Phosphorylation Of Focal Adhesion Proteins Research Articles

Abstract 2406: Mechanism of mSin1 regulation of cell migration

Abstract Studies have shown that mSin1, a major component of mTORC2, plays an important role in tumor cell migration and invasion as well as cancer metastasis. However, how mSin1 regulates cell migration is poorly understood. In this study, we found that knockout of mSin1 suppressed cell spreading and migration in mouse embryo fibroblasts (MEFs). Similarly, knockdown of mSin1 also inhibited cell spreading and migration in rhabdomyosarcoma (Rh30) and cervical cancer (HeLa) cells. Furthermore, we observed that knockout or knockdown of mSin1 reduced the activities of the small GTPases (RhoA and Rac1) in the cells. Ectopic expression of constitutively active Rac1, but not RhoA, partially attenuated the reduced cell spreading and migration induced by mSin1 deficiency. In addition, depletion of mSin1 remarkedly inhibited the phosphorylation of focal adhesion proteins, including focal adhesion kinase (FAK) at Tyr397 and paxillin at Tyr118 in the cells. The results suggest that mSin1 controls cell spreading and migration at least partly by mediating the activity of Rac1 and the phosphorylation of FAK. (Supported by the Feist-Weiller Cancer Center, LSU Health Sciences Center, Shreveport, LA, USA) Citation Format: Shile Huang, Lei Liu, Yan Luo. Mechanism of mSin1 regulation of cell migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2406.

Abstract 480: Non-muscle Myosin II Regulates Aortic Stiffness via Tension-dependent Phosphorylation of Specific Focal Adhesion Proteins

Non-muscle myosin II plays a role in many fundamental cellular processes including cell adhesion, migration, and cytokinesis. However, its role in vascular function is not well understood. Here, we investigated the function of non-muscle myosin II in the biomechanical properties of mouse proximal aorta. We found that blebbistatin, a specific inhibitor of non-muscle myosin II decreases agonist-induced aortic stress and stiffness in a dose-dependent manner. We also specifically demonstrate, in freshly isolated contractile aortic smooth muscle cells, using deconvolution microscopy that the NM myosin IIA isoform co-localizes with contractile filaments in the core of the cell as well as in the non-muscle cell cortex. However, the NM myosin IIB isoform is only colocalized with contractile filaments, and is excluded from the cell cortex. Furthermore, both the siRNA knockdown of NMIIA and NMIIB isoforms in a differentiated smooth muscle cell line A7r5 and blebbistatin-mediated inhibition of NM myosin II suppresses agonist-activated increases in phosphorylation of FAK Y925 and paxillin Y118. Thus, in the present study, we show, for the first time, that NM myosin II regulates aortic stiffness and that this regulation is mediated at least in part through the tension-dependent phosphorylation of focal adhesion proteins FAK and paxillin.

Migration regulates cellular mechanical states.

Recent studies indicate that adherent cells are keenly sensitive to external physical environment, such as substrate rigidity and topography, and internal physical states, such as cell shape and spreading area. Many of these responses are believed to involve coupled output and input of mechanical forces, which may constitute the key sensing mechanism to generate downstream regulatory signals for cell growth and differentiation. Here, we show that the state of cell migration also plays a regulatory role. Compared with migrating cells, stationary cells generate stronger, less dynamic, and more peripherally localized traction forces. These changes are coupled to reduced focal adhesion turnover and enhanced paxillin phosphorylation. Further, using cells migrating along checkerboard micropatterns, we show that the appearance of new focal adhesions directly in front of existing focal adhesions is associated with the down-regulation of existing focal adhesions and associated traction forces. Together, our results imply a mechanism where cell migration regulates traction forces by promoting dynamic turnover of focal adhesions, which may then regulate processes such as wound healing and embryogenesis where cell differentiation must coordinate with migration state and proper localization.

Selective Role of Vinculin in Contractile Mechanisms of Endothelial Permeability.

