Fibronectin-binding integrins participate in the control of GEF-H1 activation and its role in breast cancer

Abstract

Rho family of small GTPases plays a crucial role in several biolog- ical processes and cycles between on and off states. This switch is controlled by GEFs (Rho-activators) and GAPs (Rho-inhibitors). We have previously identified by Mass Spectrometry (MS) analysis that GEF-H1 is the major GEF that activates RhoA and we observed a significant GEF-H1 overexpression in human breast cancer biop- sies compared with normal breast tissue. The aim of this work is to study the role of GEF-H1 depletion in tumor development and the mechanism of GEF-H1 fibronectin (FN)-binding integrins de- pendent activation. Using CRISPR/Cas9 technology, we generated GEF-H1-knock out (KO) cells in a murine invasive breast cancer cell line (LM3). GEF-H1-KO cells were implanted subcutaneously in the mammary fat pad of BALB/c mice, showing a significant decrease in tumor formation (n=14, p<0.0004), lung metastasis development (n=12, p<0.0348) and an increase in apoptosis (BAX staining n=12, p<0.0001) compared with mice inoculated with WT cancer cells. In order to study the mechanism of GEF-H1 activation, we used mouse fibroblasts that only express FN-binding integrins (pKO-α5β1 or -αvβ3). After immunostaining we observed that αvβ3 integrins trig- gers the dissociation of GEF-H1 from microtubules, leading to RhoA activation, stress fibers (SF) formation and focal adhesions (FA) maturation. The opposite effects were observed after GEF-H1 de- pletion, suggesting that αvβ3 integrins control RhoA activity thought GEF-H1 activation. After analyzing pKO-fibroblast MS-phosphopro- teomics data and looking in several phospho-sites databases, com- bined with different kinase inhibitors assays, we found MARK2/3 as a candidate for GEF-H1 phosphorylation. Indeed, S151 was found as the main site involved in the regulation of GEF-H1 localization and activity. These findings indicate that activation of GEF-H1/RhoA is orchestrated in FN-adherent cells in an integrin-specific manner and promotes tumor and metastatic processes.