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.
Highlights
We have previously shown that Pdgfb-iCreertinducible endothelial cell (EC)-specific homozygous deletion of Focal adhesion kinase (FAK) leads to reduced primary tumour growth and angiogenesis [2]
Immunohistochemical analysis of tumour sections from ECCre−;FAKDM/double mutant (DM) and ECCre+;FAKDM/DM mice again showed no significant differences in blood vessel perfusion or NG2-positive pericyte association (Figure 5A, B), to the results shown in Figure 4A, B
We showed previously that endothelial FAK is involved in tumour growth and angiogenesis [2]
Summary
We have previously shown that Pdgfb-iCreertinducible endothelial cell (EC)-specific homozygous deletion of FAK leads to reduced primary tumour growth and angiogenesis [2]. Other reports have indicated that hemizygous EC-specific FAK kinase-dead (KD) mice show no effect on primary tumour growth, but show reduced vascular endothelial growth factor (VEGF)-stimulated vascular permeability [6] and metastasis [7]. These data have supported the development of pharmacological FAK kinase inhibitors. Mice that are haploinsufficient for FAK expression show increased tumour growth and angiogenesis, indicating a dose-dependent role for FAK [9], and this has raised the notion that homozygous FAK KD animals may show a different tumour angiogenic phenotype in vivo
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