Focal Adhesion Kinase In Endothelial Cells Research Articles

Elucidating the role of the kinase activity of endothelial cell focal adhesion kinase in angiocrine signalling and tumour growth.

A common limitation of cancer treatments is chemotherapy resistance. We have previously identified that endothelial cell (EC)-specific deletion of focal adhesion kinase (FAK) sensitises tumour cells to DNA-damaging therapies, reducing tumour growth in mice. The present study addressed the kinase activity dependency of EC FAK sensitisation to the DNA-damaging chemotherapeutic drug, doxorubicin. FAK is recognised as a therapeutic target in tumour cells, leading to the development of a range of inhibitors, the majority being ATP competitive kinase inhibitors. We demonstrate that inactivation of EC FAK kinase domain (kinase dead; EC FAK-KD) in established subcutaneous B16F0 tumours improves melanoma cell sensitisation to doxorubicin. Doxorubicin treatment in EC FAK-KD mice reduced the percentage change in exponential B16F0 tumour growth further than in wild-type mice. There was no difference in tumour blood vessel numbers, vessel perfusion or doxorubicin delivery between genotypes, suggesting a possible angiocrine effect on the regulation of tumour growth. Doxorubicin reduced perivascular malignant cell proliferation, while enhancing perivascular tumour cell apoptosis and DNA damage in tumours grown in EC FAK-KD mice 48 h after doxorubicin injection. Human pulmonary microvascular ECs treated with the pharmacological FAK kinase inhibitors defactinib, PF-562,271 or PF-573,228 in combination with doxorubicin also reduced cytokine expression levels. Together, these data suggest that targeting EC FAK kinase activity may alter angiocrine signals that correlate with improved acute tumour cell chemosensitisation. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

MG53 inhibits angiogenesis through regulating focal adhesion kinase signalling.

Mitsugumin 53 (MG53), which is expressed predominantly in striated muscle, has been demonstrated to be a myokine/cardiokine secreted from striated muscle under specific conditions. The important roles of MG53 in non‐striated muscle tissues have also been examined in multiple disease models. However, no previous study has implicated MG53 in the control of endothelial cell function. In order to explore the effects of MG53 on endothelial cells, human umbilical vein endothelial cells (HUVECs) were stimulated with recombinant human MG53 (rhMG53). Then, rhMG53 uptake, focal adhesion kinase (FAK)/Src/Akt/ERK1/2 signalling pathway activation, cell migration and tube formation were determined in vitro. The efficacy of rhMG53 in regulating angiogenesis was also detected in postnatal mouse retinas. The results demonstrated that rhMG53 directly entered into endothelial cells in a cholesterol‐dependent manner. The uptake of rhMG53 directly bound to FAK in endothelial cells, which resulted in a significant decrease in FAK phosphorylation at Y397. Accompanied by the dephosphorylation of FAK, rhMG53 uncoupled FAK‐Src interaction and reduced the phosphorylation of Src at Y416. Consequently, the activation of FAK/Src downstream signalling pathways, such as Akt and ERK1/2, was also significantly inhibited by rhMG53. Furthermore, rhMG53 remarkably decreased HUVEC migration and tube formation in vitro and postnatal mouse retinal angiogenesis in vivo. Taken together, these data indicate that rhMG53 inhibits angiogenesis through regulating FAK/Src/Akt/ERK1/2 signalling pathways. This may provide a novel molecular mechanism for the impaired angiogenesis in ischaemic diseases.

Abstract 1997: FAK inhibition induced FAK nuclear localization targets both tumor growth and angiogenesis

