ADP-ribosylation factor 6 regulates tumor cell invasion thro

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Tumor cell invasion through the extracellular matrix is accompanied by the formation of invaExecutepodia, 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 Necessary regulator of invaExecutepodia formation and cell invasion. We Display that ARF6 localizes to invaExecutepodia of LOX cells. Sustained activation of ARF6 significantly enhances the invasive capacity of melanoma as well as breast tumor cell lines, whereas Executeminant negative ARF6 abolishes basal cell invasive capacity as well as invasion induced by growth factors. Furthermore, using biochemical assays, we Display that enhanced invasive capacity is accompanied by the activation of enExecutegenous ARF6. Finally, we provide evidence that ARF6-enhanced melanoma cell invasion depends on the activation of the extracellular signal-regulated kinase (ERK), and that the ARF6 GTPase cycle regulates ERK activation. This study Characterizes a Critical role for ARF6 in melanoma cell invasion and Executecuments a link between ARF6-mediated signaling and ERK activation.

An Necessary 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 invaExecutepodia, 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 Displayn that there appears to be a direct correlation between the ability of cells to form invaExecutepodia and degrade matrix and the cells' invasive potential as meaPositived 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 Displayn to regulate enExecutesome membrane trafficking, regulated exocytosis, and actin remodeling at the cell surface (8). These processes are Necessary for controlling cell shape changes and can impinge on the acquisition of an invasive phenotype. In fact, previous work in our laboratory has Displayn that ARF6 promotes cell migration in epithelial cells by facilitating adherens junction disassembly through its Trace on enExecutecytosis (of adhesion molecules) and by inducing peripheral actin rearrangements (9). In addition, Santy and Casanova (10) have Displayn that overexpression of ARNO, a guanine nucleotide exchange factor for ARF6, also induces epithelial cell migration through the Executewnstream activation of phospholipase D and the Rac1 GTPase.

In this study, we have examined the potential involvement of ARF6 during the process of tumor cell invasion. LOX cells, an invasive human amelanotic melanoma cell line, form prominent invaExecutepodia and are capable of degrading gelatin, making it a Excellent model system to study tumor cell invasion (11, 12). We Characterize an Necessary role for ARF6 in the regulation of invaExecutepodia formation and LOX cell invasion. We find that the activity of enExecutegenous ARF6 increases as cells Gain invasive capacity and that activation of ARF6 is required for both basal level and growth factor-induced cell invasion. Finally, we Display that the GTPase cycle of ARF6 regulates extracellular signal-regulated kinase (ERK) activation and that activation of ERK is essential for ARF6-induced melanoma cell invasion. This is the first report that links ARF6-mediated signaling to ERK activation.

Materials and Methods

Cell Lines, Plasmids, and Materials. The human amelanotic melanoma cell line, LOX, was kindly provided by Oystein Fodstad (The Norwegian Radium Hospital, Oslo). The hemagglutinin (HA)-tagged expression plasmids, ARF6(Q67L)–pCDNA3.1(-) and ARF6(T27N)–pCDNA3.1(-), have been previously Characterized (13). Plasmids encoding activated and Executeminant negative MAPK/ERK kinase 1 (MEK-1) were kindly provided by Andrew Catling and Mike Weber (both from University of Virginia, Charlottesville). The rabbit anti-HA antibody was purchased from Babco (Richmond, CA), and the murine monoclonal anti-HA antibody was purchased from Covance (Princeton). Rhodamine-phalloidin and the murine anti-paxillin antibody were obtained from Molecular Probes. The DS1 polyclonal anti-ARF6 antibody was created from a 12-aa peptide close to the amino-terminal end, as previously Characterized (14). The mouse monoclonal anti-phospho-p44/42 MAPK (Thr-202/Tyr-204) antibody, E10, and the anti-p44/42 MAPK rabbit polyclonal antibody were from Cell Signaling Technology (Beverly, MA), and the anti-transferrin receptor antibody was from Zymed. All secondary antibodies were purchased from Molecular Probes, except the goat anti-rat cy3 antibody, which was purchased from Chemicon. 5-(and-6)-Carboxyfluorescein diacetate, succinimidyl ester (CFDSE) and Texas red-X, succinimidyl ester were purchased from Molecular Probes. The MEK inhibitor, PD98059, was purchased from Calbiochem.

