Inhibition of endothelial FAK activity prevents tumor metastasis by enhancing barrier function.

Pharmacological focal adhesion kinase (FAK) inhibition prevents tumor growth and metastasis, via actions on both tumor and stromal cells. In this paper, we show that vascular endothelial cadherin (VEC) tyrosine (Y) 658 is a target of FAK in tumor-associated endothelial cells (ECs). Conditional kinase-dead FAK knockin within ECs inhibited recombinant vascular endothelial growth factor (VEGF-A) and tumor-induced VEC-Y658 phosphorylation in vivo. Adherence of VEGF-expressing tumor cells to ECs triggered FAK-dependent VEC-Y658 phosphorylation. Both FAK inhibition and VEC-Y658F mutation within ECs prevented VEGF-initiated paracellular permeability and tumor cell transmigration across EC barriers. In mice, EC FAK inhibition prevented VEGF-dependent tumor cell extravasation and melanoma dermal to lung metastasis without affecting primary tumor growth. As pharmacological c-Src or FAK inhibition prevents VEGF-stimulated c-Src and FAK translocation to EC adherens junctions, but FAK inhibition does not alter c-Src activation, our experiments identify EC FAK as a key intermediate between c-Src and the regulation of EC barrier function controlling tumor metastasis.

Nuclear-localized focal adhesion kinase regulates inflammatory VCAM-1 expression.

Vascular cell adhesion molecule-1 (VCAM-1) plays important roles in development and inflammation. Tumor necrosis factor-α (TNF-α) and focal adhesion kinase (FAK) are key regulators of inflammatory and integrin-matrix signaling, respectively. Integrin costimulatory signals modulate inflammatory gene expression, but the important control points between these pathways remain unresolved. We report that pharmacological FAK inhibition prevented TNF-α-induced VCAM-1 expression within heart vessel-associated endothelial cells in vivo, and genetic or pharmacological FAK inhibition blocked VCAM-1 expression during development. FAK signaling facilitated TNF-α-induced, mitogen-activated protein kinase activation, and, surprisingly, FAK inhibition resulted in the loss of the GATA4 transcription factor required for TNF-α-induced VCAM-1 production. FAK inhibition also triggered FAK nuclear localization. In the nucleus, the FAK-FERM (band 4.1, ezrin, radixin, moesin homology) domain bound directly to GATA4 and enhanced its CHIP (C terminus of Hsp70-interacting protein) E3 ligase-dependent polyubiquitination and degradation. These studies reveal new developmental and anti-inflammatory roles for kinase-inhibited FAK in limiting VCAM-1 production via nuclear localization and promotion of GATA4 turnover.

VEGF-induced vascular permeability is mediated by FAK.

Endothelial cells (ECs) form cell-cell adhesive junctional structures maintaining vascular integrity. This barrier is dynamically regulated by vascular endothelial growth factor (VEGF) receptor signaling. We created an inducible knockin mouse model to study the contribution of the integrin-associated focal adhesion tyrosine kinase (FAK) signaling on vascular function. Here we show that genetic or pharmacological FAK inhibition in ECs prevents VEGF-stimulated permeability downstream of VEGF receptor or Src tyrosine kinase activation in vivo. VEGF promotes tension-independent FAK activation, rapid FAK localization to cell-cell junctions, binding of the FAK FERM domain to the vascular endothelial cadherin (VE-cadherin) cytoplasmic tail, and direct FAK phosphorylation of ß-catenin at tyrosine-142 (Y142) facilitating VE-cadherin-ß-catenin dissociation and EC junctional breakdown. Kinase inhibited FAK is in a closed conformation that prevents VE-cadherin association and limits VEGF-stimulated ß-catenin Y142 phosphorylation. Our studies establish a role for FAK as an essential signaling switch within ECs regulating adherens junction dynamics.

Phosphatidylinositol 3,4,5-triphosphate-dependent Rac exchanger 1 (P-Rex-1), a guanine nucleotide exchange factor for Rac, mediates angiogenic responses to stromal cell-derived factor-1/chemokine stromal cell derived factor-1 (SDF-1/CXCL-12) linked to Rac activation, endothelial cell migration, and in vitro angiogenesis.

