ADP-ribosylation factor 6 regulates tumor cell invasion through the activation of the MEK/ERK signaling pathway

Abstract

Tumor cell invasion through the extracellular matrix is accompanied by the formation of invadopodia, which are actin-rich protrusions at the adherent surface of cells at sites of extracellular matrix degradation. Using the invasive human melanoma cell line LOX as a model system, we demonstrate that the ADP-ribosylation factor 6 (ARF6) GTPase is an important regulator of invadopodia formation and cell invasion. We show that ARF6 localizes to invadopodia of LOX cells. Sustained activation of ARF6 significantly enhances the invasive capacity of melanoma as well as breast tumor cell lines, whereas dominant negative ARF6 abolishes basal cell invasive capacity as well as invasion induced by growth factors. Furthermore, using biochemical assays, we show that enhanced invasive capacity is accompanied by the activation of endogenous ARF6. Finally, we provide evidence that ARF6-enhanced melanoma cell invasion depends on the activation of the extracellular signalregulated kinase (ERK), and that the ARF6 GTPase cycle regulates ERK activation. This study describes a vital role for ARF6 in melanoma cell invasion and documents a link between ARF6-mediated signaling and ERK activation.A n important characteristic of metastasizing cells is their ability to degrade and invade the extracellular matrix. Matrix degradation and cell invasion also occur during normal physiological processes, such as development and differentiation (1). The process of cell invasion is tightly regulated by a number of cell-signaling proteins, such as tyrosine kinases, Ras-related GTPases, and mitogen-activated protein kinase (MAPK) family proteins (2, 3). As an invading cell moves through the extracellular matrix, it extends actin-rich membrane protrusions into the matrix. These protrusions, called invadopodia, contain a number of actin-binding proteins and recruit various proteinases, including matrix metalloproteinases and serine proteases, which degrade matrix proteins at sites of cell invasion (4, 5). Studies on breast cancer and melanoma progression have shown that there appears to be a direct correlation between the ability of cells to form invadopodia and degrade matrix and the cells' invasive potential as measured by in vitro and in vivo assays for motility and invasion (4, 6, 7).ADP-ribosylation factor 6 (ARF6) is a member of the Ras superfamily of small GTPases, and like most GTPases, ARF6 alternates between its active GTP-bound and inactive GDP-bound conformations. The ARF6 GTPase cycle has been shown to regulate endosome membrane trafficking, regulated exocytosis, and actin remodeling at the cell surface (8). These processes are important for controlling cell shape changes and can impinge on the acquisition of an invasive phenotype. In fact, previous work in our laboratory has shown that ARF6 promotes cell migration in epithelial cells by facilitating adherens junction disassembly through its effect on endocytosis (of adhesion molecules) and by inducing peripheral actin rearrangements (9). In addition, Santy and Casanova (10) have shown that...

ADP-ribosylation Factor 1 Controls the Activation of the Phosphatidylinositol 3-Kinase Pathway to Regulate Epidermal Growth Factor-dependent Growth and Migration of Breast Cancer Cells

“…Furthermore, AMAP1 was reported to act as an effector of ARF6-GTP during invasion of glioblastomas and lung tumors (17). It was suggested that the regulation of invasion by ARF6 is dependent on the activation of the extracellular signal regulated kinase (Erk) (18). Collectively, these findings highlight the importance of ARF6 during cancer progression.…”

“…The role of ARF6 in regulating cellular invasiveness has been previously demonstrated (15,18). In this study, we therefore focused on ARF1 and next investigated whether this ARF isoform can also act to control cell migration.…”

Src-dependent repression of ARF6 is required to maintain podosome-rich sealing zones in bone-digesting osteoclasts

“…In this respect, ARF6 function seems to be versatile but always following a mechanistic principle, namely supporting membrane mass supply for membrane extension. In tumor cells, this basic feature of active ARF6 is thought to promote their invasive capacities, probably by plasma membrane extension to facilitate invadopodia formation (30,31). In polarized epithelial cells, active ARF6 facilitates the turnover of adherens junctions by promoting endocytosis of E-cadherin or ␤1-integrins, which leads to depolarization and cell spreading, as seen during epithelial-to-mesenchymal transition (32)(33)(34).…”

EMT, the cytoskeleton, and cancer cell invasion

“…Invadopodia have been identified in numerous cancer cell lines, including malignant melanoma, breast cancer, glioma, and head and neck cancer [193][194][195][196]. The formation of invadopodia can be initiated by various signals, for example by EGF, HGF or TGFβ-induced signal transduction [193,197,198] or by α 6 β 1 -integrin engagement [199].…”