Increased vascular endothelial cell (EC) permeability is a result of intercellular gap formation that may be induced by contraction-dependent and contraction-independent mechanisms. This study investigated a role of the adaptor protein vinculin in EC permeability induced by contractile (thrombin) and noncontractile (IL-6) agonists. Although thrombin and IL-6 caused a similar permeability increase in human pulmonary ECs and disrupted the association between vinculin and vascular endothelial-cadherin, they induced different patterns of focal adhesion (FA) arrangement. Thrombin, but not IL-6, caused formation of large, vinculin-positive FAs, phosphorylation of FA proteins, FA kinase and Crk-associated substrate, and increased vinculin-talin association. Thrombin-induced formation of talin-positive FA and intercellular gaps were suppressed in ECs with small interfering RNA-induced vinculin knockdown. Vinculin knockdown and inhibitors of Rho kinase and myosin-II motor activity also attenuated thrombin-induced EC permeability. Importantly, ectopic expression of the vinculin mutant lacking the F-actin-binding domain decreased thrombin-induced Rho pathway activation and EC permeability. In contrast, IL-6-induced EC permeability did not involve RhoA- or myosin-dependent mechanisms but engaged Janus kinase/signal transducer and activator of transcription-mediated phosphorylation and internalization of vascular endothelial-cadherin. This process was vinculin independent but Janus kinase/tyrosine kinase Src-dependent. These data suggest that vinculin participates in a contractile-dependent mechanism of permeability by integrating FA with stress fibers, leading to maximal RhoA activation and EC permeability response. Vinculin inhibition does not affect contractile-independent mechanisms of EC barrier failure. This study provides, for the first time, a comparative analysis of two alternative mechanisms of vascular endothelial barrier dysfunction and defines a specific role for vinculin in the contractile type of permeability response.

The ubiquitin-proteasome system regulates focal adhesions at the leading edge of migrating cells.

Cell migration requires the cyclical assembly and disassembly of focal adhesions. Adhesion induces phosphorylation of focal adhesion proteins, including Cas (Crk-associated substrate/p130Cas/BCAR1). However, Cas phosphorylation stimulates adhesion turnover. This raises the question of how adhesion assembly occurs against opposition from phospho-Cas. Here we show that suppressor of cytokine signaling 6 (SOCS6) and Cullin 5, two components of the CRL5SOCS6 ubiquitin ligase, inhibit Cas-dependent focal adhesion turnover at the front but not rear of migrating epithelial cells. The front focal adhesions contain phospho-Cas which recruits SOCS6. If SOCS6 cannot access focal adhesions, or if cullins or the proteasome are inhibited, adhesion disassembly is stimulated. This suggests that the localized targeting of phospho-Cas within adhesions by CRL5SOCS6 and concurrent cullin and proteasome activity provide a negative feedback loop, ensuring that adhesion assembly predominates over disassembly at the leading edge. By this mechanism, ubiquitination provides a new level of spatio-temporal control over cell migration.

Substrate stiffness modulates lung cancer cell migration but not epithelial to mesenchymal transition.

Biomechanical properties of the tumor microenvironment, including matrix/substrate stiffness, play a significant role in tumor evolution and metastasis. Epithelial to Mesenchymal Transition (EMT) is a fundamental biological process that is associated with increased cancer cell migration and invasion. The goal of this study was to investigate (1) how substrate stiffness modulates the migration behaviors of lung adenocarcinoma cells (A549) and (2) if stiffness-induced changes in cell migration correlate with biochemical markers of EMT. Collagen-coated polydimethylsiloxane (PDMS) substrates and an Ibidi migration assay were used to investigate how substrate stiffness alters the migration patterns of A549 cells. RT-PCR, western blotting and immunofluorescence were used to investigate how substrate stiffness alters biochemical markers of EMT, that is, E-cadherin and N-cadherin, and the phosphorylation of focal adhesion proteins. Increases in substrate stiffness led to slower, more directional migration but did not alter the biochemical markers of EMT. Interestingly, growth factor (i.e., Transforming Growth Factor-β) stimulation resulted in similar levels of EMT regardless of substrate stiffness. We also observed decreased levels of phosphorylated focal adhesion kinase (FAK) and paxillin on stiffer substrates which correlated with slower cell migration. These results indicate that substrate stiffness modulates lung cancer cell migration via focal adhesion signaling as opposed to EMT signaling.

