ADP-Ribosylation Factor 6 Regulates Glioma Cell Invasion through the IQ-Domain GTPase-Activating Protein 1-Rac1–Mediated Pathway

Malignant human gliomas display rapid proliferation, extensive neovascularization, and apoptosis resistance, diffuse invasion into the surrounding brain parenchyma, and eventually gives rise to recurrent tumors despite radical surgery, irrespective of their histologic type and grade of malignancy (1, 2). Glioma cell invasion is a highly complex process, and mechanisms regulating cell motility may represent key elements of the invasive cascade (3). ADP-ribosylation factor 6 (ARF6), which belongs to the ARF family of small GTP-binding proteins with multiple roles in fundamental biological processes, such as actin remodeling (4–6), has been shown to play an important role in tumor cell invasion (4–6). Studies showed that ARF6 is expressed at high levels in highly invasive breast cancer cells, and ARF6 activation increases the invasive potential of melanoma cells and breast cancer cells (7–9). In gliomas, exogenous expression of EFA6A, a guanine nucleotide exchange factor (GEF) that promotes ARF6 activation, enhanced cell motility and invasiveness in in vitro assays for invasion and migration (10).

The requirement of ARF6 for cell spreading, Rac1-induced ruffling, cell migration, and wound healing has been recently described (5, 6). Although ARF6-dependent modulation of Rac1 activation seems to be important, molecular mechanisms by which ARF6 regulates cell motility remain unclear. Several studies showed that ARF6 stimulates actin reorganization and membrane ruffling, promoting the acquisition of a migratory phenotype through activation of Rac1 (11–13). Rac1 is critical for changes that facilitate cell motility. Rac1 directs actin assembly, which results in the formation of lamellipodia at the leading edge of migrating cells and, thus, is a key player in cell movement (14). Depletion of Rac1 by small interfering RNA (siRNA) decreases cell migration and invasion in tumor cells, including glioma cells (2, 15, 16). Although Rac1 has been described as a downstream target of ARF6, the ARF6-dependent Rac1 regulation seems to be complex and varies with cell types. For example, in epithelial cells an ARF6-GEF, ARNO promotes Rac1 activation by recruiting a Rac1-GEF complex, Dock180/ELMO1 (17). A Rac1-GEF, β-PIX, is relocated to the plasma membrane, and Rac1 activation is enhanced in both ARF6-depleted HEK 293 and HepG2 cells (18). In addition, in NIH 3T3 cells, another key regulator of cell motility, IQ-domain GTPase-activating protein 1 (IQGAP1) was found in the macropinosomes induced by ARF6 near sites of membrane ruffling (19). IQGAP1 plays important roles in various aspects of cell physiology, particularly in cell migration, through its interaction with various proteins (20–22). Although IQGAP1 has been reported to function as either a target or a regulator of Rac1, IQGAP1 regulates cell adhesion and migration by binding to Rac1 and stabilizing Rac1-GTP (22). Similar to ARF6 activation modulated by upstream growth factors (7, 8, 11, 23), IQGAP1 participates in signaling cascades that are also stimulated by growth factors, such as epidermal growth factor (EGF) and HGF (20, 22). IQGAP1 localizes at the leading edge of migrating cells, and knockdown of IQGAP1 in breast cancer cells decreases cell motility and invasion (24). In human cancers, IQGAP1 is overexpressed in colorectal carcinomas and associated with invasion fronts (25).

In the current study, we examined the involvement of ARF6 in glioma cell invasion using in vitro, ex vivo, and in vivo models. Our data show that ARF6 is abundantly expressed in various highly invasive glioma cell lines and is required for enhanced glioma cell migration. Knockdown of ARF6 expression by siRNAs suppressed glioma cell invasion ex vivo and in the brain of mice. Furthermore, ARF6 mediates Rac1 activation in glioma cells upon external stimulation with serum and HGF. We show that IQGAP1 is required for the ARF6-mediated activation of Rac1, a signaling pathway that seems to be essential for glioma cell invasion.