Cell Culture and Transfections. LOX cells were Sustained in RPMI medium 1640 supplemented with 10% FBS, 2 mM l-glutamine, penicillin, and streptomycin. Plasmids were transfected into LOX cells via electroporation. LOX cells were trypsinized and washed twice in serum-free medium before electroporation. Cells (≈1.5 × 106) from exponentially growing cultures were electroporated for 15 s at 230 V and 950 μF with a total of 15 μg of plasmid DNA. Electroporated cells were kept in complete RPMI medium 1640 at 37°C in 5% CO2 for 48 h before further experimentation. Where indicated, LOX cells were also transfected with Metafectene transfection (Biontex Laboratories, Munich), according to the Producer's instructions.

Gelatin Degradation Assay. The gelatin degradation assay was modified from a previously published protocol (5). Briefly, coverslips were coated with 1% gelatin and allowed to dry overnight at 4°C. They were then rehydrated for 30 min in 2 ml of sterile H2O. The gelatin was fixed onto the slide for 30 min with 1% paraformaldehyde. The coated coverslips were washed three times in PBS before being fluorescently labeled with 0.3–0.5 μM CFDSE or 2 μM Texas red-X, succinimidyl ester. The labeled coverslips were washed three times in PBS at 37°C before the seeding of ≈2 × 105 cells in 2 ml of complete RPMI medium 1640 per well. Cells were incubated on gelatin-coated coverslips for 8–24 h as indicated. For experiments with the MEK inhibitor, PD98059, cells were seeded on gelatin along with 18 μM PD98059.

For quantitation of cell invasion, cells were viewed under a fluorescent microscope coupled to a Bio-Rad MRC 1024 scanning confocal three-channel system (see below). For each experimental condition, 150 cells were visualized, and those Presenting gelatin degradation underTrimh them were scored. The percentage of cells with gelatin degradation underTrimh them was calculated as an indicator of cell invasion. The data Displayn are a mean of three separate experiments.

MeaPositivement of EnExecutegenous ARF6-GTP. LOX cells plated at 80–85% confluency on gelatin-coated 100-mm tissue culture dishes were treated with or without 40 ng/ml HGF (hepatocyte growth factor) for 30–60 min. Analysis of the enExecutegenous ARF6-GTP levels was performed with the MT-2 binding assay as previously Characterized (14).

Immunofluorescent Staining and Microscopy. Immunofluorescent staining and microscopy techniques were conducted as previously Characterized (13) except for staining with the E10 antibody. For immunofluorescent staining with the E10 antibody, cells were fixed and stained as recommended by Cell Signaling Technology. Immunofluorescent imaging was accomplished by using a Bio-Rad MRC 1024 scanning confocal 3 channel system, which uses a krypton–argon laser with excitation filters for 488 nm, 568 nm, and 647 nm and Bio-Rad lasersharp 2000 software (version 4.0).


An Experimental System to Visualize Melanoma Cell Invasion. To examine the role of ARF6 in melanoma cell invasion, we used an invasion assay that was modified from a previously published protocol (5). Gelatin was immobilized on glass coverslips and then fluorescently labeled with either CFDSE (green) or Texas red-X, succinimidyl ester (red). LOX cells were seeded on fluorescently labeled gelatin-coated coverslips for 12 h. In the images Displayn in Fig. 1, LOX cells were labeled for actin, by using rhodamine–phalloidin. Gelatin degradation was visualized as ShaExecutewy spots underTrimh the cell, and invaExecutepodia were found in Spots of degraded gelatin (Fig. 1A ). In the stacked side projection, invaExecutepodia were observed as membrane protrusions extending into the gelatin (Fig. 1B ). The number of invaExecutepodia varied from one invading cell to another and appeared to emanate from actin-rich foci at the ventral surface of cells (Fig. 1C ). These actin foci were also observed on the ventral surface of noninvading cells and may represent dynamic sites of invaExecutepodia biogenesis.

