additionally, ERK phosphorylation is enhanced in the presence of LPA in WT Hic-5 cells. A pharmacological inhibitor of MEK activity inhibits LPA-stimulated WT Hic-5 cell migration and ERK phosphorylation, suggesting Hic-5 enhances migration via MEK activation of ERK. Together, these studies indicate that Hic-5, a focal adhesion protein in endothelial cells, is recruited to the pseudopodia in the presence of LPA and enhances migration.     

Agrin regulates CLASP2-mediated capture of microtubules at the neuromuscular junction synaptic membrane

Transforming growth factor β (TGF-β)-stimulated epithelial-mesenchymal transition (EMT) is an important developmental process that has also been implicated in increased cell invasion and metastatic potential of cancer cells. Expression of the focal adhesion protein Hic-5 has been shown to be up-regulated in epithelial cells in response to TGF-β. Herein, we demonstrate that TGF-β-induced Hic-5 up-regulation or ectopic expression of Hic-5 in normal MCF10A cells promoted increased extracellular matrix degradation and invasion through the formation of invadopodia. Hic-5 was tyrosine phosphorylated in an Src-dependent manner after TGF-β stimulation, and inhibition of Src activity or overexpression of a Y38/60F nonphosphorylatable mutant of Hic-5 inhibited matrix degradation and invasion. RhoC, but not RhoA, was also required for TGF-β- and Hic-5-induced matrix degradation. Hic-5 also induced matrix degradation, cell migration, and invasion in the absence of TGF-β via Rac1 regulation of p38 MAPK. These data identify Hic-5 as a critical mediator of TGF-β-stimulated invadopodia formation, cell migration, and invasion.     

Expression of Focal Adhesion Proteins in the Developing Rat Kidney

Focal adhesions play a critical role as centers that transduce signals by cell-matrix interactions and regulate fundamental processes such as proliferation, migration, and differentiation. Focal adhesion kinase (FAK), paxillin, integrin-linked kinase (ILK), and hydrogen peroxide–inducible clone-5 (Hic-5) are major proteins that contribute to these events. In this study, we investigated the expression of focal adhesion proteins in the developing rat kidney. Western blotting analysis revealed that the protein levels of FAK, p-FAK³⁹⁷, paxillin, p-paxillin¹¹⁸, and Hic-5 were high in embryonic kidneys, while ILK expression persisted from the embryonic to the mature stage. Immunohistochemistry revealed that FAK, p-FAK³⁹⁷, paxillin, and p-paxillin¹¹⁸ were strongly expressed in condensed mesenchymal cells and the ureteric bud. They were detected in elongating tubules and immature glomerular cells in the nephrogenic zone. Hic-5 was predominantly expressed in mesenchymal cells as well as immature glomerular endothelial and mesangial cells, suggesting that Hic-5 might be involved in mesenchymal cell development. ILK expression was similar to that of FAK in the developmental stages. Interestingly, ILK was strongly expressed in podocytes in mature glomeruli. ILK might play a role in epithelial cell differentiation as well as kidney growth and morphogenesis. In conclusion, the temporospatially regulated expression of focal adhesion proteins during kidney development might play a role in morphogenesis and cell differentiation.     

DOCK10-mediated Cdc42 activation is necessary for amoeboid invasion of melanoma cells

BACKGROUND: Tumor cells can move in a three-dimensional (3D) environment in either mesenchymal-type or amoeboid modes. In mesenchymal-type movement, cells have an elongated morphology with Rac-induced protrusions at the leading edge. Amoeboid cells have high levels of actomyosin contractility, and movement is associated with deformation of the cell body through the matrix without proteolysis. Because signaling pathways that control the activation of GTPases for amoeboid movement are poorly understood, we sought to identify regulators of amoeboid movement by screening an siRNA library targeting guanine nucleotide exchange factors (GEFs) for Rho-family GTPases. RESULTS: We identified DOCK10, a Cdc42 GEF, as a key player in amoeboid migration; accordingly, we find that expression of activated Cdc42 induces a mesenchymal-amoeboid transition and increases cell invasion. Silencing DOCK10 expression promotes conversion to mesenchymal migration and is associated with decreased MLC2 phosphorylation and increased Rac1 activation. Consequently, abrogating DOCK10 and Rac1 expression suppresses both amoeboid and mesenchymal migration and results in decreased invasion. We show that the Cdc42 effectors N-WASP and Pak2 are required for the maintenance of the rounded-amoeboid phenotype. Blocking Cdc42 results in loss of mesenchymal morphology, arguing that Cdc42 is also involved in mesenchymal morphology through different activation and effector pathways. CONCLUSIONS: Previous work has identified roles of Rho and Rac signaling in tumor cell movement, and we now elucidate novel roles of Cdc42 signaling in amoeboid and mesenchymal movement and tumor cell invasion.     