Glioma cell lines, antibodies, and reagents. Human LN18, LN229, U118MG, U87MG, and T98G glioma cells were obtained from American Type Culture Collection. U251MG, U373MG, LN215, and LNZ308 glioma cells were from our collection (26). SNB19 cells were from Dr. Y-H. Zhou (University of California). D54MG and A172 cells were from Dr. D. Bigner (Duke University). Normal human astrocytes (NHA) were from Lonza. The following reagents were used in our studies: mouse anti-ARF6 (3A-1), rabbit anti-IQGAP1 (H-109), mouse anti-Dock180 (H-4), goat anti–β-PIX (L-17), and goat anti–β-actin antibodies (Santa Cruz Biotechnology), rabbit anti–extracellular signal-regulated kinase 1/2 (ERK1/2) and anti–phosphorylated ERK1/2 antibodies (Cell Signaling), a mouse anti-Rac1 antibody (BD Bioscience Pharmingen), rabbit anti-ELMO1 antibody (27), an Alexa Fluor 488–conjugated rabbit anti-GFP antibody (Molecular Probes), and Rac1/Cdc42 activation assay kit (Upstate Technology). Cell culture media and other reagents were from Hyclone, Invitrogen/Bethesda Research Laboratories, Sigma Chemicals, and Fisher Scientific.

Vectors, siRNAs, and transfections. Human wild-type ARF6 (ARF6-WT), constitutively active ARF6 (Q67L) in pcDNA3.1 or pcDNA3.1/neo (7), was separately transfected into U87MG cells using an Effectene reagent (Qiagen). G418 (900 μg/mL)–resistant cells were selected, and expression of ARF6 in various cells was determined by immunoblotting with a mouse anti-ARF6 antibody. siRNAs were synthesized by Dharmacon. The target sequences were ARF6 5′-GCACCGCAUUAUCAAUGACCG-3′ (18), Rac1 5′-AAGGAGAUUGGUGCUGUAAAA-3′ (15), and IQGAP1 5′-AAAGUUCUACGGGAAGUAA-3′ (28).

Transfection of glioma cells with the aforementioned siRNA was performed using Lipofectamine 2000, as previously described (16). Mock transfection was done in parallel using the Stealth RNAi Negative Control Med GC (Invitrogen). After 24 h, the siRNA/lipid complexes were removed, and the cells were maintained in complete medium for an additional 48 h. The inhibition of protein expression was determined by immunoblotting analyses.

An ARF6 siRNA was constructed into a pG-SUPER/GFP vector (from Dr. G. Borisy at Northwestern University; ref. 29) to synthesize the 19-bp double-stranded ARF6 target sequence as an ARF6 short hairpin RNA (shRNA). Either pG-SUPER/ARF6-GFP or pG-SUPER/GFP was cotransfected with pcDNA3.1/neo vector at a ratio of 10:1 into SNB19 cells using an Effectene reagent. G418 (700 mg/mL)–resistant cell clones were reselected by GFP expression via fluorescence-activated cell sorting, and knockdown of ARF6 in these cells was validated by immunoblotting analyses.

Immunoblotting, coimmunoprecipitation, and Rac1 activation assays. Immunoblotting and Rac1 activation analyses were performed, as previously described (16). Coimmunoprecipitation, followed by immunoblotting, analyses for association of ARF6 and IQGAP1 were performed, as previously described (30), using an anti-ARF6 antibody or an isotype-matched IgG control for immunoprecipitation and indicated antibodies for immunoblotting analyses.

