Alexopoulou Annika N, Lees Delphine M, Bodrug Natalia, Lechertier Tanguy, Fernandez Isabelle, D'Amico Gabriela, Dukinfield Matthew, Batista Silvia, Tavora Bernardo, Serrels Bryan, Hodivala-Dilke Kairbaan
Department of Molecular Oncology, BSRC Alexander Fleming, Athens, Greece.
Centre for Tumour Biology, Barts Cancer Institute, Queen Mary University of London, London, UK.
J Pathol. 2017 Jul;242(3):358-370. doi: 10.1002/path.4911. Epub 2017 Jun 1.
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 -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 , ECCre+;FAK and ECCre+;FAK mice were injected subcutaneously with syngeneic B16F0 melanoma cells. Tumour growth and tumour blood vessel functions were unchanged between ECCre+;FAK and ECCre-;FAK control mice. In contrast, tumour growth and vessel density were decreased in ECCre+;FAK and ECCre+;FAK 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 and ECCre+;FAK mice. Furthermore, although ECCre+;FAK mice showed reduced blood vessel leakage, ECCre+;FAK and ECCre-;FAK mice showed no difference in leakage. Mechanistically, fibronectin-stimulated Y397 autophosphorylation was reduced in Cre+;FAK ECs as compared with Cre+;FAK cells, with no change in phosphorylation of the known Src targets FAK-Y577, FAK-Y861, FAK-Y925, paxillin-Y118, p130Cas-Y410. Cre+;FAK ECs showed decreased Src target phosphorylation levels, suggesting that the Y397E substitution actually disrupted Src activation. Reduced VE-cadherin-pY658 levels in Cre+;FAK ECs were rescued in Cre+FAK ECs, corresponding with the rescue in vessel leakage in the ECCre+;FAK mice. We show that EC-specific FAK kinase activity is required for tumour growth, angiogenesis, and vascular permeability. The ECCre+;FAK mice restored the KD-dependent tumour vascular leakage observed in ECCre+;FAK 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.
粘着斑激酶（FAK）抑制剂已被开发为潜在的抗癌药物，目前正在进行临床试验。FAK激酶结构域的体外激活会触发Y397的自磷酸化、Src激活以及随后其他FAK酪氨酸残基的磷酸化。然而，FAK Y397突变如何影响体内肿瘤血管生成中FAK激酶失活（KD）表型尚不清楚。我们构建了三种由Pdgfb-iCre驱动的内皮细胞（EC）特异性、他莫昔芬诱导的纯合突变小鼠品系：FAK野生型（WT）、FAK KD和FAK双突变体（DM），即带有假定磷酸模拟Y397E突变的KD。将这些ECCre+;FAK、ECCre+;FAK和ECCre+;FAK小鼠皮下注射同基因B16F0黑色素瘤细胞。ECCre+;FAK和ECCre-;FAK对照小鼠之间的肿瘤生长和肿瘤血管功能没有变化。相比之下，与Cre-同窝小鼠相比，ECCre+;FAK和ECCre+;FAK小鼠的肿瘤生长和血管密度降低。尽管两种基因型中灌注血管百分比或周细胞覆盖没有变化，但ECCre+;FAK和ECCre+;FAK小鼠的肿瘤缺氧情况有所升高。此外，虽然ECCre+;FAK小鼠的血管渗漏减少，但ECCre+;FAK和ECCre-;FAK小鼠在渗漏方面没有差异。从机制上讲，与Cre+;FAK细胞相比，Cre+;FAK内皮细胞中纤连蛋白刺激的Y397自磷酸化减少，已知的Src靶点FAK-Y577、FAK-Y861、FAK-Y925、桩蛋白-Y118、p130Cas-Y410的磷酸化没有变化。Cre+;FAK内皮细胞显示Src靶点磷酸化水平降低，表明Y397E替代实际上破坏了Src激活。Cre+;FAK内皮细胞中降低的VE-钙粘蛋白-pY658水平在Cre+FAK内皮细胞中得到挽救，这与ECCre+;FAK小鼠血管渗漏的挽救相对应。我们表明，内皮细胞特异性FAK激酶活性是肿瘤生长、血管生成和血管通透性所必需的。ECCre+;FAK小鼠在体内恢复了ECCre+;FAK小鼠中观察到的KD依赖性肿瘤血管渗漏。这项研究开辟了体内FAK信号传导的新领域。© 2017作者。《病理学杂志》由John Wiley & Sons Ltd代表大不列颠及爱尔兰病理学会出版。
