GEP100 Mediated Cancer Cell Migration Driven By Epidermal Growth Factor-stimulated ERK1/2-Rac1 Signaling

Epidermal growth factor (EGF)-induced cancer cell migration is key to tumor invasion and metastasis. Recent studies indicated that GEP100, a guanine nucleotide exchanging factor (GEF) that preferentially activates Arf6 in vitro and binds directly to Tyr1068/1086-phosphorylated EGFR to activate Arf6 through its pleckstrin homology domain. In addition, formation of E-cadherin-mediated cell-cell adhesion is one of the major properties that maintain the non-invasive phenotypes of various carcinomas . Formation of E-cadherin-mediated cell-cell adhesion in MCF7 cells was substantially impaired in the presence of EGF by co-overexpression of GEP100 and Arf6. Likewise, over the past few years, GEP100, via its well-established roles in the regulation of Arf6 activation, has been shown in some tumor cells to regulate cell adhesion and organization of actin cytoskeleton , both of which are correlated with tumor cell migration.Given these potential connections between GEP100 and different aspects of cancer progression, we sought to investigate whether GEP100 functioned in the EGF-signaling pathways operational in cancer cell migration and metastasis by using human breast cancer cell line MDA-MB-231 and human liver carcinoma cell line HepG2. We first found that GEP100 regulated EGF-induced cell migration of both MDA-MB-231 and HepG2 cells. We then found that not only GEP100 regulated tumor cell migration through the activation of Arf6 and extracellular signal-regulated kinase (ERK1/2), but that GEP100-dependent ERK1/2 activation was also required for Rac1 activation during EGF-induced tumor cell migration. We further showed that the urokinase-type plasminogen activator receptor (uPAR) signaling downstream of the GEP100-ERK1/2 pathway was critical for Rac1 activation. Using athymic nude mice as a model, we further found that the pleckstrin homology domain deletion mutant of GEP100 (GEP100-â–³P H) blocked tumor metastasis in vivo. The findings described here document for the first time an important role for GEP100 in regulating human breast and liver cancer cell migration in vitro, and delineate a signaling pathway downstream of EGF signaling that is essential to cell migration of different tumor types.