Immunofluorescent staining. After 48 h of transfection with siRNA for ARF6, IQGAP1, or a control, the cells were grown in chamber slides in the serum-free medium for 48 h and then treated with or without 20 ng/mL HGF for 30 min. The cells on slides were fixed with 4% PFA and permeablized with 0.2% Triton X-100. The cells were then dual-stained with mouse anti-ARF6 and rabbit anti-IQGAP1 antibodies followed by incubation with both Alexa Fluor 488–conjugated goat anti-mouse IgG and Alexa Fluor 596–conjugated goat anti-rabbit IgG antibodies (Molecular Probes). Cell nuclei were stained with Hoechst.

Wound healing, migration, and ex vivo brain slice invasion assays and quantification. As previously described (16), various cells were seeded on six-well plates with DMEM containing 10% fetal bovine serum (FBS) and grown to confluence. The cells were scratched with a sterile 200-μL pipette tip to create artificial wounds. At 0 and 24 h after wounding, phase-contrast images of the wound healing process were photographed digitally using an inverted Olympus IX50 microscope with a 10× objective. Eight images per treatment were analyzed to determine averaging position of the migrating cells at the wound edges by digitally drawing lines using the Image-Pro Plus software (Media Cybernetics). The cell migration distance was determined by measuring the width of the wound divided by 2 and by subtracting this value from the initial half-width of the wound (31). In vitro cell migration and ex vivo brain slice invasion assays were performed, as previously described (16). Lateral cell migration on brain slices was assessed by epifluorescent examination of GFP-expressing cells using a stereomicroscope (SZX12, Olympus) at 10× magnification. Haptotatic cell migration assay was performed in the presence or absence of serum or HGF in the bottom wells by using Boyden chambers through an 8-μm pore size membrane. Statistical analyses using one-way ANOVA followed by Newman-Keuls posttest were performed, as previously described (16). P < 0.05 was considered statistically significant.

Intracranial brain tumor xenografts. SNB19/ARF6 siRNA 10 and 12 cells and SNB19/control siRNA cells (1 × 10⁶ in 5 μL PBS) were stereotactically implanted into the brain of an individual nude mouse with five mice per group. The glioma-bearing mice were sacrificed 2 wk postimplantation. Their brains were removed, processed, and analyzed, as previously described (30). The GFP signal of various SBN19 tumors was enhanced by immunostaining with an Alexa Fluor 488–conjugated rabbit anti-GFP antibody.

Cellular depletion of ARF6 expression by siRNA suppresses glioma cell motility. To determine whether ARF6 plays a role in glioma cell motility, we examined the expression of ARF6 in various human glioma cell lines. As shown in Supplementary Fig. S1 (see supplementary data), all glioma cell lines, as well as NHAs examined, expressed endogenous ARF6 proteins, albeit to varying degrees. LN229, SNB19, and U373MG cells, which are invasive glioma cell lines and express relatively higher levels of ARF6, were chosen to evaluate the effect of silencing ARF6 on FBS-stimulated or HGF-stimulated Rac1 activity and cell motility using siRNA knockdown (9). After 48 h, treatment of various cells with ARF6 siRNA effectively suppressed ARF6 expression to levels <10% of those cells treated with control siRNA (Fig. 1A) and without affecting cell viability (data not shown). Knockdown of ARF6 by siRNA significantly impaired Rac1 activity stimulated by 10% FBS or 20 ng/mL HGF (Fig. 1A,, bottom). To test whether suppression of ARF6 affects glioma cell motility, we performed in vitro migration assays and found that knocking down ARF6 markedly inhibited the ability of LN229, SNB19, and U373MG cells to migrate toward chemoattractants, 10% FBS, or 20 ng/mL HGF (Fig. 1B).

To determine whether knockdown of ARF6 inhibits glioma cell invasion, we used a physiologically relevant murine brain ex vivo model (16, 31). GFP-expressing SNB19 and U373MG cells were transfected with either ARF6 siRNA or with control scrambled siRNA. After 48 h, the cells were placed bilaterally onto the putamen of a 500-μm-thick murine brain slice and allowed to invade into the brain tissue for an additional 48 h. As shown in Fig. 1C, analyses by confocal laser scanning microscopy revealed a significant inhibition of intrinsic invasiveness between control siRNA-treated and ARF6 siRNA-treated glioma cells. Taken together, these results indicate that ARF6 is required for glioma cell migration toward chemoattractants in vitro and glioma cell invasion ex vivo.