Displacement of p130Cas from focal adhesions links actomyosin contraction to cell migration

Cell adhesion complexes provide platforms where cell-generated forces are transmitted to the extracellular matrix (ECM). Tyrosine phosphorylation of focal adhesion proteins is crucial for cells to communicate with the extracellular environment. However, the mechanisms that transmit actin cytoskeletal motion to the extracellular environment to drive cell migration are poorly understood. We find that the movement of p130Cas (Cas, also known as BCAR1), a mechanosensor at focal adhesions, correlates with actin retrograde flow and depends upon actomyosin contraction and phosphorylation of the Cas substrate domain (CasSD). This indicates that CasSD phosphorylation underpins the physical link between Cas and the actin cytoskeleton. Fluorescence recovery after photobleaching (FRAP) experiments reveal that CasSD phosphorylation, as opposed to the association of Cas with Src, facilitates Cas displacement from adhesion complexes in migrating cells. Furthermore, the stabilization of Src-Cas binding and inhibition of myosin II, both of which sustain CasSD phosphorylation but mitigate Cas displacement from adhesion sites, retard cell migration. These results indicate that Cas promotes cell migration by linking actomyosin contractions to the adhesion complexes through a dynamic interaction with Src as well as through the phosphorylation-dependent association with the actin cytoskeleton.

Displacement of p130Cas from focal adhesions links actomyosin contraction to cell migration.

Cell adhesion complexes provide platforms where cell-generated forces are transmitted to the extracellular matrix (ECM). Tyrosine phosphorylation of focal adhesion proteins is crucial for cells to communicate with the extracellular environment. However, the mechanisms that transmit actin cytoskeletal motion to the extracellular environment to drive cell migration are poorly understood. We find that the movement of p130Cas (Cas, also known as BCAR1), a mechanosensor at focal adhesions, correlates with actin retrograde flow and depends upon actomyosin contraction and phosphorylation of the Cas substrate domain (CasSD). This indicates that CasSD phosphorylation underpins the physical link between Cas and the actin cytoskeleton. Fluorescence recovery after photobleaching (FRAP) experiments reveal that CasSD phosphorylation, as opposed to the association of Cas with Src, facilitates Cas displacement from adhesion complexes in migrating cells. Furthermore, the stabilization of Src-Cas binding and inhibition of myosin II, both of which sustain CasSD phosphorylation but mitigate Cas displacement from adhesion sites, retard cell migration. These results indicate that Cas promotes cell migration by linking actomyosin contractions to the adhesion complexes through a dynamic interaction with Src as well as through the phosphorylation-dependent association with the actin cytoskeleton.

Heparin‐integrin interaction in endothelial cells: Downstream signaling and heparan sulfate expression

Endothelial cells (ECs) are a source of physiologically important molecules that are synthesized and released to the blood and/or to the subendothelial extracellular matrix such as a heparan sulfate proteoglycan (HSPG) with antithrombotic properties. Previously, we have shown that heparin stimulates the synthesis and modifies the sulfation pattern of this HSPG. Here the molecular mechanisms involved in the up-regulation of HSPG synthesis by heparin in endothelial cells were decoded. The cells were stimulated with heparin and the expression of HSPG and intracellular pathways were evaluated by a combination of methods involving confocal microscopy, flow cytometry, Western blotting analyses, and [(35) S]-sulfate metabolically labeling of the cells. We observed that the up-regulation of HSPG synthesis evoked by heparin is dependent on the interaction of heparin with integrin since RGD peptide abolishes the effect. The activation of integrin leads to tyrosine-phosphorylation of focal adhesion-associated proteins such as FAK, Src, and paxillin. In addition, heparin induces ERK1/2 phosphorylation and inhibitors of Ras and MEK decreased heparin-dependent HSPG synthesis. Moreover, heparin also induced intracellular Ca(2+) release, PLCγ1 (phospholipase Cγ1) and CaMKII (calcium calmodulin kinase II) activation, as well as an increase in nitric oxide (NO) production. Finally, an intracellular Ca(2+) chelator, Ca(2+) signaling inhibitors, and an endothelial NO synthase inhibitor were all able to abolish the effect in heparan sulfate synthesis. In conclusion, the heparin-induced up-regulation of HSPG expression is associated with the phosphorylation of focal adhesion proteins and Ras/Raf/MEK/ERK MAP and Ca(2+) /NO pathways.