Stromal cell-derived factor-1 (SDF-1/CXCL-12) and vascular endothelial growth factor (VEGF), which can be secreted by hypoxic tumors, promote the generation of new blood vessels. These potent angiogenic factors stimulate endothelial cell migration via the activation of Rho GTPases and the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway. Thus, characterization of guanine nucleotide exchange factors critical in the angiogenic signaling cascades offers the possibility of identifying novel molecular targets. We demonstrated previously that mammalian target of rapamycin, an important effector and regulator of PI3K/AKT, activates phosphatidylinositol 3,4,5-triphosphate-dependent Rac exchanger 1 (P-Rex1), a Rac guanine nucleotide exchange factor identified as a target of G betagamma and PI3K, via direct interactions. In this study, we tested the hypothesis that P-Rex1 is involved in the angiogenic responses elicited by SDF-1 and VEGF. Using a knockdown approach, we demonstrate that P-Rex1 is indeed required for SDF-1 promoted signaling pathway, because there is decreased Rac activation, cell migration, and in vitro angiogenesis in P-Rex1 knockdown cells stimulated with SDF-1. In contrast, P-Rex1 knockdown does not affect responses to VEGF, and signaling to extracellular signal-regulated kinase in response to either angiogenic factor is not sensitive to P-Rex1 knockdown. We also demonstrate that in endothelial cells, VEGF promotes an increase in the expression of endogenous P-Rex1 and the SDF-1 receptor CXCR4, In addition, VEGF-pretreated cells show an increased migratory and angiogenic response to SDF-1, suggesting that VEGF stimulation can complement SDF-1/CXCR4 signaling to induce angiogenesis. We conclude that P-Rex1 is a key element in SDF-1-induced angiogenic responses and a potential target for therapeutic intervention.

G protein-coupled receptors go extracellular: RhoA integrates the integrins.

The identification of downstream effectors of G protein-coupled receptors (GPCRs) is critical for understanding the interactions between signaling cascades and for developing new pharmacological approaches for controlling GPCR-mediated responses. RhoA is a small G protein that serves as a proximal downstream effector of numerous GPCRs and regulates a variety of basic cell functions, including migration, survival, and proliferation. Intriguingly, GPCR ligands such as thrombin, sphingosine-1-phosphate, and lysophosphatidic acid, which signal through G(12/13) and activate RhoA, have recently been shown to induce the expression of the extracellular matrix protein Cyr61 (i.e., CCN1). Cyr61 is secreted and interacts with cell surface integrins to activate kinase and transcriptional cascades that are also known to contribute to cell migration, survival, and proliferation. The GPCR/RhoA/Cyr61/integrin pathway defines a novel convergence mechanism for integrating GPCR-and integrin-dependent signaling cascades that may contribute to sustained and pathophysiological responses to GPCR activation.

Thrombin receptor and RhoA mediate cell proliferation through integrins and cysteine-rich protein 61.

A subset of G-protein coupled receptors (GPCRs), including the thrombin receptor (PAR1), elicits mitogenic responses. Thrombin also activates Ras homolog gene family member A (RhoA) and activating protein (AP-1) -mediated gene expression in 1321N1 astrocytoma cells, whereas the nonmitogenic agonist carbachol does not. Transcriptomic analysis was used to explore differential gene induction by these agonists and revealed that the matricellular protein cysteine-rich 61 (Cyr61/CCN1) is selectively induced by thrombin. The ability of GPCR agonists to induce Cyr61 parallels their ability to activate RhoA; agonist-stimulated Cyr61 expression is inhibited by C3 toxin. When Cyr61 is down-regulated using short interfering RNA (siRNA) or short-hairpin RNA (shRNA), thrombin-induced DNA synthesis is significantly attenuated. When Cyr61 expression is induced, it appears in the extracellular compartment and on the cell surface. Extracellular Cyr61 interacts with alpha(5), alpha(6), and beta(1) integrins on these cells, and monoclonal antibodies directed against alpha(5) and beta(1) integrins inhibit thrombin-induced DNA synthesis. Functional blockade of Cyr61 with soluble heparin or anti-Cyr61 antibodies also inhibits thrombin-induced DNA synthesis. Thus Cyr61 is a highly inducible, secreted extracellular factor through which GPCR and RhoA signaling pathways engage integrins that contribute to GPCR-mediated proliferation.