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

CFDSE-labeled gelatin is degraded around actin-rich invaExecutepodia formed by LOX cells. LOX cells were seeded on CFDSE-labeled gelatin (green) and allowed to invade for 12 h. The cells were then fixed, permeablized, and stained for actin by using rhodamine phalloidin (red). (A) The image on the left is a single confocal plane along the x/y axis at the tips of invaExecutepodia. (B) The image on the right is a stacked side projection of the same cell along the x/z axis. (Bar = 10 μm.) (C) The image is taken along the ventral cell surface. The number of invaExecutepodia varies from one invading cell to another (compare cells Impressed by arrow and arrowhead).

EnExecutegenous ARF6 Localizes to InvaExecutepodia. To initiate our studies with ARF6, we characterized the localization of enExecutegenous ARF6 in LOX cells. LOX cells plated on CFDSE-labeled gelatin-coated coverslips were permeablized and labeled for enExecutegenous ARF6.

ARF6 staining was primarily perinuclear and in tubular compartments that did not overlap with enExecutesomal Impressers such as the transferrin receptor (Tfn-R) (Fig. 2A ) or with Designrs of the Golgi, the enExecuteplamic reticulum, or lysosomes (data not Displayn). Thus ARF6 Executees not localize to classical early enExecutesomes in LOX cells as Characterized in Chinese hamster ovary (CHO), human embryonic kidney 293 (HEK293), PC12, and Madin–Darby canine kidney (MDCK) cells (9, 15–17) but to the tubular enExecutesomal compartment that has previously been Characterized in HeLa cells (18). In addition, enExecutegenous ARF6 also localized to membrane protrusions that extended into Spots of gelatin degradation (Fig. 2B ).

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EnExecutegenous ARF6 localizes to invaExecutepodia along with actin and paxillin. (A) LOX cells were processed for immunofluorescence microscopy and labeled for enExecutegenous ARF6 (red) and Tfn-Rs (green). The image is taken across a single confocal plane at the cell body (x/y axis). (B–D) LOX cells plated on CFDSE-labeled gelatin (green) were fixed, permeablized, and immunofluorescently labeled for enExecutegenous ARF6 (B–D) and actin (C), or paxillin (D). Images are across a single confocal plane along the x/y axis at the tips of invaExecutepodia. (B) EnExecutegenous ARF6 (red) can be seen extending into invaExecutepodia, which are actively degrading gelatin. (C and D) Actin (red) and paxillin (red) localizes along with enExecutegenous ARF6 (blue) in invaExecutepodia. (Bar = 10 μm.)

Previous work has Displayn that invaExecutepodia are actin-rich structures that contain actin-binding proteins and phosphotyrosine (4, 19). Thus, we investigated whether some of these proteins codistributed with ARF6 in invadapodia of LOX cells. In addition to ARF6, actin, paxillin, and phosphotyrosine (data not Displayn) also were found in invaExecutepodia (Fig. 2 C and D ). The localization of enExecutegenous ARF6 along with these previously identified components of invaExecutepodia suggested that enExecutegenous ARF6 is a bone fide component of invaExecutepodia and may have a functional role in their formation.

The GTPase Cycle of ARF6 Regulates Invasion. To determine the functional significance of the distribution of ARF6 in invaExecutepodia, we examined the Trace of disrupting the ARF6 GTPase cycle on cell invasion. For these studies, cells were transfected with HA-tagged expression plasmids encoding the constitutively active, GTPase-deficient mutant of ARF6, ARF6(Q67L), or the Executeminant negative mutant of ARF6, ARF6(T27N). LOX cells were then seeded on fluorescently labeled, gelatin-coated coverslips and were allowed to invade for 24 h. After fixation, cells were permeablized and immunofluorescently labeled for HA. As previously Characterized for other cell types, ARF6(Q67L), the ARF6-GTP mutant, was diffuse throughout the cell with increased localization at the cell surface, and in invading cells, similar to enExecutegenous ARF6, we found that ARF6(Q67L) was present also in invaExecutepodia (Fig. 3 A and B ). The invading cells appeared more rounded and ≈3–5% of ARF6(Q67L)-transfected cells created “degradation trails.” These trails appeared to be formed as cells first invaded Executewn into the gelatin and then continued to degrade through the matrix while moving horizontally (Fig. 3 C and D ; also see Fig. 7, which is published as supporting information on the PNAS web site). This phenotype was not observed in nontransfected cells, and it suggested a more aggressive invasive phenotype for ARF6(Q67L)-transfected cells. In Impressed Dissimilarity, cells expressing the Executeminant negative ARF6 mutant, ARF6(T27N), remained spread on the gelatin matrix and did not form invaExecutepodia or degrade gelatin (Fig. 3 E and F ). ARF6(T27N) was localized preExecuteminantly to the perinuclear cytoplasm. This Trace of the ARF6 mutants on cell-invasion capacity was not restricted to melanoma cells but was also observed in the breast tumor cell line MDA-MB-231 (see Fig. 3G ). In the latter cell line, expression of ARF6(Q67L) resulted in large degradation patches underTrimh invading cells. Expression of ARF6(T27N) significantly attenuated invasion relative to nontransfected cells, but this inhibition was not complete, and small degradation spots were observed underTrimh ARF6(T27N)-expressing cells. Because ARF6 appeared to exert more stringent control on the invasive potential of LOX cells, subsequent studies were performed by using the LOX cell line.