Phosphorylation of RACK1 on Tyrosine 52 by c-Abl Is Required for Insulin-like Growth Factor I-mediated Regulation of Focal Adhesion Kinase

Focal Adhesion Kinase (FAK) activity is controlled by growth factors and adhesion signals in tumor cells. The scaffolding protein RACK1 (receptor for activated C kinases) integrates insulin-like growth factor I (IGF-I) and integrin signaling, but whether RACK1 is required for FAK function is unknown. Here we show that association of FAK with RACK1 is required for both FAK phosphorylation and dephosphorylation in response to IGF-I. Suppression of RACK1 by small interfering RNA ablates FAK phosphorylation and reduces cell adhesion, cell spreading, and clonogenic growth. Peptide array and mutagenesis studies localize the FAK binding interface to blades I-III of the RACK1 beta-propeller and specifically identify a set of basic and hydrophobic amino acids (Arg-47, Tyr-52, Arg-57, Arg-60, Phe-65, Lys-127, and Lys-130) as key determinants for association with FAK. Mutation of tyrosine 52 alone is sufficient to disrupt interaction of RACK1 with FAK in cells where endogenous RACK1 is suppressed by small interfering RNA. Cells expressing a Y52F mutant RACK1 are impaired in adhesion, growth, and foci formation. Comparative analyses of homology models and crystal structures for RACK1 orthologues suggest a role for Tyr-52 as a site for phosphorylation that induces conformational change in RACK1, switching the protein into a FAK binding state. Tyrosine 52 is further shown to be phosphorylated by c-Abl kinase, and the c-Abl inhibitor STI571 disrupts FAK interaction with RACK1. We conclude that FAK association with RACK1 is regulated by phosphorylation of Tyr-52. Our data reveal a novel mechanism whereby IGF-I and c-Abl control RACK1 association with FAK to facilitate adhesion signaling.

Rho signaling, ROCK and mDia1, in transformation, metastasis and invasion

The Rho subgroup of the Rho GTPases consisting of RhoA, RhoB and RhoC induces a specific type of actin cytoskeleton and carry out a variety of functions in the cell. mDia and ROCK are downstream effectors of Rho mediating Rho action on the actin cytoskeleton; mDia produces actin filaments by nucleation and polymerization and ROCK activate myosin to cross-link them for induction of actomyosin bundles and contractility. mDia is potentially linked to Rac activation and membrane ruffle formation through c-Src-induced phosphorylation of focal adhesion proteins, and ROCK antagonizes this mDia action. Thus, cell morphogenesis, adhesion, and motility can be determined by the balance between mDia and ROCK activities. Though they are not oncogenes by themselves, overexpression of RhoA and RhoC are often found in clinical cancers, and RhoC has been repeatedly identified as a gene associated with metastasis. The Rho-ROCK pathway is implicated in Ras-mediated transformation, the amoeboid movement of tumor cells in the three-dimensional matrix, and transmigration of tumor cells through the mesothelial monolayer. On the other hand, the Rho-mDia1 pathway is implicated in Src-mediated remodeling of focal adhesions and migration of tumor cells. There is also an indication that the Rho pathway other than ROCK is involved in Src-mediated induction of podosome and regulation of matrix metalloproteases. Thus, Rho mediates various phenotypes of malignant transformation by Ras and Src through its effectors, ROCK and mDia.