Inhibition of ARF6 by siRNA suppresses glioma cell invasion in the brain. To directly assess the effect of knockdown of ARF6 on glioma cell invasion, we used a murine brain tumor in vivo model. We first generated a vector, pG-SUPER/ARF6/GFP, that simultaneously expressed GFP and an ARF6 siRNA. This siRNA/GFP vector was cotransfected with a pcDNA3.0/(neo) vector into SNB19 cells. Previous studies showed that orthotopic SNB19 glioma cells invade into normal brain parenchyma along the vasculature and white matter tracks in single cells and cell clusters, reminiscent to the invasive phenotype of human primary glioma specimens (32). We selected several G418-resistant cell clones that stably express GFP and display effective suppression of ARF6 to similar extents, as was seen with ARF6 siRNA (Fig. 1A). As shown in Fig. 2A, SNB19 ARF6 siRNA cell clones 4, 10, and 12 showed significant decrease in ARF6 expression and 10% FBS–stimulated or 20 ng/mL HGF–stimulated Rac1 activation than that with control siRNA (bottom). The knockdown mediated by pG-SUPER/ARF6/GFP was maintained over 3 months without affecting cell viability under identical culture conditions (data not shown).

Next, we examined the effect of shRNA inhibition of ARF6 on cell motility of SNB19 cells using in vitro and in mouse brain ex vivo assays. As shown in Fig. 2B, in wound healing assays, cell motility of ARF6 siRNA cell clones 4, 10, and 12 was significantly inhibited compared with control siRNA cells. At 24 h after wounding, average distances of the migrating ARF6 siRNA cell clones at the wound edges (mean ± SE) were ∼72 ± 11.3, 50 ± 3.3, and 56 ± 6.5 μm in 10% FBS; 82 ± 7.3, 75 ± 3.7, and 84 ± 4.7 μm in 20 ng/mL HGF compared with control siRNA cells of 125 ± 0.5 μm in both conditions (the wounds were closed). Similar inhibition of cell migration by ARF6 siRNA was also observed when cell clones were analyzed in Boyden chamber migration assays (data not shown). Next, we determined whether ARF6 depletion in SNB19 cells abrogated glioma cell lateral migration/invasion and downward invasion into the brain tissues using the ex vivo murine brain tissue slice assay. As shown in Fig. 2C, inhibition of ARF6 by siRNA in SNB19 cells displayed less lateral migration/invasion on the brain tissue slices compared with control siRNA cells (Fig. 2C , top, white arrows in images with green fluorescence). Analyses by confocal laser scanning microscopy showed that glioma cell invasiveness into the brain tissues was significantly attenuated in ARF6 siRNA cell clones (mean ± SE; clone 10, 121.6 ± 4.5 mm/48 h; clone 12, 123.1 ± 4.1 mm/48 h) when compared with control siRNA cells (172.1 ± 3.9 mm/48 h).

Finally, an orthotopic brain glioma model was used to determine whether inhibition of ARF6 expression by siRNA could suppress glioma cell infiltration into the brain parenchyma of animals. As shown in Fig. 2D, mice that received ARF6 siRNA cell clones developed much less invasive gliomas (Fig. 2D, H&E in b and c and GFP in e and f) compared with the brains that received control siRNA glioma cells (Fig. 2D, H&E in a and GFP in d). Taken together, these results suggest that ARF6 is required in the process of glioma cell invasion in the brain.