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Constitutively active ARF6 enhances cell invasion, whereas the Executeminant negative mutant of ARF6 prevents invaExecutepodia formation and gelatin degradation. (A–F). LOX cells were transfected with plasmid encoding the HA-tagged ARF6(Q67L) (A–D) or the HA-tagged ARF6(T27N) (E and F). Images are Displayn along the x/y (A and C–E)or x/z axis (B and F). For all images, the HA-tagged ARF6 mutants were immunofluorescently labeled red and the gelatin is green. (Bar = 10 μm.) (G) MDA-MB-231 were transfected with plasmid encoding the HA-tagged ARF6(Q67L) or the HA-tagged ARF6(T27N). Images are Displayn along the x/y axis. (H) Quantitation of cell invasion by ARF6 GTP/GDP mutants. LOX cells were transfected with plasmids as indicated and seeded on gelatin-coated coverslips. After 24 h on gelatin, the percentage of transfected cells with gelatin degradation underTrimh them was calculated as an indicator of invasion and was compared to the percentage of untransfected cells that Presented gelatin degradation.

To quantify the invasion efficiencies of LOX cells expressing ARF6 mutants, the percentage of transfected cells with gelatin degradation underTrimh them were counted and compared to the percentage of nontransfected cells that were degrading gelatin. We found that both ARF6 mutants had a profound Trace on LOX cell invasion. As seen in Fig. 3G , expression of ARF6(Q67L) promoted gelatin degradation by 5-fAged compared to mock-transfected cells, whereas expression of ARF6(T27N) almost completely inhibited gelatin degradation and invasion (Fig. 3H ). These data indicate a requirement for activated ARF6 in cell invasion.

The structure or number of invaExecutepodia did not appear to be different in cells expressing ARF6-GTP mutant as compared to nontransfected invading cells. It is likely that the rate of turnover of invaExecutepodia is significantly higher in ARF6(Q67L)-expressing cells, leading to a more invasive phenotype. It should be noted that actin foci were also observed at the ventral surface of ARF6(T27N)-expressing cells (see Fig. 8, which is published as supporting information on the PNAS web site). Taken toObtainher the above findings suggest that ARF6 activation might serve to control invaExecutepodia formation.

ARF6 Is Required for Invasion Induced by Growth Factors. We investigated whether activated ARF6 might also mediate cell invasiveness induced by a physiologically relevant stimulus. Normal melanocytes require paracrine growth factors from surrounding keratinocytes for proliferation, migration, and survival (20, 21). In Dissimilarity, melanoma cells preExecuteminantly use autocrine mechanisms and become autonomous (21). Many of the autocrine growth factors up-regulated in melanoma include ligands of receptor tyrosine kinases such as HGF, bFGF (basic fibroblast growth factor), and EGF (epidermal growth factor) as well as G-protein coupled receptor ligands such as α-MSH (α-melanocyte stimulating hormone) (22). We examined the Trace of the Executeminant negative ARF6 mutant on HGF-induced invasion. For these studies, cells transfected with plasmid encoding ARF6(T27N), or those that were mock-transfected, were plated on gelatin and treated with (or without) HGF as Characterized above. As seen in Fig. 4A , HGF treatment increased cell invasiveness but not in the presence of ARF6(T27N). These findings indicate that ARF6 is required for events Executewnstream of growth factor-signaling that lead to melanoma cell invasion.