Rapamycin inhibits F-actin reorganization and phosphorylation of focal adhesion proteins

An early event of cell migration is characterized as the rapid reorganization of the actin cytoskeleton. Recently, we have demonstrated that rapamycin inhibits tumor cell motility. To understand the underlying mechanism, this study was set to determine whether rapamycin inhibition of cell motility is related to its prevention of F-actin reorganization. We found that rapamycin prevented type I insulin-like growth factor (IGF-I)-stimulated F-actin reorganization in human rhabdomyosarcoma (Rh30), Ewing sarcoma (Rh1), glioblastoma (U-373) and prostate carcinoma (PC-3) cells, and concurrently inhibited phosphorylation of focal adhesion proteins, including focal adhesion kinase (FAK), paxillin and p130(Cas) in the cells. The effect of rapamycin was blocked by expression of a rapamycin-resistant mutant of mTOR (mTORrr), but not a kinase-dead mTORrr. Downregulation of raptor mimicked the effect of rapamycin. Cells infected with a recombinant adenovirus expressing constitutively active and rapamycin-resistant mutant of p70 S6 kinase 1 (S6K1) conferred to resistance to rapamycin. Further, IGF-I failed to stimulate F-actin reorganization and phosphorylation of the focal adhesion proteins in the S6K1-downregulated cells. Expression of constitutively hypophosphorylated eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1-5A) inhibited IGF-I-stimulated F-actin reorganization, but did not alter the cellular protein or phosphorylation levels of the focal adhesion proteins. The results suggest that rapamycin inhibits IGF-I-induced F-actin reorganization and phosphorylation of the focal adhesion proteins by disruption of mTOR-raptor complex. Both S6K1 and 4E-BP1 pathways, mediated by the mTOR-raptor complex, are involved in the regulation of IGF-I-stimulated F-actin reorganization, but only the former controls IGF-I-stimulated phosphorylation of the focal adhesion proteins.

Fasudil inhibits vascular endothelial growth factor–induced angiogenesisin vitroandin vivo

Vascular endothelial growth factor (VEGF)-induced endothelial cell migration is an important component of tumor angiogenesis. Rho and Rho-associated kinase (ROCK) are key regulators of focal adhesion, stress fiber formation, and thus cell motility. Inhibitors of this pathway have been shown to inhibit endothelial cell motility and angiogenesis. In this study, we investigated the antiangiogenic effect of fasudil, one of the ROCK inhibitors. Fasudil inhibited VEGF-induced endothelial cell migration, viability, and tube formation in vitro in human umbilical vein endothelial cells. VEGF-induced endothelial cell migration was reduced by fasudil associated with loss of stress fiber formation, focal adhesion assembly, and with the suppression of tyrosine phosphorylation of focal adhesion proteins. Furthermore, fasudil inhibited VEGF-induced phosphorylation of myosin light chain, which is one of the main substrates of ROCK. Therefore, the effect of fasudil was suggested to be ROCK dependent. Fasudil not only inhibited VEGF-induced cell proliferation but also reversed the protective effect of VEGF on apoptosis, which resulted in the decrease of cell viability. Moreover, fasudil inhibited VEGF-induced angiogenesis in a directed in vivo angiogenesis assay. These data are the first demonstration that fasudil has antiangiogenic properties. Therefore, fasudil might be useful for the treatment of angiogenesis-related diseases, especially cancer.

STAT3-independent inhibition of lysophosphatidic acid-mediated upregulation of connective tissue growth factor (CTGF) by cucurbitacin I

Cucurbitacins are recognised as anti-tumour agents because of their interference with STAT3 signalling, but may also affect the integrity of the actin cytoskeleton. In the present study the effect of cucurbitacin I was investigated in fibroblasts. In these cells, cucurbitacin I interfered with lysophosphatidic acid (LPA) signalling. It inhibited tyrosine phosphorylation of focal adhesion proteins and induction of connective tissue growth factor (CTGF), a potent profibrotic protein. Inhibition of Src family kinases with PP2, but not the inactive analogue PP3, also interfered with LPA-mediated tyrosine phosphorylation and induction of CTGF. Jak2–STAT3 signalling seemed to be the connecting link, because CTGF induction was sensitive to AG490, an inhibitor of Jak2, and cucurbitacin I, an inhibitor of Jak2 and STAT3. However, LPA did not activate tyrosine phosphorylation of STAT3. Furthermore, cucurbitacin I was as effective in STAT3 knock out cells as in control cells. Therefore, the inhibitory effect of cucurbitacin I was not related to inhibition of STAT3. Immunocytochemical analysis of cucurbitacin I-treated cells revealed disassembly of F-actin fibres, reorganisation into F-actin patches and resolution of focal adhesions. The phenotypic changes resembled changes observed after treatment of the cells with cytochalasin D, which has been shown to interfere with CTGF induction. Concentrations of cucurbitacin I, which have been shown to target Jak2–STAT3 signalling, thus, profoundly affect the actin cytoskeleton and may therefore modulate cell morphology, migration, adherence and gene expression also in non-tumour cells.