Specific decrease in the level of Hic-5, a focal adhesion protein, during immortalization of mouse embryonic fibroblasts, and its association with focal adhesion kinase

Hic-5 is a paxillin homologue with four LIM domains in its C-terminal region, localized mainly in focal adhesions in normal fibroblasts. Hic-5 is also known to associate with focal adhesion kinase (FAK) or the related CAKbeta, and with vinculin. In the present study, we examined changes in Hic-5 and paxillin protein levels in primary mouse embryo fibroblasts (MEF) during mortal and immortal stages. The Hic-5 level was markedly decreased when cells became immortalized, whereas that of paxillin was increased. The vinculin level was not changed significantly. Hic-5 was mainly localized in focal adhesion plaques of mortal MEF but was localized in the nuclear periphery in the immortalized MEF; the number of focal adhesion plaques was decreased in these cells. Mouse Hic-5 contains three LD domains in its N-terminal half, and the first LD domain (LD1) appears to be involved in interaction with FAK. However, this interaction was not essential for recruitment of Hic-5 to focal adhesions, since its subcellular localization was similar in FAK(-/-) cells. Forced expression of Hic-5 decreased colony forming ability of MEF from FAK(+/+) mice, but not of FAK(-/-) cells. These observations suggested the involvement of Hic-5 in determination of cellular proliferative capacity in collaboration with other cytoskeletal components.     

Hic-5, a paxillin homologue, binds to the protein-tyrosine phosphatase PEST (PTP-PEST) through its LIM 3 domain

The Hic-5 protein is encoded by a transforming growth factor-beta1- and hydrogen peroxide-inducible gene, hic-5, and has striking similarity to paxillin, especially in their C-terminal LIM domains. Like paxillin, Hic-5 is localized in focal adhesion plaques in association with focal adhesion kinase in cultured fibroblasts. We carried out yeast two-hybrid screening to identify cellular factors that form a complex with Hic-5 using its LIM domains as a bait, and we identified a cytoplasmic tyrosine phosphatase (PTP-PEST) as one of the partners of Hic-5. These two proteins are associated in mammalian cells. From in vitro binding experiments using deletion and point mutations, it was demonstrated that the essential domain in Hic-5 for the binding was LIM 3. As for PTP-PEST, one of the five proline-rich sequences found on PTP-PEST, Pro-2, was identified as the binding site for Hic-5 in in vitro binding assays. Paxillin also binds to the Pro-2 domain of PTP-PEST. In conclusion, Hic-5 may participate in the regulation of signaling cascade through its interaction with distinct tyrosine kinases and phosphatases.     

Initial steps of metastasis: cell invasion and endothelial transmigration

Metastasis is the leading cause of cancer mortality. The metastatic cascade represents a multi-step process which includes local tumor cell invasion, entry into the vasculature followed by the exit of carcinoma cells from the circulation and colonization at the distal sites. At the earliest stage of successful cancer cell dissemination, the primary cancer adapts the secondary site of tumor colonization involving the tumor-stroma crosstalk. The migration and plasticity of cancer cells as well as the surrounding environment such as stromal and endothelial cells are mandatory. Consequently, the mechanisms of cell movement are of utmost relevance for targeted intervention of which three different types have been reported. Tumor cells can migrate either collectively, in a mesenchymal or in an amoeboid type of movement and intravasate the blood or lymph vasculature. Intravasation by the interaction of tumor cells with the vascular endothelium is mechanistically poorly understood. Changes in the epithelial plasticity enable carcinoma cells to switch between these types of motility. The types of migration may change depending on the intervention thereby increasing the velocity and aggressiveness of invading cancer cells. Interference with collective or mesenchymal cell invasion by targeting integrin expression or metalloproteinase activity, respectively, resulted in an amoeboid cell phenotype as the ultimate exit strategy of cancer cells. There are little mechanistic details reported in vivo showing that the amoeboid behavior can be either reversed or efficiently inhibited. Future concepts of metastasis intervention must simultaneously address the collective, mesenchymal and amoeboid mechanisms of cell invasion in order to advance in anti-metastatic strategies as these different types of movement can coexist and cooperate. Beyond the targeting of cell movements, the adhesion of cancer cells to the stroma in heterotypic circulating tumor cell emboli is of paramount relevance for anti-metastatic therapy.     

