The β3-integrin endothelial adhesome regulates microtubule dependent cell migration

Abstract

Integrin β3 is seen as a key anti-angiogenic target for cancer treatment due to its expression on neovasculature, but the role it plays in the process is complex; whether it is pro-or anti-angiogenic depends on the context in which it is expressed. To understand precisely β3's role in regulating integrin adhesion complexes in endothelial cells, we characterised, by mass spectrometry, the β3-dependent adhesome. We show that depletion of β3-integrin in this cell type leads to changes in microtubule behaviour that control their migration. β3-integrin regulates microtubule stability in endothelial cells through Rcc2/Anxa2 driven control of Rac1 activity. Our findings reveal that angiogenic processes, both in vitro and in vivo, are more sensitive to microtubule targeting agents when β3-integrin levels are reduced. Affliations:1 School of Biological Sciences, University of East Anglia, Norwich, UK. (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/145839 doi: bioRxiv preprint first posted online Jun. 3, 2017; 2 INTRODUCTIONAngiogenesis, the formation of new blood vessels from those that already exist, plays an essential role in tumour growth (Hanahan and Weinberg, 2011). As such, targeting angiogenesis is seen as crucial in many anti-cancer strategies (Zhao and Adjei, 2015).Therapies directed against vascular endothelial growth factor (VEGF) and its major receptor, VEGF-receptor-2 (VEGFR2), whilst effective in a number of cancers, are not without side-effects due to the role this signaling pathway plays in vascular homeostasis (Chen and Hung, 2013). Fibronectin (FN)-binding endothelial integrins, especially αvβ3-and α5β1-integrins, have emerged as alternative anti-angiogenic targets because of their expression in neovasculature (Brooks et al., 1994;Kim et al., 2000). However, neither global nor conditional knockouts of these integrins block tumour angiogenesis long-term (Murphy et al., 2015;Reynolds et al., 2002;Steri et al., 2014), and clinical trials of blocking antibodies and peptides directed against these extracellular matrix (ECM) receptors have been disappointing (Schaffner et al., 2013; Stupp et al., 2014).To gain novel insight into how αvβ3-integrin regulates outside-in signal transmission (Hynes, 2002), we have undertaken an unbiased analysis of the molecular composition of the mature endothelial adhesome (the network of structural and signaling proteins involved in regulating cell-matrix adhesions (Zaidel-Bar et al., 2007)), and profiled changes that occur when β3-integrin function or expression are manipulated. In so doing, we have uncovered β3-integrin dependent changes in microtubule behaviour that regulate cell migration. RESULTS AND DISCUSSIONThe isolation and analysis of integrin adhesion complexes (IACs) by massspectrometry (MS) is difficult because of the low affinity and transient nature of the molecular interactions occurring at these sites. However, usi...

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