Contribution of Src-FAK signaling to the induction of connective tissue growth factor in renal fibroblasts

Expression of connective tissue growth factor (CTGF) is sensitive to reorganization of the actin cytoskeleton, but also to alterations in cell morphology due to extracellular forces, for example, cyclic stretching or mechanical loading. Dynamic alterations of focal adhesion proteins were thus proposed to modulate CTGF induction. Immortalized human renal fibroblasts were cultured in or on top of preformed collagen-1 gels. Proteins were detected by immunofluorescence and quantified by Western blotting. Fibroblasts cultured in/on collagen gels resembled cells in vivo by their spindle-like morphology, absence of actin stress fibers, small punctiform focal contacts, and low levels of CTGF expression. Disassembly of microtubules by short-term treatment with colchicine induced cell rounding, cortical recruitment of patchy F-actin, reorganization of focal contacts into strong clusters, and upregulation of CTGF, all of which were dependent on RhoA-Rho-kinase signaling. Clustering of focal adhesion sites activated Src-family kinases and focal adhesion kinase (FAK). Interference with Src activity by PP2 had no effect on the morphological alterations but decreased tyrosine phosphorylation of focal adhesion proteins and almost completely prevented upregulation of CTGF. Furthermore, inhibition of phosphatidylinositol 3-kinase reduced CTGF expression. On the other hand, when the fibroblasts were cultured on a rigid matrix, that is collagen-coated plates, strong focal complexes prevented the dynamic alterations, and RhoA-mediated upregulation of CTGF expression was independent of Src-FAK signaling. Assembly of focal adhesion proteins regulates CTGF expression, providing a link between actin network, adhesion receptors, and CTGF-mediated functions such as synthesis of extracellular matrix proteins.

Hyperoxia combined with high tidal volume mechanical ventilation reduces alveolar type II cell adhesion: reduced phosphorylation of focal adhesion proteins and effect of KGF

Hyperoxia combined with high tidal volume mechanical ventilation reduces alveolar type II cell adhesion: reduced phosphorylation of focal adhesion proteins and effect of KGF

Geranylgeranylacetone inhibits lysophosphatidic acid‐induced invasion of human ovarian carcinoma cells in vitro

Lysophosphatidic acid (LPA) induced a dose-dependent increase of cancer cell invasion by promoting Rho/Rho-associated kinase signaling. Prenylation of Rho is essential for regulating cell growth, motility, and invasion. Geranylgeranylacetone (GGA), an isoprenoid compound, is used clinically as an antiulcer drug. Recent findings suggested that GGA might inhibit the small GTPase activation by suppressing prenylation. The authors hypothesized that the anticancer effects of GGA result from the inhibition of Rho activation. The authors examined the effect of GGA using an in vitro invasion assay in human ovarian carcinoma cells, and analyzed the mechanism of the GGA effect on Rho activation, stress fiber formation and focal adhesion assembly, which are essential processes for cell invasion. The induction of ovarian carcinoma cell invasion by LPA was inhibited by the addition of GGA in a dose-dependent manner. Treatment of cancer cells with GGA resulted in inactivation of Rho, changes in cell morphology, loss of stress fiber formation and focal adhesion assembly, and the suppression of tyrosine phosphorylation of focal adhesion proteins. The effect of GGA on cancer cells was partially prevented by the addition of geranylgeraniol, which is an intermediate of geranylgeranyl pyrophosphate and compensates geranylgeranylation of Rho. The inhibition of LPA-induced invasion by GGA was, at least in part, derived from suppressed Rho activation by preventing geranylgeranylation.

