Endothelial specific inactivation of FAK-Y397 and FAK-Y861 phosphorylation in tumour growth and angiogenesis in vivo
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Tumour angiogenesis is a hallmark of cancer. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase involved in endothelial cells (ECs) survival, proliferation and migration. FAK has several tyrosine phosphorylation sites thought to be involved in FAK function but the requirement of phosphorylation of these residues in vivo is unknown. We have generated mice where endogenous FAK is deleted simultaneously with the expression of nonphosphorylatable FAK-Y397F or FAK-Y861F mutated or wild type forms of FAK in adult endothelium in order to test this. My data show that EC-FAK-Y397FKI mice present with decreased tumour angiogenesis (in sygeneic B16F0, CMT19T and LLC) but impaired B16F0 and CMT19T tumour growth only, with increased tumour hypoxia. FAK-Y397F tumour endothelium is not perfusion, leakage or vascular maturation defective. This mutation affects VEGF-, PlGF- and bFGF-driven angiogenesis in vivo and VEGF+Ang2 administration is able to partially rescue this phenotype ex vivo. In contrast, endothelial FAK-Y861F mutation leads to an initial delay in B16F0 tumour angiogenesis, that subsequently resolves, and does not affect B16F0 tumour growth. LLC and CMT19T tumour growth and angiogenesis are not affected by the endothelial FAK-Y861F mutation; neither are tumour blood vessel perfusion, leakage, vascular maturation or tumour hypoxia. VEGF-, PlGFand bFGF-driven angiogenesis in vivo and ex vivo was not affected by the endothelial FAK-Y861F mutation, whereas increased in vivo angiogenesis was triggered by Ang2 administration. Lastly, to understand whether cytokine profiles that might affect angiocrine signalling are affected differentially in FAK-Y397F vs FAK-Y861F endothelial cells, I show that CCL1 and CCL2 are increased in FAK-Y397F but IL- 13, IL-1F3, CCL4, IL-1F1, CCL2 and others are increased in FAK-Y861F endothelial cells. Overall my data indicates that endothelial-specific FAK mutations on two phosphorylation sites has different effects on tumour angiogenesis, tumour growth, growth factor stimulated angiogenesis in vivo and ex-vivo and cytokine production.
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