Investigating the role of ADP-ribosylation factor 6 in tumor cell invasion and extracellular signal-regulated kinase activation

Tumor cell invasion is a coordinated process involving the formation of invadopodia and the localized degradation of the extracellular matrix (ECM). The process of cell invasion is regulated by cell-signaling proteins such as Ras-related GTPases and members of the mitogen-activated protein kinase (MAPK) family. Our studies have focused on the role of the ADP-ribosylation factor 6 (ARF6) GTPase in the process of tumor cell invasion. Using activated and dominant negative mutants of ARF6 in a tumor cell culture model, our laboratory has demonstrated that the GTPase cycle of ARF6 regulates invadopodia formation and matrix degradation. Furthermore, ARF6-mediated cell invasion was found to be dependent on the activation of the extracellular signal-regulated kinase (ERK). These findings demonstrate a critical role for ARF6 in ERK activation and tumor cell invasion. To investigate the role of ARF6 in tumor cell invasion and ERK activation, a number of methods were employed. These procedures include transfection of LOX cells, in vitro matrix-degradation assays, immunofluorescence microscopy, and biochemical assays. These approaches can be applied effectively to measure the degree of invasiveness fostered by ARF6 and/or other GTPases and to examine the subcellular distribution of the molecular players that are trafficked or recruited to sites of cell invasion.