The activated insulin-like growth factor I receptor induces depolarization in breast epithelial cells characterized by actin filament disassembly and tyrosine dephosphorylation of FAK, Cas, and paxillin.

Insulin-like growth factor I (IGF-I) promotes the motility of different cell types. We investigated the role of IGF-I receptor (IGF-IR) signaling in locomotion of MCF-7 breast cancer epithelial cells overexpressing the wild-type IGF-IR (MCF-7/IGF-IR). Stimulation of MCF-7/IGF-IR cells with 50 ng/ml IGF-I induced disruption of the polarized cell monolayer followed by morphological transition toward a mesenchymal phenotype. Immunofluorescence staining of the cells with rhodamine-phalloidin revealed rapid disassembly of actin fibers and development of a cortical actin meshwork. Activation of phosphatidylinositol (PI)3-kinase downstream of the IGF-IR was necessary for this process, as blocking PI 3-kinase activity with the specific inhibitor LY 294002 at 10 microM prevented disruption of the filamentous actin. In parallel, IGF-IR activation induced rapid and transient tyrosine dephosphorylation of focal adhesion proteins p125 focal adhesion kinase (FAK), p130 Crk-associated substrate (Cas), and paxillin. This process required phosphotyrosine phosphatase (PTP) activity, since pretreatment of the cells with 5 microM phenylarsine oxide (PAO), an inhibitor of PTPs, rescued FAK and its associated proteins Cas and paxillin from IGF-I-induced dephosphorylation. In addition, PAO-pretreated cells were refractory to IGF-I-induced morphological transition. Thus, our findings reveal a new function of the IGF-IR, the ability to depolarize epithelial cells. In MCF-7 cells, mechanisms of IGF-IR-mediated cell depolarization involve PI 3-kinase signaling and putative PTP activities.     

Hic-5 interacts with GIT1 with a different binding mode from paxillin

Hic-5, a member of the paxillin family of adaptor molecules, is localized at focal adhesion and implicated in integrin-mediated signaling. Hic-5 and paxillin exhibit structural homology and share interacting factors, however, diverse functions are suggested for them. In this study, we carried out yeast two-hybrid screening to identify Hic-5 interacting factors using its LD3-4 region, which includes the Hic-5-specific amino acid sequence, as a bait. Through the screening, we identified GIT1, an Arf GTPase-activating protein, as a Hic-5 binding protein. The interaction of these two proteins was mediated by the LD3 motif of Hic-5 and the C-terminal region, which includes a paxillin-binding subdomain, of GIT1. Although GIT1 is known as a paxillin-binding protein, we only observed weak association of paxillin with GIT1 in the overexpression system. In contrast, Hic-5 firmly bound to GIT1 under the same conditions. In addition, the paxillin/GIT1 complex contained PIX, a guanine nucleotide exchange factor, whereas the Hic-5/GIT1 complex contained a smaller amount of PIX. These results suggested that paxillin and Hic-5 associate with GIT1 with different binding modes, and that the Hic-5 complex possesses static features compared with the paxillin complex, which contains both positive and negative regulators of GTPases involved in actin dynamics. Moreover, Hic-5-mediated inhibition of cell spreading was restored by co-expression of the C-terminal fragment of GIT1, which perturbs the interaction of Hic-5 with endogenous GIT1. Thus, it was demonstrated that Hic-5 and GIT1 interact functionally in addition to showing a physical association.     

