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‐ iCre ert ‐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 +; FAK WT / WT , ECCre +; FAK KD / KD and ECCre +; FAK DM / DM mice were injected subcutaneously with syngeneic B16F0 melanoma cells. Tumour growth and tumour blood vessel functions were unchanged between ECCre +; FAK WT / WT and ECCre −; FAK WT / WT control mice. In contrast, tumour growth and vessel density were decreased in ECCre +; FAK KD / KD and ECCre +; FAK DM / 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 +; FAK KD / KD and ECCre +; FAK DM / DM mice. Furthermore, although ECCre +; FAK KD / KD mice showed reduced blood vessel leakage, ECCre +; FAK DM / DM and ECCre −; FAK DM / DM mice showed no difference in leakage. Mechanistically, fibronectin‐stimulated Y397 autophosphorylation was reduced in Cre+; FAK KD / KD ECs as compared with Cre+; FAK WT / WT cells, with no change in phosphorylation of the known Src targets FAK‐Y577 , FAK‐Y861 , FAK‐Y925 , paxillin‐ Y118 , p130Cas‐Y410. Cre+; FAK DM / DM ECs showed decreased Src target phosphorylation levels, suggesting that the Y397E substitution actually disrupted Src activation. Reduced VE ‐cadherin‐ pY658 levels in Cre+; FAK KD / KD ECs were rescued in Cre+ FAK DM / DM ECs , corresponding with the rescue in vessel leakage in the ECCre +; FAK DM / DM mice. We show that EC ‐specific FAK kinase activity is required for tumour growth, angiogenesis, and vascular permeability. The ECCre+;FAK DM/DM mice restored the KD‐dependent tumour vascular leakage observed in ECCre+;FAK KD/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.