Abstract Tumors are not a cell-autonomous event, as most tumors include non-neoplastic stromal cells, so-called tumor microenvironment (TME). The TME is the tumor's immediate surroundings that comprise various types of cells and soluble factors, which stimulate bidirectionally via dynamic paracrine signaling supporting primary tumor progression. Among them, endothelial cells (ECs) significantly contribute to promoting tumor growth and metastasis through their ability to establish tumor vessels. Most cancer therapies have been centered on targeting altered signaling in tumor cells to prevent cancer growth, largely ignoring the importance of the TME. Identifying key signaling pathways that simultaneously prevent both tumor growth and angiogenesis may provide a new therapeutic strategy for various cancers. Focal adhesion kinase (FAK) is an integrin associated protein tyrosine kinase, which is frequently overexpressed in advanced human cancers. Small molecule FAK catalytic inhibitors (FAK-I) are currently being tested in clinical trials as anticancer agents. We found that kinase-inhibited FAK forces FAK nuclear localization in cancer cells. Although the role of nuclear FAK is not fully understood, several studies have shown that nuclear FAK may regulate gene expression. Using B16 melanoma ear tumor model, we found that FAK-I efficiently reduced B16 tumor lesions and tumor angiogenesis. While vehicle-treated mice exhibited high cytoplasmic FAK levels, FAK-I-treated mice exhibited robust FAK nuclear localization within B16 tumor cells and the TME. As FAK-I affects the FAK activity in both tumor cells and the TME, we attempted to dissect respective molecular mechanisms by which nuclear FAK contributes to the reduction in tumor growth and angiogenesis. From RNA sequencing data analysis from FAK-I treated cancer cell lines, we found that FAK inhibition significantly upregulated gene sets involved in cell cycle regulation, including CDK inhibitor p21. Increased expression of p21 in FAK-I treated B16 cells was confirmed using RT-qPCR, immunoblotting, and immunostaining. Prolonged FAK-I treatment increased B16 cell apoptosis both in vitro and in vivo compared to vehicle group. To address if FAK-I also blocks angiogenesis in vivo, we performed a Matrigel plug assay and found that FAK-I significantly reduced VEGF/FGF-stimulated angiogenesis. FAK-I treatment promoted nuclear FAK localization in vitro and in vivo ECs, suggesting that nuclear FAK in ECs inhibits cell proliferation and migration required for angiogenic activity. Using EC-specific FAK kinase-dead mouse, we confirmed that nuclear FAK in ECs is responsible for reducing angiogenic activity in the Matrigel plug model. Together, these results indicate that nuclear FAK induced by FAK-I can be used as a promising therapeutic strategy simultaneously targeting both tumor growth and angiogenesis. Citation Format: Yelitza Rodriguez, James Murphy, Kyuho Jeong, Ahn Erin, Steve Lim. FAK inhibition induced FAK nuclear localization targets both tumor growth and angiogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1997.

Tumor Angiogenesis Is Differentially Regulated by Phosphorylation of Endothelial Cell Focal Adhesion Kinase Tyrosines-397 and -861.

Expression of focal adhesion kinase (FAK) in endothelial cells (EC) is essential for angiogenesis, but how FAK phosphorylation at tyrosine-(Y)397 and Y861 regulate tumor angiogenesis in vivo is unknown. Here, we show that tumor growth and angiogenesis are constitutively reduced in inducible, ECCre+;FAKY397F/Y397F -mutant mice. Conversely, ECCre+;FAKY861F/Y861F mice exhibit normal tumor growth with an initial reduction in angiogenesis that recovered in end-stage tumors. Mechanistically, FAK-Y397F ECs exhibit increased Tie2 expression, reduced Vegfr2 expression, decreased β1 integrin activation, and disrupted downstream FAK/Src/PI3K(p55)/Akt signaling. In contrast, FAK-Y861F ECs showed decreased Vegfr2 and Tie2 expression with an enhancement in β1 integrin activation. This corresponds with a decrease in Vegfa-stimulated response, but an increase in Vegfa+Ang2- or conditioned medium from tumor cell-stimulated cellular/angiogenic responses, mimicking responses in end-stage tumors with elevated Ang2 levels. Mechanistically, FAK-Y861F, but not FAK-Y397F ECs showed enhanced p190RhoGEF/P130Cas-dependent signaling that is required for the elevated responses to Vegfa+Ang2. This study establishes the differential requirements of EC-FAK-Y397 and EC-FAK-Y861 phosphorylation in the regulation of EC signaling and tumor angiogenesis in vivo. SIGNIFICANCE: Distinct motifs of the focal adhesion kinase differentially regulate tumor blood vessel formation and remodeling.