Regulation of Focal Adhesion Kinase Activation,Breast Cancer Cell Motility,and Amoeboid Invasion by the RhoA Guanine Nucleotide Exchange Factor Net1

Net1 is a RhoA guanine nucleotide exchange factor (GEF) that is overexpressed in a subset of human cancers and contributes to cancer cell motility and invasion in vitro. However, the molecular mechanism accounting for its role in cell motility and invasion has not been described. In the present work, we show that expression of both Net1 isoforms in breast cancer cells is required for efficient cell motility. Although loss of Net1 isoform expression only partially blocks RhoA activation, it inhibits lysophosphatidic acid (LPA)-stimulated migration as efficiently as knockdown of RhoA itself. However, we demonstrate that the Net1A isoform predominantly controls myosin light-chain phosphorylation and is required for trailing edge retraction during migration. Net1A interacts with focal adhesion kinase (FAK), localizes to focal adhesions, and is necessary for FAK activation and focal adhesion maturation during cell spreading. Net1A expression is also required for efficient invasion through a Matrigel matrix. Analysis of invading cells demonstrates that Net1A is required for amoeboid invasion, and loss of Net1A expression causes cells to shift to a mesenchymal phenotype characterized by high β₁-integrin activity and membrane type 1 matrix metalloproteinase (MT1-MMP) expression. These results demonstrate a previously unrecognized role for the Net1A isoform in controlling FAK activation during planar cell movement and amoeboid motility during extracellular matrix (ECM) invasion.     

Reduced expression of the ROCK inhibitor Rnd3 is associated with increased invasiveness and metastatic potential in mesenchymal tumor cells

Background

Mesenchymal and amoeboid movements are two important mechanisms adopted by cancer cells to invade the surrounding environment. Mesenchymal movement depends on extracellular matrix protease activity, amoeboid movement on the RhoA-dependent kinase ROCK. Cancer cells can switch from one mechanism to the other in response to different stimuli, limiting the efficacy of antimetastatic therapies.

Methodology and Principal Findings

We investigated the acquisition and molecular regulation of the invasion capacity of neoplastically transformed human fibroblasts, which were able to induce sarcomas and metastases when injected into immunocompromised mice. We found that neoplastic transformation was associated with a change in cell morphology (from fibroblastic to polygonal), a reorganization of the actin cytoskeleton, a decrease in the expression of several matrix metalloproteases and increases in cell motility and invasiveness. In a three-dimensional environment, sarcomagenic cells showed a spherical morphology with cortical actin rings, suggesting a switch from mesenchymal to amoeboid movement. Accordingly, cell invasion decreased after treatment with the ROCK inhibitor Y27632, but not with the matrix protease inhibitor Ro 28-2653. The increased invasiveness of tumorigenic cells was associated with reduced expression of Rnd3 (also known as RhoE), a cellular inhibitor of ROCK. Indeed, ectopic Rnd3 expression reduced their invasive ability in vitro and their metastatic potential in vivo.

Conclusions

These results indicate that, during neoplastic transformation, cells of mesenchymal origin can switch from a mesenchymal mode of movement to an amoeboid one. In addition, they point to Rnd3 as a possible regulator of mesenchymal tumor cell invasion and to ROCK as a potential therapeutic target for sarcomas.     

Tyrosine-phosphorylated Hic-5 inhibits epidermal growth factor-induced lamellipodia formation

The focal adhesion protein Hic-5, a homologue to paxillin, has been shown to be tyrosine-phosphorylated in fibroblasts in response to stimuli such as osmotic stress, serum, LPA and endothelin. However, the function of this modification to Hic-5 is unclear. Herein, we show that Hic-5 is tyrosine-phosphorylated on residues 38 and 60 following epidermal growth factor (EGF) treatment of COS-7 cells, coincident with an increase in peripheral actin reorganization. To explore the role of Hic-5 phosphorylation in this process, we introduced wild-type (WT) and mutant Hic-5 constructs into COS-7 cells and determined that EGF-induced lamellipodia formation was suppressed by WT Hic-5. This effect required localization to focal adhesions as well as phosphorylation of Hic-5 as overexpression of both a non-targeting and a non-phosphorylatable Hic-5 failed to inhibit peripheral actin reorganization. Interestingly, overexpression of non-phosphorylatable Y31/118F or WT paxillin did not affect lamellipodia formation, indicating that this effect is specific to Hic-5. The EGF-induced lamellipodia were Rac-dependent and overexpressed WT Hic-5, but not non-phosphorylatable Hic-5 inhibited Rac activation. Our data suggest that Hic-5 tyrosine phosphorylation functions to regulate signaling associated with lamellipodia formation, a process fundamental to cell motility.     

