Focal Adhesion Kinase (FAK) tyrosine 397E mutation restores the vascular leakage defect in endothelium‐specific FAK‐kinase dead mice
View/ Open
Date
2017-07-01ICR Author
Author
Alexopoulou, AN
Lees, DM
Bodrug, N
Lechertier, T
Fernandez, I
D'Amico, G
Dukinfield, M
Batista, S
Tavora, B
Serrels, B
Hodivala‐Dilke, K
Type
Journal Article
Metadata
Show full item recordAbstract
<jats:title>Abstract</jats:title><jats:p>Focal adhesion kinase (<jats:styled-content style="fixed-case">FAK</jats:styled-content>) inhibitors have been developed as potential anticancer agents and are undergoing clinical trials. <jats:italic>In vitro</jats:italic> activation of the <jats:styled-content style="fixed-case">FAK</jats:styled-content> kinase domain triggers autophosphorylation of <jats:styled-content style="fixed-case">Y397</jats:styled-content>, Src activation, and subsequent phosphorylation of other <jats:styled-content style="fixed-case">FAK</jats:styled-content> tyrosine residues. However, how <jats:styled-content style="fixed-case">FAK Y397</jats:styled-content> mutations affect <jats:styled-content style="fixed-case">FAK</jats:styled-content> kinase‐dead (<jats:styled-content style="fixed-case">KD</jats:styled-content>) phenotypes in tumour angiogenesis <jats:italic>in vivo</jats:italic> is unknown. We developed three Pdgfb‐<jats:styled-content style="fixed-case">iCre<jats:sup>ert</jats:sup></jats:styled-content>‐driven endothelial cell (<jats:styled-content style="fixed-case">EC</jats:styled-content>)‐specific, tamoxifen‐inducible homozygous mutant mouse lines: <jats:styled-content style="fixed-case">FAK</jats:styled-content> wild‐type (<jats:styled-content style="fixed-case">WT</jats:styled-content>), <jats:styled-content style="fixed-case">FAK KD</jats:styled-content>, and <jats:styled-content style="fixed-case">FAK</jats:styled-content> double mutant (<jats:styled-content style="fixed-case">DM</jats:styled-content>), i.e. <jats:styled-content style="fixed-case">KD</jats:styled-content> with a putatively phosphomimetic <jats:styled-content style="fixed-case">Y397E</jats:styled-content> mutation. These <jats:styled-content style="fixed-case">ECCre</jats:styled-content>+;<jats:styled-content style="fixed-case">FAK<jats:sup>WT</jats:sup></jats:styled-content><jats:sup>/</jats:sup><jats:styled-content style="fixed-case"><jats:sup>WT</jats:sup></jats:styled-content>, <jats:styled-content style="fixed-case">ECCre</jats:styled-content>+;<jats:styled-content style="fixed-case">FAK<jats:sup>KD</jats:sup></jats:styled-content><jats:sup>/</jats:sup><jats:styled-content style="fixed-case"><jats:sup>KD</jats:sup></jats:styled-content> and <jats:styled-content style="fixed-case">ECCre</jats:styled-content>+;<jats:styled-content style="fixed-case">FAK<jats:sup>DM</jats:sup></jats:styled-content><jats:sup>/</jats:sup><jats:styled-content style="fixed-case"><jats:sup>DM</jats:sup></jats:styled-content> mice were injected subcutaneously with syngeneic <jats:styled-content style="fixed-case">B16F0</jats:styled-content> melanoma cells. Tumour growth and tumour blood vessel functions were unchanged between <jats:styled-content style="fixed-case">ECCre</jats:styled-content>+;<jats:styled-content style="fixed-case">FAK<jats:sup>WT</jats:sup></jats:styled-content><jats:sup>/</jats:sup><jats:styled-content style="fixed-case"><jats:sup>WT</jats:sup></jats:styled-content> and <jats:styled-content style="fixed-case">ECCre</jats:styled-content>−;<jats:styled-content style="fixed-case">FAK<jats:sup>WT</jats:sup></jats:styled-content><jats:sup>/</jats:sup><jats:styled-content style="fixed-case"><jats:sup>WT</jats:sup></jats:styled-content> control mice. In contrast, tumour growth and vessel density were decreased in <jats:styled-content style="fixed-case">ECCre</jats:styled-content>+;<jats:styled-content style="fixed-case">FAK<jats:sup>KD</jats:sup></jats:styled-content><jats:sup>/</jats:sup><jats:styled-content style="fixed-case"><jats:sup>KD</jats:sup></jats:styled-content> and <jats:styled-content style="fixed-case">ECCre</jats:styled-content>+;<jats:styled-content style="fixed-case">FAK<jats:sup>DM</jats:sup></jats:styled-content><jats:sup>/</jats:sup><jats:styled-content style="fixed-case"><jats:sup>DM</jats:sup></jats:styled-content> 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 <jats:styled-content style="fixed-case">ECCre</jats:styled-content>+;<jats:styled-content style="fixed-case">FAK<jats:sup>KD</jats:sup></jats:styled-content><jats:sup>/</jats:sup><jats:styled-content