Focal Adhesion Kinase (FAK) tyrosine 397E mutation restores the vascular leakage defect in endothelium‐specific FAK‐kinase dead mice

Abstract

Focal adhesion kinase (FAK) inhibitors have been developed as potential anticancer agents and are undergoing clinical trials. In vitro activation of the FAK kinase domain triggers autophosphorylation of Y397, Src activation, and subsequent phosphorylation of other FAK tyrosine residues. However, how FAK Y397 mutations affect FAK kinase‐dead (KD) phenotypes in tumour angiogenesis in vivo is unknown. We developed three Pdgfb‐iCreert‐driven endothelial cell (EC)‐specific, tamoxifen‐inducible homozygous mutant mouse lines: FAK wild‐type (WT), FAK KD, and FAK double mutant (DM), i.e. KD with a putatively phosphomimetic Y397E mutation. These ECCre+;FAKWT / WT, ECCre+;FAKKD / KD and ECCre+;FAKDM / DM mice were injected subcutaneously with syngeneic B16F0 melanoma cells. Tumour growth and tumour blood vessel functions were unchanged between ECCre+;FAKWT / WT and ECCre−;FAKWT / WT control mice. In contrast, tumour growth and vessel density were decreased in ECCre+;FAKKD / KD and ECCre+;FAKDM / DM mice, as compared with Cre − littermates. Despite no change in the percentage of perfused vessels or pericyte coverage in either genotype, tumour hypoxia was elevated in ECCre+;FAKKD / KD and ECCre+;FAKDM / DM mice. Furthermore, although ECCre+;FAKKD / KD mice showed reduced blood vessel leakage, ECCre+;FAKDM / DM and ECCre−;FAKDM / DM mice showed no difference in leakage. Mechanistically, fibronectin‐stimulated Y397 autophosphorylation was reduced in Cre+;FAKKD / KD ECs as compared with Cre+;FAKWT / WT cells, with no change in phosphorylation of the known Src targets FAK‐Y577, FAK‐Y861, FAK‐Y925, paxillin‐Y118, p130Cas‐Y410. Cre+;FAKDM / DM ECs showed decreased Src target phosphorylation levels, suggesting that the Y397E substitution actually disrupted Src activation. Reduced VE‐cadherin‐pY658 levels in Cre+;FAKKD / KD ECs were rescued in Cre+FAKDM / DM ECs, corresponding with the rescue in vessel leakage in the ECCre+;FAKDM / DM mice. We show that EC‐specific FAK kinase activity is required for tumour growth, angiogenesis, and vascular permeability. The ECCre+;FAKDM/DM mice restored the KD‐dependent tumour vascular leakage observed in ECCre+;FAKKD/KD mice in vivo. This study opens new fields in in vivo FAK signalling. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

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

“…We next sought to investigate how FAK-Y397F and FAK-Y861F mutations differentially affect downstream signaling and compare these responses with our previously published FAK-kinase dead (FAK-KD) mutant ECs (32). Western blot analysis of FAK-Y397F and FAK-KD ECs showed reduced FAK phosphorylation at Y397, Y577, Y861, and Y925.…”

The roles of nuclear focal adhesion kinase (FAK) on Cancer: a focused review

“…Furthermore, FAK is essential for maintaining vascular functions in tumor angiogenesis. Lees et al [16] found that FAK recovered the vascular leakage defect through the activation of kinase domain. And it is a fact that cytokines induce vascular growth factor expression by the FAK signaling pathway.…”

Oridonin inhibits the migration and epithelial‐to‐mesenchymal transition of small cell lung cancer cells by suppressing FAK‐ERK1/2 signalling pathway

“…FAK is a 125 kDa non‐receptor protein tyrosine kinase which is located at sites of integrin clustering in focal adhesions; therefore, FAK plays a central role in cellular processes that include migration and adhesion . FAK has many phosphorylated tyrosine sites, and these sites are key for the signal transduction function of FAK.…”