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Focal Adhesion Kinase (FAK) tyrosine 397E mutation restores the vascular leakage defect in endothelium‐specific FAK‐kinase dead mice

dc.contributor.authorAlexopoulou, AN
dc.contributor.authorLees, DM
dc.contributor.authorBodrug, N
dc.contributor.authorLechertier, T
dc.contributor.authorFernandez, I
dc.contributor.authorD'Amico, G
dc.contributor.authorDukinfield, M
dc.contributor.authorBatista, S
dc.contributor.authorTavora, B
dc.contributor.authorSerrels, B
dc.contributor.authorHodivala‐Dilke, K
dc.date.accessioned2018-06-28T09:08:00Z
dc.date.issued2017-07-01
dc.identifierhttp://onlinelibrary.wiley.com/doi/10.1002/path.4911/abstract
dc.identifier.citationJOURNAL OF PATHOLOGY, 2017, 242 (3), pp. 358 - 370
dc.identifier.issn0022-3417
dc.identifier.urihttps://repository.icr.ac.uk/handle/internal/1959
dc.description.abstract<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 &amp; Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.</jats:p>
dc.format.extent358 - 370
dc.languageeng
dc.language.isoeng
dc.publisherWiley
dc.rights.urihttps://creativecommons.org/licenses/by/4.0
dc.subjecttumour angiogenesis focal adhesion kinase SMOOTH-MUSCLE-CELLS SRC-FAMILY KINASES TUMOR ANGIOGENESIS BARRIER FUNCTION PHOSPHORYLATION PERMEABILITY ACTIVATION CANCER AUTOPHOSPHORYLATION LOCALIZATION
dc.titleFocal Adhesion Kinase (FAK) tyrosine 397E mutation restores the vascular leakage defect in endothelium‐specific FAK‐kinase dead mice
dc.typeJournal Article
rioxxterms.licenseref.urihttps://creativecommons.org/licenses/by/4.0
rioxxterms.licenseref.startdate2017
rioxxterms.typeJournal Article/Review
dc.relation.isPartOfJOURNAL OF PATHOLOGY
pubs.issue3
pubs.notesISI Document Delivery No.: EX7US Times Cited: 0 Cited Reference Count: 44 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 Cancer Research UK [8218/A18673] We thank Julie Holdsworth and Bruce Williams for their help with in vivo experiments, George Elia for histological expertise, and Elisabetta DeJana for the polyclonal antibody against VECAD-pY658. Funding was received from Cancer Research UK 8218/A18673. 0 WILEY HOBOKEN J PATHOL none 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(ert)-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(WT/WT), ECCre+;FAK(KD/KD) and ECCre+;FAK(DM/DM) mice were injected subcutaneously with syngeneic B16F0 melanoma cells. Tumour growth and tumour blood vessel functions were unchanged between ECCre+;FAK(WT/WT) and ECCre-;FAK(WT/WT) control mice. In contrast, tumour growth and vessel density were decreased in ECCre+;FAK(KD/KD) and ECCre+;FAK(DM/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+;FAK(KD/KD) and ECCre+;FAK(DM/DM) mice. Furthermore, although ECCre+;FAK(KD/KD) mice showed reduced blood vessel leakage, ECCre+;FAK(DM/DM) and ECCre-;FAK(DM/DM) mice showed no difference in leakage. Mechanistically, fibronectin-stimulated Y397 autophosphorylation was reduced in Cre+;FAK(KD/KD) ECs as compared with Cre+;FAK(WT/WT) cells, with no change in phosphorylation of the known Src targets FAK-Y577, FAK-Y861, FAK-Y925, paxillin-Y118, p130Cas-Y410. Cre+;FAK(DM/DM) ECs showed decreased Src target phosphorylation levels, suggesting that the Y397E substitution actually disrupted Src activation. Reduced VE-cadherin-pY658 levels in Cre+;FAK(KD/KD) ECs were rescued in Cre+ FAK(DM/DM) ECs, corresponding with the rescue in vessel leakage in the ECCre+;FAK(DM/DM) mice. We show that EC-specific FAK kinase activity is required for tumour growth, angiogenesis, and vascular permeability. The ECCre+;FAK(DM/DM) mice restored the KD-dependent tumour vascular leakage observed in ECCre+;FAK(KD/KD) mice in vivo. This study opens new fields in in vivo FAK signalling. (C) 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
pubs.notesNot known
pubs.organisational-group/ICR
pubs.organisational-group/ICR/Primary Group
pubs.organisational-group/ICR/Primary Group/ICR Divisions
pubs.organisational-group/ICR/Primary Group/ICR Divisions/Cancer Therapeutics
pubs.organisational-group/ICR/Primary Group/ICR Divisions/Cancer Therapeutics/Signal Transduction & Molecular Pharmacology
pubs.organisational-group/ICR
pubs.organisational-group/ICR/Primary Group
pubs.organisational-group/ICR/Primary Group/ICR Divisions
pubs.organisational-group/ICR/Primary Group/ICR Divisions/Cancer Therapeutics
pubs.organisational-group/ICR/Primary Group/ICR Divisions/Cancer Therapeutics/Signal Transduction & Molecular Pharmacology
pubs.volume242
pubs.embargo.termsNot known
icr.researchteamSignal Transduction & Molecular Pharmacology
dc.contributor.icrauthorAires Batista, Silvia


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