Tumour vascular leakage defect due to endothelium‐targeted FAK‐kinase dead mutation is abrogated by FAK‐Y397E expression

Focal adhesion kinase (FAK) inhibitors have been developed as potential anti-cancer agents and are in clinical trials. In vitro activation of FAK kinase domain triggers autophosphorylation of tyrosine 397 (Y397), Src activation and subsequent phosphorylation of other FAK tyrosine residues. However, how FAK-Y397 mutations affect FAK-kinase-dead 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 kinase-dead (KD), and double mutant (DM); 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, compared to 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 showed no difference in leakage. Mechanistically, fibronectin-stimulated Y397 autophosphorylation was reduced in Cre+;FAKKD/KD ECs when compared with Cre+;FAKWT/WT cells, with no change in phosphorylation of known Src targets FAK(Y577), FAK(Y861), FAK(Y925), paxillin(Y118) or Cas(Y410). Cre+;FAKDM/DM ECs showed decreased Src target phosphorylation levels, suggesting that the Y397E substitution actually disrupted Src activation. Reduced VECAD-pY658 levels in Cre+;FAKKD/KD ECs was 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.