Role of RHOA-activator in breast cancer progression

Abstract

Cells express different classes of fibronectin (FN)-binding integrins, which induce signalling pathways that integrate Rho-GTPases to convert integrin signals into specific effector functions. The aim of this work is to study the relationship between differential expression of integrins and specific activation of Rho-GTPases in cancer. Using genetically engineered cells, we observed that α5β1-integrins pro- moted the formation of small adhesions and low RhoA activation, while αVβ3-expressing cells showed large adhesions, thick stress fibers and high RhoA activation. To further analyse these cellular phenotypes, we looked for specific RhoA activators (GEFs). For this purpose, we performed Mass Spectrometry analysis follow by bio- chemical assays and observed that GEF-H1 activation is αVβ3-in- tegrin dependent. By bioinformatic analysis using a mRNA dataset (Oncomine) we found high GEF-H1 expression in human breast cancer compared with non-tumoral breast tissue. In addition, high GEF-H1 expression correlated with a lower patient survival (n= 65, p=0.0071). We also observed by immunohistochemistry a significant GEF-H1 over-ex- pression in human breast cancer biopsies compared with normal tissue (n=72, p=0.0201). Furthermore, GEF-H1 protein expression levels correlate with the invasive potential of human and murine breast cancer cell lines. Using CRISPR/Cas9 technology, we gen- erated GEF-H1-knock out (KO) clones in a murine invasive breast cancer cell. We observed a significant decrease in the migration and invasion rates in GEF-H1-KO cells (p<0.001) and a correlation with a decrease in focal adhesion formation and signal- ing. These results showed that GEF-H1-RhoA activation may mediate the signaling involved in controlling cell structure, proliferation, mi- gration and invasion of breast cancer cells. In addition, the studies in human tumor samples suggest that GEF-H1 might be a potential tumoral biomarker.