Quantitative image analysis of intra-tumoral bFGF level as a molecular marker of paclitaxel resistance.

BACKGROUND: The role of basic fibroblast growth factor (bFGF) in chemoresistance is controversial; some studies showed a relationship between higher bFGF level and chemoresistance while other studies showed the opposite finding. The goal of the present study was to quantify bFGF levels in archived tumor tissues, and to determine its relationship with chemosensitivity. METHODS: We established an image analysis-based method to quantify and convert the immunostaining intensity of intra-tumor bFGF to concentrations; this was accomplished by generating standard curves using human xenograft tumors as the renewable tissue source for simultaneous image analysis and ELISA. The relationships between bFGF concentrations and tumor chemosensitivity of patient tumors (n = 87) to paclitaxel were evaluated using linear regression analysis. RESULTS: The image analysis results were compared to our previous results obtained using a conventional, semi-quantitative visual scoring method. While both analyses indicated an inverse relationship between bFGF level and tumor sensitivity to paclitaxel, the image analysis method, by providing bFGF levels in individual tumors and therefore more data points (87 numerical values as opposed to four groups of staining intensities), further enabled the quantitative analysis of the relationship in subgroups of tumors with different pathobiological properties. The results show significant correlation between bFGF level and tumor sensitivity to the antiproliferation effect, but not the apoptotic effect, of paclitaxel. We further found stronger correlations of bFGF level and paclitaxel sensitivity in four tumor subgroups (high stage, positive p53 staining, negative aFGF staining, containing higher-than-median bFGF level), compared to all other groups. These findings suggest that the relationship between intra-tumoral bFGF level and paclitaxel sensitivity was context-dependent, which may explain the previous contradictory findings on the merit of using plasma or urine bFGF level as a prognostic indicator. CONCLUSION: The present study established a quantitative image analysis method that enabled the measurement of intratumoral bFGF level in archived tissues. The ability to quantify a potential biomarker provided the opportunity to study the relationship between the biomarker and chemosensitivity in tumor subgroups and thereby enabled hypothesis generation for additional translational research.

Tumor necrosis factor-alpha-stimulated cell proliferation is mediated through sphingosine kinase-dependent Akt activation and cyclin D expression.

Tumor necrosis factor-alpha (TNF-alpha) has been shown to activate sphingosine kinase (SphK) in a variety of cell types. The extent to which SphK signaling mediates the pleiotropic effects of TNF-alpha is not entirely clear. The current study examined the role of SphK activity in TNF-alpha-stimulated cell proliferation in 1321N1 glioblastoma cells. We first demonstrated that pharmacological inhibitors of SphK markedly decrease TNF-alpha-stimulated DNA synthesis. Signaling mechanisms through which SphK mediated the effect of TNF-alpha on DNA synthesis were then examined. Inhibition of Rho proteins with C3 exoenzyme or of Rho kinase with Y27632 attenuated TNF-alpha-stimulated DNA synthesis. However, RhoA activation by TNF-alpha was not blocked by SphK inhibition. ERK activation was also required for TNF-alpha-stimulated DNA synthesis but likewise TNF-alpha-induced ERK activation was not blocked by inhibition of SphK. Thus, neither RhoA nor ERK activation are the SphK-dependent transducers of TNF-alpha-induced proliferation. In contrast, TNF-alpha-stimulated Akt phosphorylation, which was also required for DNA synthesis, was attenuated by SphK inhibition or SphK1 knockdown by small interfering RNA. Furthermore, cyclin D expression was increased by TNF-alpha in a SphK- and Akt-dependent manner. Additional studies demonstrated that TNF-alpha effects on DNA synthesis, ERK, and Akt phosphorylation are not mediated through cell surface Gi -coupled S1P receptors, because none of these responses were inhibited by pertussis toxin. We conclude that SphK-dependent Akt activation plays a significant role in TNF-alpha-induced cyclin D expression and cell proliferation.