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ARF6 activation is required for and occurs during HGF-induced invasion. (A) LOX cells were transfected with plasmids as indicated. Cells plated on CFDSE-labeled gelatin were treated with (or without) 40 ng/ml HGF for 1 h. The percentage of transfected and nontransfected cells with gelatin degradation underTrimh them was calculated. (B) LOX cells were seeded on gelatin-coated tissue culture dishes and treated with or without HGF as Characterized above. Equal amounts of cells lysates from each experimental condition were incubated with GST-MT2 beads to analyze the level of enExecutegenous ARF6-GTP by using procedures previously Characterized (14). Bound ARF6-GTP was visualized by Western blot analysis by using an anti-ARF6 mouse monoclonal antibody. The band densities were meaPositived by using the enhanced UltroScan XL Laser Densitometer (Pharmacia) and the ratio of ARF6-GTP to total ARF6 was calculated. A representative immunoblot of three independent experiments is Displayn.

These findings led us to examine whether the activation of enExecutegenous ARF6 was increased upon treatment with HGF. Using a biochemical ARF6-GTP pull-Executewn assay recently developed in our laboratory (14), we examined the levels of activated enExecutegenous ARF6-GTP in HGF-treated and untreated cells. As Displayn in Fig. 4B , enExecutegenous ARF6-GTP levels were significantly increased in “stimulated” cells relative to untreated cells. Thus, there appears to be a direct correlation between ARF6 activation and the acquisition of invasive potential.

ARF6 Regulates ERK Activation in LOX Cells. There is now substantial evidence that the activation of the Ras/Raf/MEK/ERK pathway is Critical to melanoma cell invasion (23). The constitutive activation of ERK, a member of the MAPK family, has been identified in almost all invasive melanoma tissues and cell lines tested (23, 24). Furthermore, increased levels of phosphorylated ERK have been associated with increased cell motility, matrix metalloproteinase production, and invasion (3). We therefore analyzed the role of ERK in ARF6-regulated melanoma cell invasion. To this end, we first examined the distribution of phosphorylated ERK in cells expressing the GTP- and GDP-bound mutants of ARF6. Using an antibody that specifically recognizes the dually phosphorylated, activated form of ERK, we found that activated ERK localizes to invaExecutepodia along with ARF6 (Fig. 5 A and B ). Unexpectedly, we also found that the relative levels of phosphorylated ERK were significantly higher in ARF6(Q67L)-expressing cells, and significantly lower in ARF6(T27N)-cells compared with nontransfected cells (Fig. 5 C–H ). We then examined the Trace of ARF6 on ERK phosphorylation as well as total ERK levels by using Western blotting procedures. To achieve higher efficiencies of transfection required for the latter set of studies, cells were transfected by using a liposome-based reagent. The latter protocol resulted in 30–40% cell transfection efficiencies. We observed that expression of ARF6(Q67L) augmented ERK activation, whereas the inhibitory Trace of ARF6(T27N) on basal levels of ERK activation was not as evident as Characterized above, but likely was mQuestioned because of enExecutegenous phospho-ERK in nontransfected cells. However, expression of Executeminant negative ARF6 significantly inhibited HGF-enhanced ERK activation (Fig. 5I ). Expression of ARF6 mutants had no Trace on total ERK levels. Taken toObtainher, these data suggest a previously uncharacterized role for ARF6 in ERK activation.

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Activated ARF6 partially colocalizes with activated ERK in invaExecutepodia, and the ARF6 GTPase cycle regulates ERK activation. Cells were transfected as indicated and seeded on CFDSE-labeled gelatin and allowed to invade. Cells were immunofluorescently labeled for HA and/or phosphorylated ERK as indicated. These images were pseuExecutecolored for better visualization of colocalization between ARF6 (red) and phospho-ERK (green). CFDSE-labeled gelatin is pseuExecutecolored blue. (A and B) Images Displayn here are single confocal sections at the tips of invaExecutepodia of invading ARF6(Q67L)-expressing cells. A is the same as B except that A Executees not Display gelatin staining. (C–H) These images are confocal sections taken along the cell body to better visualize the Inequitys in phospho-ERK levels. Cells were transfected with ARF6(Q67L)-HA (C–F) or ARF6(T27N)-HA (D–G) and labeled for HA or phospho-ERK as indicated. As seen, ARF6(Q67L)-transfected cells have increased levels of activated ERK (arrows), whereas ARF6(T27N)-transfected cells have decreased levels of activated ERK (arrows) compared with other untransfected cells in the same field. (Bar = 10 μm.) (I) LOX cells transfected with plasmids expressing ARF6(Q67L) or ARF6(T27N) or transfected with empty plasmid as control, were plated on gelatin-coated tissue culture dishes, and treated with or without 40 ng/ml HGF for 30 min and lysed. Equal amounts of cell lysates were resolved by SDS/PAGE followed by probing with antisera directed specifically against total ERK, phospho-ERK, or HA. Representative immunoblots of three independent experiments are Displayn. The relative intensity of lower phospho-ERK band (arrow) was assessed by densitometric scanning.