Abstract A04: Endothelial FAK activity controls vascular permeability and tumor metastasis

Abstract Tumor spread remains a primary cause of cancer mortality. Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase co-activated by integrin and growth factor receptors in the control of vascular permeability. As FAK inhibitors are being tested in clinical trials, it is important to determine whether FAK signaling plays distinct roles in tumor versus stromal cell biology. Herein, we elucidate a new mechanism for endothelial cell (EC) FAK activity in the regulation of blood vessel barrier integrity and the passage of tumor cells through an EC barrier. We show that vascular endothelial cadherin (VEC) tyrosine (Y) 658 is phosphorylated by FAK in tumor-associated 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. Although it is known that Src tyrosine kinase activity is important in controlling VEC tyrosine phosphorylation downstream of VEGF, we find that pharmacological Src inhibition prevents FAK activation. Moreover, FAK inhibition prevents both Src and FAK translocation to EC adherens junctions, but does not alter Src activation. Together, these results identify EC FAK as a key intermediate between Src and the regulation of EC barrier function controlling tumor metastasis. Citation Format: Christine Jean, Xiao Lei Chen, Isabelle Tancioni, Sean Uryu, Christine Lawson, Nichol Miller, Patric Turowski, Elisabetta Dejana, Sara Weis, David Cheresh, David D. Schlaepfer. Endothelial FAK activity controls vascular permeability and tumor metastasis. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A04. doi:10.1158/1538-7445.CHTME14-A04

Focal adhesion kinase

This investigation describes the clinical significance of phosphorylated focal adhesion kinase (FAK) at the major activating tyrosine site (Y397) in epithelial ovarian cancer (EOC) cells and tumor-associated endothelial cells. FAK gene amplification as a mechanism for FAK overexpression and the effects of FAK tyrosine kinase inhibitor VS-6062 on tumor growth, metastasis, and angiogenesis were examined. FAK and phospho-FAKY397 were quantified in tumor (FAK-T; pFAK-T) and tumor-associated endothelial (FAK-endo; pFAK-endo) cell compartments of EOCs using immunostaining and qRT-PCR. Associations between expression levels and clinical variables were evaluated. Data from The Cancer Genome Atlas were used to correlate FAK gene copy number and expression levels in EOC specimens. The in vitro and in vivo effects of VS-6062 were assayed in preclinical models. FAK-T and pFAK-T overexpression was significantly associated with advanced stage disease and increased microvessel density (MVD). High MVD was observed in tumors with elevated endothelial cell FAK (59%) and pFAK (44%). Survival was adversely affected by FAK-T overexpression (3.03 vs 2.06 y, P = 0.004), pFAK-T (2.83 vs 1.78 y, P < 0.001), and pFAK-endo (2.33 vs 2.17 y, P = 0.005). FAK gene copy number was increased in 34% of tumors and correlated with expression levels (P < 0.001). VS-6062 significantly blocked EOC and endothelial cell migration as well as endothelial cell tube formation in vitro. VS-6062 reduced mean tumor weight by 56% (P = 0.005), tumor MVD by 40% (P = 0.0001), and extraovarian metastasis (P < 0.01) in orthotopic EOC mouse models. FAK may be a unique therapeutic target in EOC given the dual anti-angiogenic and anti-metastatic potential of FAK inhibitors.

Inhibitory effects of Yangzheng Xiaoji on angiogenesis and the role of the focal adhesion kinase pathway

Angiogenesis is an essential event during the excessive growth and metastatic spread of solid tumours. Anti-angiogenic agents have become a new choice of therapy for patients with cancer. In the present study, we investigated the potential effect of Yangzheng Xiaoji, a traditional Chinese medicinal formula presently used in the treatment of several solid tumours including liver cancer and gastric cancer, on angiogenesis, invitro. The human vascular endothelial cell line HECV was used. A Matrigel-based sandwich tubule formation assay was employed to assess invitro angiogenesis, a colorimetric method for assessing invitro cell growth. Electric cell-substrate impedance sensing (ECIS) was used to evaluate the adhesion and migration of endothelial cells. The effects on activation of focal adhesion kinase (FAK) were evaluated using western blotting and immunofluorescence methods. YangzhenXiaoji extract DME25 significantly inhibited tube formation (p=0.046 vs control). This was seen together with a concentration-dependent inhibition on cell-matrix adhesion and cellular migration. It was demonstrated that the focal adhesion kinase (FAK) inhibitor PF557328 had a significant synergistic effect on DME25-induced inhibition of cell adhesion, migration and tube formation. The study showed that DME25 inhibited the phosphorylation of FAK in endothelial cells. In conclusion, Yangzhen Xiaoji has a marked effect on angiogenesis, invitro and that this effect is at least partly mediated by the focal adhesion kinase (FAK) pathway.

