Abstract 389: Mice with kinase-dead FAK mutation in endothelial cells are protected from VEGF-induced permeability and are resistant to tumor cell metastasis
Authors: Nam Ju-Ock , Acevedo Lisette M. , Goka Erik , Chen Xiao Lei , Walsh Colin , Tanjoni Isabelle , Guan Jun-Lin , Weis Sara M. , Cheresh David A. , Schlaepfer David D. 
Published: February 2011

Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that facilitates signaling downstream of integrin and growth factor cell surface receptors. During development, complete or conditional inactivation of FAK expression results in embryonic lethality associated with blood vessel defects. We previously reported that tamoxifen-inducible, Cre-mediated FAK deletion from adult endothelial cells (ECs) (i-EC-FAK-KO mice) is surprisingly not lethal due to functional compensation by the FAK-related proline-rich tyrosine kinase 2 (Pyk2) (Weis et al. J. Cell Biol. 181: 43-50, 2008).

Integrins and FAK participate in vascular endothelial growth factor (VEGF)-induced EC responses, but the role of FAK signaling downstream of VEGF remains undetermined. Rather than knocking out FAK expression, we created a knockin mouse with a kinase-dead (KD) point mutation in FAK by homologus recombination. Homozygous FAK KD knockin is embryonic lethal (Lim et al., unpublished) whereas heterozygous WT/KD mice grow to adulthood. By crossing i-EC-FAK-KO mice with FAK WT/KD mice, we obtained i-EC-FAK-KO/WT (termed WT) and i-EC-FAK-KO/KD (termed KD) mice whereby tamoxifen-inducible Cre expression results in adult ECs expressing WT or KD FAK in a hemizygous manner with no compensatory changes in Pyk2 expression or activity.

To test the requirement for FAK activity in VEGF signaling, tail vein injections of VEGF were performed and heart lysates were analyzed for signaling events after 2 min. Whereas VEGF Receptor 2 was activated in both WT and KD mice, tyrosine phosphorylation of FAK and FAK substrates including p130Cas, paxillin, c-Src, and β-catenin were decreased in KD mice. Identical results were obtained in cultured human ECs (HUVEC) upon VEGF stimulation in the presence of a small molecule FAK inhibitor (PF-271). Notably, inhibition of FAK activity prevented the dissociation of VE-cadherin and β-catenin complexes after VEGF stimulation in vivo and in vitro. As these junctional proteins regulate EC barrier function, WT and KD mice were tested for the ability of VEGF to induce vascular permeability in vivo. KD mice showed significantly less (p&lt;0.02, n=11) vascular leak than WT mice indicating that FAK activity is required for EC permeability induced by VEGF.

As vascular leak can accompany and enhance tumor cell extravasation and metastasis, murine B16F10 melanoma cells were injected intravenously in WT and KD mice. Analyses revealed significantly fewer experimental lung metastases in KD mice (2-fold difference, p&lt;0.001). Taken together, our results provide the first in vivo evidence that FAK activity is required for VEGF-induced permeability, supporting the notion that small molecule FAK inhibitors may represent a novel approach to block the permeability-inducing effects of VEGF that exacerbate the progression of cancer, metastasis, or ischemic disease.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 389.