IQGAP1 is involved in ARF6-mediated Rac1 activation and glioma cell migration upon stimulation. Having shown that the critical role of ARF6 in glioma cell invasion in vitro, ex vivo, and in animal brains, we investigated the mechanism by which ARF6 regulates glioma cell migration. Previous studies of ARF6 in melanoma cells, breast cancer cells, and Madin-Darby canine kidney (MDCK) cells showed that HGF and EGF induce protein phosphorylation of ERK1/2 through activation of ARF6, leading to an increase in cell migration (7, 33). In glioma cells, EFA6A-enhanced cell motility and invasiveness also require ERK1/2 activation (10). Thus, we examined whether ARF6 is required for HGF stimulation of ERK1/2 in glioma cells. As shown in Fig. 3, 20 ng/mL of HGF strongly stimulated ERK1/2 protein phosphorylation and Rac1 activation in LN229, SNB39, and U373MG glioma cells. Moreover, inhibition of ARF6 protein by siRNA knockdown markedly impaired HGF-stimulated Rac1 activation but had no effect on HGF-stimulated protein phosphorylation of ERK1/2 in these three glioma cell lines. It should be noted that in the experiments shown in Figs. 1 and 3, same batches of glioma cells were transiently transfected with control and ARF6 siRNA and subjected to analyses of immunoblot for knockdown of ARF6 (Figs. 1A and 3), in vitro cell migration (Fig. 1B), ex vivo cell invasion (Fig. 1C), and phosphorylation of ERK1/2 (Fig. 3). Together, these data suggest that activation of ERK1/2 by HGF may not play a prominent role in ARF6-mediated glioma cell migration.

Recent studies showed that Rac1 is a downstream target of ARF6, and several regulatory proteins are likely to be involved in mediating signals between ARF6 and Rac1 (33). Using coimmunoprecipitation methods, we investigated whether ARF6 and Rac1 form a complex upon HGF stimulation and whether other Rac1-GEFs, such as Dock180/ELMO1 (17) and β-PIX (34), or other regulatory/scaffold proteins, such as IQGAP1 (20), could be recruited along with ARF6 upon HGF stimulation. As shown in Fig. 4A, ARF6, Rac1, ELMO1, and Dock180 were expressed at high levels whereas β-PIX was found at low levels in SNB19 cells, as well as several other glioma cell lines examined (data not shown). These cells were treated with 20 ng/mL of HGF, and total cell lysates were immunoprecipitated with an anti-ARF6 antibody or an isotype-matched IgG control. We found that association of ARF6 with Rac1 became evident in 5 minutes upon HGF stimulation and further increased with more prolonged HGF treatment. Residual association of ARF6 with IQGAP1 was also observed without stimulation, but the association was induced rapidly upon a 5-minute HGF stimulation. In contrast, we could not detect ELMO1, Dock180, or β-PIX proteins in the complex with ARF6 and Rac1 (left). These coimmunoprecipitated protein complexes were specific because coimmunoprecipitations using an isotype-matched IgG control did not result in any pull down of indicated proteins under identical conditions (right).

To further examine the connection between ARF6 and IQGAP1, we determined whether ARF6 and IQGAP1 are colocalized in the invasion fronts of HGF-stimulated SNB19 cells by immunofluorescent staining after ARF6 and IQGAP1 were knocked down. As shown in Fig. 4B, in serum-starved control siRNA-treated SNB19 cells, most of ARF6 (green) and IQGAP1 (red) were localized in the cytosol. HGF treatment induced membrane ruffling and formation of cellular protrusions and triggered redistribution of both ARF6 and IQGAP1 from the cytoplasm to membrane protrusions. Colocalization studies revealed that ARF6 is localized to the leading edge with IQGAP1 (Fig. 4B,, middle, arrows). Moreover, effective knockdown of ARF6 or IQGAP1 by siRNAs (Fig. 4C) reduced formation of extended ruffles on the plasma membrane, and the majority of IQGAP1 or ARF6 stayed in the cytoplasm (Fig. 4B , bottom two rows). When colocalization of ARF6 and IQGAP1 was quantified, we found that, without serum and HGF, ∼8.7 ± 3.5% (mean ± SE in 10 random fields) control cells showed colocalization of these two proteins whereas, with 20 ng/mL HGF stimulation, 74 ± 3.7% control cells exhibited costaining of ARF6 and IQGAP1 at lamellipodia. When ARF6 or IQGAP1 was knocked down, only 9.2 ± 3.7% and 7.8 ± 4.6% cells showed overlapping ARF6 and IQGAP labeling. Together, these data suggest that, upon HGF stimulation, ARF6 translocation to the membrane triggers redistribution of IQGAP1 from the cytoplasm to the leading edges of membrane ruffling.