LIM-kinase is critical for the mesenchymal-to-amoeboid cell morphological transition in 3D matrices

Tumor cells can migrate in 3D matrices in either a mesenchymal-like or amoeboid mode. HT1080 fibrosarcoma cells cultured in 3D collagen gels change their morphology from mesenchymal-like (elongated) to amoeboid (round) following protease inhibitor (PI) treatment or active Rho or ROCK expression. In this study, we examined the role of LIM-kinase 1 (LIMK1) in the PI- or Rho/ROCK-induced cell morphological change. We showed that LIMK1 was activated after PI treatment of HT1080 cells in 3D collagen gels and this activation was blocked by a ROCK inhibitor. While overexpression of LIMK1 induced cell rounding, knockdown of LIMK1 or the expression of kinase-inactive LIMK1 suppressed PI- or Rho/ROCK-induced cell rounding. These results suggest that LIMK1 plays an essential role in the PI- or Rho/ROCK-induced mesenchymal-to-amoeboid cell morphological transition of HT1080 cells cultured in 3D collagen gels. Furthermore, LIMK1 knockdown suppressed the invasive activity of HT1080 cells in collagen gels with or without PIs, indicating that LIMK1 mediates both the mesenchymal and amoeboid modes of invasion of HT1080 cells.     

ROCK signaling mediates the adoption of different modes of migration and invasion in human mammary epithelial tumor cells

For the invasive migration of tumor cells, at least two mechanisms are currently discussed: (1) the mesenchymal mode depending on extracellular proteolysis and (2) the proteolysis-independent amoeboid mode depending on the activity of the Rho kinase ROCK. The ability of tumor cells to switch between different modes of motility has been shown to limit the efficiency of agents aimed to reduce invasion. Here we show by combining 2D and 3D migration assays that human mammary tumor cells exhibited a strongly reduced migration velocity as compared to their normal counterparts indicating that high invasiveness is not necessarily correlated with high migratory capacity in 2D assays. This reduced migration was apparently due to significant differences in actin organization, decreased persistence of lamellipodia by 50% and increased cell substrate adhesion. These differences resulted from a 2.5-fold higher activity of ROCK and were mediated by its downstream effectors myosin light chain kinase and cofilin. Thus, inhibition of ROCK activity caused a marked increase in 2D migration efficiency by 40%, without, however, affecting 3D invasion. A massive reduction of invasion by 60% was achieved by the simultaneous inhibition of the ROCK-dependent amoeboid and the extracellular proteolysis-dependent mesenchymal mode. These results may point to a new efficient strategy for blocking tumor cell invasion in vivo.     

Paxillin phosphorylation and complexing with Erk and FAK are regulated by PLD activity in MDA-MB-231 cells

MDA-MB-231 cells are highly aggressive human breast adenocarcinoma cells that depend on PLD activity for survival. In response to the stress of serum withdrawal, there is increased motility and invasiveness of these cells that is associated with a rapid increase in PLD activity. In addition, PLD activity is elevated in response to most mitogenic signals. Similar to PLD, paxillin, a focal adhesion adaptor protein, and Erk, mitogen-activated protein kinase, play vital roles in cell motility through regulation of focal adhesion dynamics. Here, we addressed whether there is a functional correlation between paxillin and PLD that may influence cancer cell motility. We investigated the role of PLD activity on paxillin regulation, Erk activation and formation of a paxillin-Erk and paxillin-FAK association. Inhibition of PLD activity led to an increase in paxillin tyrosine phosphorylation, a decrease in Erk activation, as measured by phosphorylation, and enhanced association of paxillin with Erk. In addition, we found that paxillin tyrosine phosphorylation depends upon Erk activity and may be a consequence of an increased association with FAK. Taken together, these results suggest that Erk activity is governed by PLD activity and regulates the tyrosine phosphorylation of paxillin, potentially explaining its role in cell motility. This study indicated that PLD, Erk, paxillin and FAK participate in the same signaling pathway in this breast cancer cell line.