Endothelial focal adhesion kinase mediates cancer cell homing to discrete regions of the lungs via E-selectin up-regulation

Primary tumors secrete factors that alter the microenvironment of distant organs, rendering those organs as fertile soil for subsequent metastatic cancer cell colonization. Although the lungs are exposed to these factors ubiquitously, lung metastases usually develop as a series of discrete lesions. The underlining molecular mechanisms of the formation of these discrete lesions are not understood. Here we show that primary tumors induce formation of discrete foci of vascular hyperpermeability in premetastatic lungs. This is mediated by endothelial cell-focal adhesion kinase (FAK), which up-regulates E-selectin, leading to preferential homing of metastatic cancer cells to these foci. Suppression of endothelial-FAK or E-selectin activity attenuates the number of cancer cells homing to these foci. Thus, localized activation of endothelial FAK and E-selectin in the lung vasculature mediates the initial homing of metastatic cancer cells to specific foci in the lungs.

Endothelial FAK is required for tumour angiogenesis.

Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that plays a fundamental role in integrin and growth factor mediated signalling and is an important player in cell migration and proliferation, processes vital for angiogenesis. However, the role of FAK in adult pathological angiogenesis is unknown. We have generated endothelial-specific tamoxifen-inducible FAK knockout mice by crossing FAK-floxed (FAKfl/fl) mice with the platelet derived growth factor b (Pdgfb)-iCreER mice. Tamoxifen-treatment of Pdgfb-iCreER;FAKfl/fl mice results in FAK deletion in adult endothelial cells (ECs) without any adverse effects. Importantly however, endothelial FAK-deletion in adult mice inhibited tumour growth and reduced tumour angiogenesis. Furthermore, in in vivo angiogenic assays FAK deletion impairs vascular endothelial growth factor (VEGF)-induced neovascularization. In addition, in vitro deletion of FAK in ECs resulted in reduced VEGF-stimulated Akt phosphorylation and correlating reduced cellular proliferation as well as increased cell death. Our data suggest that FAK is required for adult pathological angiogenesis and validates FAK as a possible target for anti-angiogenic therapies.

Angiopoietin-2 Stimulation of Endothelial Cells Induces αvβ3 Integrin Internalization and Degradation

The angiopoietins (Ang-1 and Ang-2) have been identified as agonistic and antagonistic ligands of the endothelial receptor tyrosine kinase Tie2, respectively. Both ligands have been demonstrated to induce translocation of Tie2 to cell-cell junctions. However, only Ang-1 induces Tie2-dependent Akt activation and subsequent survival signaling and endothelial quiescence. Ang-2 interferes negatively with Ang-1/Tie2 signaling, thereby antagonizing the Ang-1/Tie2 axis. Here, we show that both Ang-1 and Ang-2 recruit beta3 integrins to Tie2. This co-localization is most prominent in cell-cell junctions. However, only Ang-2 stimulation resulted in complex formation among Tie2, alphavbeta3 integrin, and focal adhesion kinase as evidenced by co-immunoprecipitation experiments. Focal adhesion kinase was phosphorylated in the FAT domain at Ser(910) upon Ang-2 stimulation and the adaptor proteins p130Cas and talin dissociated from alphavbeta3 integrin. The alphavbeta3 integrin was internalized, ubiquitinylated, and gated toward lysosomes. Taken together, the experiments define Tie2/alphavbeta3 integrin association-induced integrin internalization and degradation as mechanistic consequences of endothelial Ang-2 stimulation.