To determine whether IQGAP1 is a downstream regulator necessary for ARF6 signaling in glioma cell migration, we ectopically expressed ARF6-WT and a constitutively active GTPase-deficient mutant of ARF6 (ARF6-Q67L) in U87MG glioma cells that showed low level of ARF6 expression (Supplementary Fig. S1). G418 drug–resistant cell populations were selected among ARF6-WT–expressing, ARF6-Q67L–expressing, and vector-expressing U87MG cell groups. Various cell groups were then transiently transfected with siRNAs of control, Rac1, and IQGAP, respectively. Exogenous expression of ARF6 or knockdown of Rac1 and IQGAP1 did not significantly affect the expression of other proteins, except the targeted molecules (Fig. 5A). We then examined whether knockdown of IQGAP1 blocks the effect of exogenously expressed ARF6-WT and ARF-Q67L to result in decreased glioma cell migration. As shown in Fig. 5B, in the absence of chemoattractants (serum-free) in the lower wells of a Boyden chamber, expression of ARF6-Q67L in U87MG cells results in an increase in cell motility by ∼2-fold compared with the vector-transfected cells (left). However, ectopic expression of ARF6-WT did not enhance cell migration under the identical conditions (data not shown). When cells were exposed to 10% FBS in the lower chambers, expression of ARF6-WT in U87MG cells promoted cell migration by 1.5-fold compared with vector-transfected cells (right). Furthermore, in the presence or absence of 10% FBS in the lower compartments, knockdown of IQGAP1 or Rac1 by siRNA significantly suppressed the motility of U87MG cells enhanced by the ARF6-Q67L expression alone (left) or ARF6-WT expression together with exposure to 10% FBS (right). These data suggest that IQGAP1 plays a critical role downstream of ARF6-mediated glioma cell migration. Lastly, we tested whether knockdown of IQGAP1 affects activation of Rac1 by ARF6 signaling. As shown in Fig. 5C, when the same batches of U87MG glioma cell clones expressing vector, ARF6-WT, or ARF6-Q67L that displayed reduced IQGAP expression by IQGAP siRNA knockdown, but not control siRNA (Fig. 5A,, third panel), were cultured in media with or without 10% FBS, ectopic expression of ARF6-Q67L or ARF6-WT increased the GTP-bound form of Rac1 compared with vector-transfected U87MG control cells in the absence or presence of 10% FBS, respectively. Depletion of IQGAP1 by IQGAP1 siRNA, but not control siRNA, significantly decreased GTP-bound forms of Rac1 to minimal levels (Fig. 5C). Taken together, these data suggest a previously unknown connection between ARF6 and IQGAP1 to Rac1 activation.