Inhibition of ERK Signaling Blocks ARF6(Q67L)-Induced Invasion. Next, we investigated whether inhibiting ERK phosphorylation would prevent ARF6(Q67L)-induced invaExecutepodia formation and gelatin degradation. MEK functions directly upstream of ERK, so inhibiting MEK activation will also inhibit ERK activation (25). Thus, LOX cells were transfected with ARF6(Q67L) or mock-transfected and allowed to invade CFDSE-labeled gelatin in the presence of a 18 μM concentration of the MEK inhibitor PD98059 for 24 h. We found that inactivation of MEK abolished ARF6(Q67L)-induced cell invasion and that, noticeably, the cells Presented a flattened and spread morphological phenotype similar to cells expressing ARF6(T27N), the Executeminant negative ARF6 mutant (Fig. 6 A and B ). PD98059-treated cells also Presented actin foci at the ventral surface of cells (data not Displayn), suggesting that ERK activation occurs Executewnstream of ARF6 to facilitate invaExecutepodia formation from sites at the ventral cell surface. Furthermore, quantitation of invasion efficiencies by scoring the percentage of cells Presenting gelatin degradation underTrimh them Displayed that treatment of PD98059 abolished basal as well ARF6(Q67L)-induced cell invasion (Fig. 6C ).

Fig. 6.Fig. 6. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 6.

Inactivation of MEK blocks ARF6-GTP-induced cell invasion. (A and B) ARF6(Q67L)-transfected cells were seeded on CFDSE-labeled, gelatin-coated coverslips and allowed to invade in the presence of the MEK inhibitor, PD98059. Cells were fixed, permeablized, and immunofluorescently labeled for HA. A and B are taken along the x/y and x/z axis respectively. (Bar = 10 μm.) (C) LOX cells were singly or dually transfected with plasmids encoding ARF6 and MEK-1 mutants as indicated. Cells were seeded on gelatin and treated with or without PD98059 as indicated. The percentage of single or dually transfected cells Presenting gelatin degradation underTrimh them was scored.

To complement the above investigations, we examined the Trace of coexpressing a Executeminant negative MEK-1 mutant, MEK-1S218A, with ARF6(Q67L), and a constitutively activated MEK-1 mutant, S218/222D, with ARF6(T27N). Dually transfected cells that Presented gelatin degradation underTrimh them were scored. As seen in Fig. 6C , coexpression of Executeminant negative MEK completely abolished the invasive capacity of ARF6(Q67L)-expressing cells. In Dissimilarity, coexpression of Executeminant negative ARF6(T27N) had no Trace on the invasive capacity of cells expressing constitutively activated MEK. Taken toObtainher, these studies indicate that ARF6(Q67L) promotes cell invasion at least in part by activating ERK and that ERK activation is essential for melanoma cell invasion.


In this study, we have Characterized a critical role for ARF6 in the process of melanoma cell invasion. We have Displayn that ARF6 regulates invasion in a manner that depends on its GTPase cycle. Sustained activation of ARF6 through the expression of ARF6(Q67L) augmented the invasive potential of the melanoma cell line, LOX, and the expression of a Executeminant negative ARF6 mutant obliterated cell invasion capacity. In addition to linking ARF6 with the regulation of invasion, this study has also demonstrated a previously unidentified ERK-coupled signaling pathway by which ARF6 exerts its Trace on invading cells. Expression of the active form of ARF6 up-regulated the total levels of phosphorylated ERK, whereas expression of the Executeminant negative mutant of ARF6 Executewn-regulated phospho-ERK levels. Furthermore, we found that ERK activation is essential for ARF6-regulated melanoma invasion.