style="fixed-case"><jats:sup>KD</jats:sup></jats:styled-content> and <jats:styled-content style="fixed-case">ECCre</jats:styled-content>+;<jats:styled-content style="fixed-case">FAK<jats:sup>DM</jats:sup></jats:styled-content><jats:sup>/</jats:sup><jats:styled-content style="fixed-case"><jats:sup>DM</jats:sup></jats:styled-content> mice. Furthermore, although <jats:styled-content style="fixed-case">ECCre</jats:styled-content>+;<jats:styled-content style="fixed-case">FAK<jats:sup>KD</jats:sup></jats:styled-content><jats:sup>/</jats:sup><jats:styled-content style="fixed-case"><jats:sup>KD</jats:sup></jats:styled-content> mice showed reduced blood vessel leakage, <jats:styled-content style="fixed-case">ECCre</jats:styled-content>+;<jats:styled-content style="fixed-case">FAK<jats:sup>DM</jats:sup></jats:styled-content><jats:sup>/</jats:sup><jats:styled-content style="fixed-case"><jats:sup>DM</jats:sup></jats:styled-content> and <jats:styled-content style="fixed-case">ECCre</jats:styled-content>−;<jats:styled-content style="fixed-case">FAK<jats:sup>DM</jats:sup></jats:styled-content><jats:sup>/</jats:sup><jats:styled-content style="fixed-case"><jats:sup>DM</jats:sup></jats:styled-content> mice showed no difference in leakage. Mechanistically, fibronectin‐stimulated <jats:styled-content style="fixed-case">Y397</jats:styled-content> autophosphorylation was reduced in Cre+;<jats:styled-content style="fixed-case">FAK<jats:sup>KD</jats:sup></jats:styled-content><jats:sup>/</jats:sup><jats:styled-content style="fixed-case"><jats:sup>KD</jats:sup> ECs</jats:styled-content> as compared with Cre+;<jats:styled-content style="fixed-case">FAK<jats:sup>WT</jats:sup></jats:styled-content><jats:sup>/</jats:sup><jats:styled-content style="fixed-case"><jats:sup>WT</jats:sup></jats:styled-content> cells, with no change in phosphorylation of the known Src targets <jats:styled-content style="fixed-case">FAK‐Y577</jats:styled-content>, <jats:styled-content style="fixed-case">FAK‐Y861</jats:styled-content>, <jats:styled-content style="fixed-case">FAK‐Y925</jats:styled-content>, paxillin‐<jats:styled-content style="fixed-case">Y118</jats:styled-content>, p130Cas‐Y410. Cre+;<jats:styled-content style="fixed-case">FAK<jats:sup>DM</jats:sup></jats:styled-content><jats:sup>/</jats:sup><jats:styled-content style="fixed-case"><jats:sup>DM</jats:sup> ECs</jats:styled-content> showed decreased Src target phosphorylation levels, suggesting that the <jats:styled-content style="fixed-case">Y397E</jats:styled-content> substitution actually disrupted Src activation. Reduced <jats:styled-content style="fixed-case">VE</jats:styled-content>‐cadherin‐<jats:styled-content style="fixed-case">pY658</jats:styled-content> levels in Cre+;<jats:styled-content style="fixed-case">FAK<jats:sup>KD</jats:sup></jats:styled-content><jats:sup>/</jats:sup><jats:styled-content style="fixed-case"><jats:sup>KD</jats:sup> ECs</jats:styled-content> were rescued in Cre+<jats:styled-content style="fixed-case">FAK<jats:sup>DM</jats:sup></jats:styled-content><jats:sup>/</jats:sup><jats:styled-content style="fixed-case"><jats:sup>DM</jats:sup> ECs</jats:styled-content>, corresponding with the rescue in vessel leakage in the <jats:styled-content style="fixed-case">ECCre</jats:styled-content>+;<jats:styled-content style="fixed-case">FAK<jats:sup>DM</jats:sup></jats:styled-content><jats:sup>/</jats:sup><jats:styled-content style="fixed-case"><jats:sup>DM</jats:sup></jats:styled-content> mice. We show that <jats:styled-content style="fixed-case">EC</jats:styled-content>‐specific <jats:styled-content style="fixed-case">FAK</jats:styled-content> kinase activity is required for tumour growth, angiogenesis, and vascular permeability. The ECCre+;FAK<jats:sup>DM/DM</jats:sup> mice restored the KD‐dependent tumour vascular leakage observed in ECCre+;FAK<jats:sup>KD/KD</jats:sup> mice <jats:italic>in vivo</jats:italic>. This study opens new fields in <jats:italic>in vivo</jats:italic> FAK signalling. © 2017 The Authors. <jats:italic>The Journal of Pathology</jats:italic> published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.</jats:p>
Collections
Subject
tumour angiogenesis focal adhesion kinase SMOOTH-MUSCLE-CELLS SRC-FAMILY KINASES TUMOR ANGIOGENESIS BARRIER FUNCTION PHOSPHORYLATION PERMEABILITY ACTIVATION CANCER AUTOPHOSPHORYLATION LOCALIZATION
Research team
Signal Transduction & Molecular Pharmacology
Language
eng
License start date
2017
Citation
JOURNAL OF PATHOLOGY, 2017, 242 (3), pp. 358 - 370
Publisher
Wiley