Role of Focal Adhesion Kinase Ser-732 Phosphorylation in Centrosome Function during Mitosis

Focal adhesion kinase (FAK) is the major cytoplasmic tyrosine kinase in focal adhesions and a critical mediator of integrin signaling in a variety of cells, including endothelial cells (ECs). Here we describe a new function for FAK in the regulation of centrosome functions in a Ser-732 phosphorylation-dependent manner during mitosis. Deletion of FAK in primary ECs causes increases in centrosome numbers, multipolar and disorganized spindles, and unaligned chromosomes during mitosis. Re-expression of wild-type FAK, but not S732A mutant, rescued these mitotic defects, suggesting a role for Ser-732 phosphorylation in the regulation of centrosomal functions. Consistent with this possibility, Ser-732-phosphorylated FAK was found to co-localize in centrosomes in mitotic cells. FAK also associated with cytoplasmic dynein in a Ser-732 phosphorylation-dependent manner. Further analysis in FAK-null primary ECs showed that S732A mutant could rescue EC migration but not proliferation or tubulogenesis in vitro. Last, we showed that deletion of FAK in ECs reduced tumor angiogenesis in vivo, which could be restored by re-expression of wild-type FAK but not S732A mutant. Together, these studies demonstrated a novel role for Ser-732 phosphorylation of FAK in the regulation of centrosome function during mitosis, which may contribute to EC proliferation and angiogenesis.

Compensatory role for Pyk2 during angiogenesis in adult mice lacking endothelial cell FAK

Focal adhesion kinase (FAK) plays a critical role during vascular development because knockout of FAK in endothelial cells (ECs) is embryonic lethal. Surprisingly, tamoxifen-inducible conditional knockout of FAK in adult blood vessels (inducible EC–specific FAK knockout [i-EC-FAK-KO]) produces no vascular phenotype, and these animals are capable of developing a robust growth factor–induced angiogenic response. Although angiogenesis in wild-type mice is suppressed by pharmacological inhibition of FAK, i-EC-FAK-KO mice are refractory to this treatment, which suggests that adult i-EC-FAK-KO mice develop a compensatory mechanism to bypass the requirement for FAK. Indeed, expression of the FAK-related proline-rich tyrosine kinase 2 (Pyk2) is elevated and phosphorylated in i-EC-FAK-KO blood vessels. In cultured ECs, FAK knockdown leads to increased Pyk2 expression and, surprisingly, FAK kinase inhibition leads to increased Pyk2 phosphorylation. Pyk2 can functionally compensate for the loss of FAK because knockdown or pharmacological inhibition of Pyk2 disrupts angiogenesis in i-EC-FAK-KO mice. These studies reveal the adaptive capacity of ECs to switch to Pyk2-dependent signaling after deletion or kinase inhibition of FAK.

Molecular mechanisms of the vascular responses to haemodynamic forces

Blood vessels are permanently subjected to mechanical forces in the form of stretch, encompassing cyclic mechanical strain due to the pulsatile nature of blood flow and shear stress. Significant variations in mechanical forces, of physiological or physiopathological nature, occur in vivo. These are accompanied by phenotypical modulation of smooth muscle cells and endothelial cells, producing structural modifications of the arterial wall. In all the cases, vascular remodelling can be allotted to a modification of the tensional strain or shear, and underlie a trend to reestablish baseline mechanical conditions. Vascular cells are equipped with numerous receptors that allow them to detect and respond to the mechanical forces generated by pressure and shear stress. The cytoskeleton and other structural components have an established role in mechanotransduction, being able to transmit and modulate tension within the cell via focal adhesion sites, integrins, cellular junctions and the extracellular matrix. Mechanical forces also initiate complex signal transduction cascades, including nuclear factor-kappaB and mitogen-activated protein kinase pathways, leading to functional changes within the cell.

Conditional knockout of focal adhesion kinase in endothelial cells reveals its role in angiogenesis and vascular development in late embryogenesis

Focal adhesion kinase (FAK) is a critical mediator of signal transduction by integrins and growth factor receptors in a variety of cells including endothelial cells (ECs). Here, we describe EC-specific knockout of FAK using a Cre-loxP approach. In contrast to the total FAK knockout, deletion of FAK specifically in ECs did not affect early embryonic development including normal vasculogenesis. However, in late embryogenesis, FAK deletion in the ECs led to defective angiogenesis in the embryos, yolk sac, and placenta, impaired vasculature and associated hemorrhage, edema, and developmental delay, and late embryonic lethal phenotype. Histologically, ECs and blood vessels in the mutant embryos present a disorganized, detached, and apoptotic appearance. Consistent with these phenotypes, deletion of FAK in ECs isolated from the floxed FAK mice led to reduced tubulogenesis, cell survival, proliferation, and migration in vitro. Together, these results strongly suggest a role of FAK in angiogenesis and vascular development due to its essential function in the regulation of multiple EC activities.