In this study, we show that ARF6 mediates glioma cell migration upon HGF and FBS stimulation in vitro and is critical for glioma cell invasion in the brain of animals. We show that ARF6 is expressed in various human glioma cell lines examined. When ARF6 is suppressed by siRNA in invasive LN229, U373MG, and SNB19 glioma cells, HGF-stimulated and FBS-stimulated Rac1 activation and cell motility were substantially attenuated in vitro. When ARF6 was knocked down by siRNA in invasive SNB19 gliomas ex vivo and in the brain, glioma cell invasion into brain parenchyma was also significantly inhibited. Our mechanistic studies reveal that ARF6 mediates HGF stimulation of glioma cell motility through Rac1 and IQGAP1. Inhibition of Rac1 and IQGAP1 by siRNA abrogates HGF-stimulated cell migration, whereas knockdown of IQGAP1 by siRNA suppressed FBS-stimulated cell migration and Rac1 activation. Our data provide convincing evidence that ARF6 regulates HGF-stimulated and FBS-stimulated glioma cell migration through formation of a complex with IQGAP1 and Rac1 in vitro and is critical for invasive behaviors of malignant glioma cells in the brain.

The role of ARF6 in mediating cell motility has been studied in several experimental model systems in vitro. Ectopic expression of ARF6-WT enhanced in MDA-MB-231 breast cancer cell migration and GTPase-deficient mutant ARF6-Q67L inhibited cell migration (8). In LOX melanoma cells and MDCK cells, expression of ARF6-Q67L sustained HGF-stimulated cell migration whereas a dominant-negative ARF6-T27N inhibited HGF stimulation (7, 33). In U373MG glioma cells, activation of ARF6 by one of its own GEF, EFA6A, also stimulated cell migration (10). In the latter three studies, it was shown that ARF6 mediates HGF stimulation of cell migration through activation of ERK1/2 and Rac1 activities. Additionally, coexpression of another GEF of ARF6, GEP100, in MCF-7 cells evoke EGF-dependent cell invasion in vitro, whereas inhibition of GEF100 blocked lung metastasis of mouse 4T1 mammary tumor cells in mice (9). Our results corroborated with these studies. We show that ARF6 is critical for HGF-stimulated glioma cell migration in vitro, cell invasion ex vivo, and in the brain of animals. However, as shown in Fig. 3, HGF stimulation of four glioma cell lines, LN229, U373MG, SNB19, and D54MG (not shown), induced significant protein phosphorylation of ERK1/2. Moreover, effective knockdown of ARF6 in these glioma cells impaired HGF-stimulated Rac1 activity but did not affect HGF-stimulated phosphorylation of ERK1/2. Additionally, in U87MG/ARF6-Q67L cells, expression of constitutively active ARF6-Q67L mutant had minimal effect on phosphorylation of ERK1/2, Akt, and c-Jun NH₂ kinase (data not shown). Although we cannot rule out the existence of compensatory pathways that lead to ERK activation upon depletion of ARF6 in glioma cells, our data suggest that ARF6-mediated HGF or FBS stimulation of glioma cell migration differs from that in breast cancer cells, melanoma cells, and MDCK cells (7, 33). The discrepancy of our results in the involvement of ERK1/2 activation in HGF-stimulated cell migration with the aforementioned studies probably reflects a cell type difference. Moreover, in addition to Rac1 activation, ERK1/2 activation could also be required for HGF-stimulated glioma cell migration. A study of the effect of inhibition of ERK1/2 activation by a MEK inhibition U0126 on HGF-stimulated cell motility is warranted to test this hypothesis. Nonetheless, our results provide the first in vivo evidence that inhibition of ARF6 by siRNA impaired glioma cell infiltration into brain parenchyma in animals, underscoring the critical role of ARF6 in modulation of glioma invasion in the brain.

Activation of Rac1 has been linked with ARF6-mediated cell migration in response to various stimuli (4). In LOX cells, MDCK cells, and U373MG cells, stimulation of ARF6 activates Rac1 through protein phosphorylation of ERK1/2 (7, 10, 33). Alternatively, in Chinese hamster ovary and MDCK cells, activation of ARF6 stimulates Rac1 through interaction with Rac1 coupled with the Dock180/ELMO1 complex (13, 17), perhaps also through binding to β-PIX (18). β-PIX was shown to be upstream of ARF6 and modulates ARF6 activity through a G protein–coupled receptor kinase-interacting protein 1 (34). Our data show that Rac1 could be effectively coprecipitated with ARF6 upon HGF stimulation in a time-dependent manner. However, we failed to detect Dock180, ELMO1, and β-PIX in the complex of ARF6/Rac1 in our glioma cell models (Fig. 4A). Additionally, knockdown of DOCK180/ELMO1 complex by siRNA (16) only partially blocked the ARF6-mediated activation of Rac1 and cell migration in LN229, U373MG, and SNB19 cells (data not shown). Thus, mechanisms that result in Rac1 activation downstream of ARF6 likely vary depending on cell types.