Endothelin Receptor Blockade Inhibits Molecular Effectors of Kaposi's Sarcoma Cell Invasion and Tumor Growth in Vivo

Endothelin-1 (ET-1) and its receptors are overexpressed in human Kaposi's sarcoma lesions. Here we show that in human KS IMM cell line ET-1 increased secretion and activation of matrix-metalloproteinase-2 (MMP-2), -3, -7, -9 and -13, as well as of membrane-type 1-MMP (MT1-MMP). ET-1 and ET-3 also enhanced the expression of tissue inhibitor of MMP-2, essential for MT1-MMP-mediated MMP-2 activation. Combined addition of both ET(B) receptor (ET(B)R) and ET(A)R antagonists completely blocked the ET-1-induced MMP activity. By immunohistochemistry, we observed that ET-1 increased MMP-2 and MT1-MMP expression and their localization at the cell surface. Treatment with both antagonists resulted also in the suppression of ET-1-induced phosphorylation of focal adhesion proteins, FAK and paxillin, which are essentials for cell motility. ET-1 induced a dose-dependent enhancement in KS IMM cell migration and MMP-dependent invasiveness that were inhibited by ET-1 receptor antagonists. The small molecule, A-182086, an orally bioavailable ET(A/B)R antagonist, completely inhibited cell proliferation and tumor growth in KS IMM xenografts. These findings demonstrate that ET-1-driven autocrine loop is crucial for enhanced invasiveness of KS IMM cells and promote tumor growth in vivo. Such activities can be blocked by the ET(A/B)R antagonists, which may be effective anti-angiogenic and anti-tumor molecules for the treatment of Kaposi's sarcoma.

C-Met: structure, functions and potential for therapeutic inhibition.

Studies on signal transduction pathways have generated various promising molecular targets for therapeutic inhibition in cancer therapy. Receptor tyrosine kinases represent an important class of such therapeutic targets. c-Met is a receptor tyrosine kinase that has been shown to be overexpressed and/or mutated in a variety of malignancies. A number of c-Met activating mutations, many of which are located in the tyrosine kinase domain, have been detected in various solid tumors and have been implicated in invasion and metastasis of tumor cells. It is known that stimulation of c-Met via its natural ligand, hepatocyte growth factor (also known as scatter factor, HGF/SF) results in a plethora of biological and biochemical effects in the cell. Activation of c-Met signaling can lead to scattering, angiogenesis, proliferation, enhanced cell motility, invasion, and eventual metastasis. In this review, the role of c-Met dysregulation in tumor progression and metastasis is discussed in detail with particular emphasis on c-Met mutations. Moreover, we summarize current knowledge on various pathways of c-Met signal transduction, highlighting the central role in the cytoskeletal functions. In this summary is included recent data in our laboratory indicating that phosphorylation of focal adhesion proteins, such as paxillin, p125FAK, and PYK2, occurs in response to c-Met stimulation in lung cancer cells. Most importantly, current data on c-Met suggest that when mutated or overexpressed in malignant cells, c-Met would serve as an important therapeutic target.

Protein phosphatase-2A restricts migration of Lewis lung carcinoma cells by modulating the phosphorylation of focal adhesion proteins.

Compared to metastatic Lewis lung carcinoma (LLC) cells, nonmetastatic LLC cells have increased levels of activity of the protein phosphatase PP-2A, which functions to limit their migration through transwell chambers. Inhibition of PP-2A in nonmetastatic LLC stimulates their transmigration to levels similar to those of metastatic LLC cells. Studies to define the signaling pathways intermediate between diminished PP-2A activity and stimulated migration showed that inhibiting PP-2A activity resulted in paxillin serine hyperphosphorylation and tyrosine dephosphorylation. Paxillin was important for the stimulated migration because the increased transmigration in response to PP-2A inhibition was dampened by expression of mutant paxillin at the LIM3 S457 and S481 residues. Inhibition of PP-2A also led to the dissolution of FAK/Src/paxillin focal adhesion complexes, which was also dependent on paxillin S457 and S481 residues. In addition, inhibition of PP-2A resulted in dephosphorylation of Src inhibitory Y527 residue, suggesting increased Src activity. The stimulated transmigration of cells with diminished PP-2A was in part dependent on this Src activity. These studies show the importance of PP-2A in limiting tumor cell migration through its modulation of proteins of the focal adhesions.