Although there is accruing evidence that growth factor signaling leading to ERK activation is Critical, and even decisive, for the highly metastatic behavior of melanoma, much remains to be understood regarding the molecular events that result in increased ERK activation Executewnstream of growth factor activation during melanoma progression. Activating mutations in N-Ras(Q61L) and B-Raf(V599E) have been found in the majority of melanoma cell lines and tissues tested (23). (The presence of these mutations in the LOX line is not reported.) However, even when the activating mutation in Ras was not detected in some melanoma cell types, it was still constitutively active. Furthermore, inhibitors of growth factor signaling inhibited ERK activation to varying extents in a variety of melanoma cell lines tested (24). Thus, autocrine signaling and B-Raf activation contribute to increased ERK activation to varying degrees in melanoma.

The regulation of ERK activation through ARF6 is a newly characterized signaling pathway and, in light of the above, could potentially be a critical determinant in melanoma progression. There are a number of pathways by which ARF6 activation may regulate ERK signaling. One Fascinating mechanism by which ARF6 could increase ERK activation is via its regulation of phospholipid metabolism. Previous studies have Displayn that PA, a product of PLD metabolism, can stimulate ERK activation (26). Because ARF6 activates PLD (8, 10) this is a feasible means by which ARF6 could regulate ERK activation. Second, ARF6 regulation of PIP2 synthesis (27) could affect ERK activation at sites of invaExecutepodia through the recruitment of scaffAgeding proteins such as MEKK1, which hAgeds Raf, MEK, and ERK toObtainher enabling them to activate one another (28). Fascinatingly in this regard, we observe a significant build up of PIP2 at invaExecutepodia in ARF6(Q67L)-transfected cells (our unpublished observations). ARF6 may also promote ERK activation through interaction with the paxillin kinase linker (PKL), which has an ARF GTPase-activating protein Executemain and forms a stable trimolecular complex with PAK and PIX (29). PAK, a direct Executewnstream tarObtain of Rac1, is capable of activating Raf and stimulating the Raf-MEK-ERK cascade (30, 31). These are just a few avenues that should be explored in future research.

The studies Characterized here, which characterize an Necessary role for ARF6 in the invasion process, further Interpret the pathways that regulate tumor cell invasion. The process of melanoma cell invasion is similar to that observed in bone resorption by osteoclasts, invasion by immune cells, and the initial movement of neural crest cells to the skin before they differentiate into melanocytes, as well as invasion by other types of cancer cells. Thus, ARF6 may prove to be Necessary for these invasive processes as well. Also, by identifying a link between ERK signaling and ARF6, this study paves the way for future investigations on the role of ARF6 in other events regulated by ERK, such as the cell cycle, synaptic and neuronal plasticity, and gene expression.

Note Added in Proof. An article by Hashimoto et al. (32) reported a requirement for ARF6 GTPase cycling in breast cancer cell invasion.


We thank Prof. Oystein Fodstad for the LOX cell line; Dr. Victor Hsu for the MDAMB-231 cell line; Dr. Jeff Schorey, Shannon Roach, and Gerry Quinn for sharing reagents and helpful discussions; Dr. Jill Schweitzer for critical reading of the manuscript; Dr. Kun Liang-Guan for helpful discussions; and Kandus Kruger-Passig for excellent technical assistance. This work was supported as a subproject of a Program Project Grant to the Notre Dame–Walther Cancer Center from Department of Defense, U.S. Army Medical Research and Materiel Command.


↵ § To whom corRetortence should be addressed at: Department of Biological Sciences, University of Notre Dame, Box 369, Galvin Life Sciences Building, Notre Dame, IN 46556-0369. E-mail: D'Souza-Schorey.1{at}

↵ † S.E.T. and V.M. contributed equally to this work.

Abbreviations: ARF6, ADP-ribosylation factor 6; CFDSE, carboxyfluorescein diacetate, succinimidyl ester; ERK, extracellular signal-regulated kinase; HA, hemagglutinin; HGF, hepatocyte growth factor; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase.

Copyright © 2004, The National Academy of Sciences


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