IQGAP1 is an effector of Rac1 and Cdc42, which regulates cell adhesion and migration by modulating actin filaments and microtubule polarization. In response to EGF and vascular endothelial growth factor, IQGAP1 forms complexes with various proteins, including Rac1 enhancing cell migration (20, 22, 35). In NIH3T3 cells, ARF6 GTPase activity is required to regulate IQGAP1-dependent recruitment of a protein complex at membrane ruffles, suggesting coordinated modulation of cell motility by ARF6 and IQGAP1 at a close proximity near membrane ruffles (19). We show that, upon HGF stimulation, IQGAP1 was rapidly recruited into a complex of ARF6 and Rac1 in glioma cells. IQGAP1 is also colocalized with ARF6 at HGF-induced membrane ruffles and cell protrusions. When ARF6 or IQGAP1 was knocked down by siRNA, recruitment of IQGAP1 into the leading edges was diminished. Furthermore, inhibition of IQGAP1 or Rac1 by siRNA in U87MG cells with ectopically expressed ARF6-WT or ARF6-Q67L attenuated ARF6-mediated HGF stimulation of cell migration and serum-stimulated Rac1 activities. Thus, these results establish for the first time a link between ARF6 and Rac1 by IQGAP1 and show an association of ARF6, Rac1, and IQGAP1 in a complex within membrane ruffles. Moreover, in breast and ovarian cancer cells, IQGAP1 directly binds to ERK2 and induces ERK2 phosphorylation. Knockdown of IQGAP1 reduced CD44-stimulated, insulin-like growth factor–stimulated, and EGF-stimulated ERK1/2 phosphorylation inhibiting cell migration (28, 36). In our study, we found that inhibition of ARF6 did not affect HGF-stimulated ERK1/2 phosphorylation, thus suggesting that ERK1/2 activation might not have a prominent role in ARF6-mediated stimulation of IQGAP1 and Rac1. Whether IQGAP1 still interacts with phosphorylated ERK1/2 contributing HGF-stimulated cell migration warrants further investigation.

In summary, the key findings of this study are that ARF6 mediates HGF-stimulated and serum-stimulated glioma cell migration through IQGAP1 and Rac1. Inhibition of ARF6 significantly inhibited glioma cell infiltration ex vivo and in the brain. Our results show for the first time a critical role of ARF6 in regulating the invasive phenotype of glioma cells in vivo and place IQGAP1 between ARF6 and Rac1 in HGF-stimulated signaling in glioma cell invasion. Further investigation of ARF6-IQGAP1-Rac1–mediated glioma cell migration could establish molecules involved in this pathway as potential therapeutic targets for treatment of patients inflicted with malignant gliomas.

No potential conflicts of interest were disclosed.

Grant support: NIH grants CA102011 and CA130966, American Cancer Society grant RSG CSM-107144 (S-Y. Cheng), NIH grant CA115316 (C. D'Souza-Schorey), and Hillman Fellows Program (S-Y. Cheng and B. Hu).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank D. Bigner for D54MG and A172 cells, Y-H. Zhou for SNB19 cells, G. Borisy for a pG-SUPER/GFP vector, K. Ravichandran for the anti-ELMO1 antibody, G. Wang for assistance using the laser confocal microscope, Y. Wan for using an inverted microscope, and N. Balass for proofreading of this manuscript.