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Cell, Tumor, and Stem Cell Biology |
1 Department of Chemistry, Open Laboratory of Chemical Biology, Institute of Molecular Technology for Drug Discovery and Synthesis, and 2 Department of Pathology, University of Hong Kong; 3 Li Ka Shing Institute of Health Sciences and Centre for Emerging Infectious Diseases, The Chinese University of Hong Kong, Shatin, Hong Kong, China; 4 Department of Gastroenterology, Nanfang Hospital; 5 Department of Neurology, The Second Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China; 6 Institute of Molecular and Chemical Biology, East China Normal University, Shanghai, China; and 7 Institut de Pharmacologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique-Unité Mixte Recherche, Valbonne, France
Requests for reprints: Marie Chia-mi Lin, Department of Chemistry, The University of Hong Kong, Kadoorie Biological Science Building, Pokfulam Road, Hong Kong, China. Phone: 852-2299-0776; Fax: 852-2817-1006; E-mail: mcllin{at}hkusua.hku.hk.
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Introduction |
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It has been shown that overexpression of the COOH-terminal region of EFA6A and EFA6B induced the lengthening of microvilli-like membrane protrusions in fibroblastic cell lines (3). Both of these exogenously expressed proteins were found to be localized to structures enriched in polymerized actin such as large membrane ruffles and microspikes on the contact-free plasma membrane (2). In nonneuronal cells, EFA6A perturbed the membrane trafficking of transferrin, suggesting that it might coordinate endocytosis with cytoskeletal rearrangements (2, 3). Moreover, it has also been found to regulate tight junction formation in kidney cells (4). In neuronal cells, Sakagami et al. (5) have recently shown that EFA6A is expressed at the somatodendritic regions of the rat hippocampal neurons and is involved in the regulation of dendritic growth. However, the functional role of EFA6A in the brain remains elusive. In the present study, we aimed to investigate the function of EFA6A in glial cells or glioma.
Human gliomas are the most common primary brain tumors. They account for >40% of all central nervous system neoplasms and have a median survival rate of <12 months (6, 7). The highly lethal nature of this tumor results from the acquisition of an invasive phenotype that allows the tumor cells to infiltrate the surrounding brain tissues (8). The mechanisms for this invasive process, however, are poorly understood. In this study, we first showed that elevated EFA6A expression was detected in a panel of human glioma patient tissue samples. We then examined the role of EFA6A in glioma cell invasion. Using the human glioblastoma cell line U373 as a model, we further showed that EFA6A significantly enhanced cell migration and invasiveness through the activation of ARF6 and extracellular signal–regulated kinase (ERK), suggesting for the first time a role of EFA6A in glioma cell movement and invasion.
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Materials and Methods |
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Measurement of the mRNA levels by semiquantitative RT-PCR. Total RNA was extracted from various human tissue samples and human glioma cells lines using Trizol reagent (Invitrogen, Carlsbad, CA). RT-PCR was done using the Superscript Preamplification System (Invitrogen). Hotstart PCR conditions were as follows: 45 seconds at 94°C, 30 seconds at 55°C, 1 minute at 72°C for 30 cycles [EFA6A and glial fibrillary acidic protein (GFAP)] or for 26 cycles [glyceraldehyde-3-phosphate dehydrogenase (GAPDH)]. The primer pairs for EFA6A were sense 5'-gcctgactctttcagttgtg-3' and antisense 5'-gaagtatcgctgggagaagt-3'; for GFAP were sense 5'-gagtcgctggaggaggagatc-3' and antisense 5'-gggactcgttcgtgccgcgc-3'; and for GAPDH were sense 5'-tgcctcctgcaccaccaact-3' and antisense 5'-cccgttcagctcagggatga-3'.
Plasmids, chemicals, and antibodies. The cDNAs encoding the NH2-terminal vesicular stomatitis virus glycoprotein (VSVG)–tagged EFA6A and its dominant negative mutant EFA6A(E242K) were cloned as previously described (2). Hemagglutinin (HA)-tagged pXS-ARF6(T27N) was kindly provided by Dr. J.G. Donaldson (National Heart, Lung, and Blood Institute, NIH, Bethesda, MD). pGEX-4T1-GST-GGA3 (1-26) was a generous gift from Dr. P. Chavrier (Centre National de la Recherche Scientifique, Institut Curie, Paris, France). The full-length cDNAs encoding the VSVG-tagged EFA6A and HA-tagged ARF6(T27N) were subcloned into the multiple cloning sites of pTRE2hyg plasmid (Clontech) to generate pTRE2hyg-VSVG-EFA6A and pcDNA3.1/Zeo-HA-ARF6(T27N) expression plasmids (Invitrogen), respectively. The sequences were verified by DNA sequencing. pTRE-luc control response plasmid was obtained from Clontech. Mouse monoclonal anti-VSVG antibody (clone P5D4) was obtained from Roche Diagnostics Corp. (Mannheim, Germany). Rabbit monoclonal anti-HA tag antibody was bought from NeoMarkers (Fremont, CA). Mouse monoclonal anti-p-ERK1/2 and anti-ARF6 were bought from Santa Cruz Biotechnology (Santa Cruz, CA). Mitogen-activated protein kinase/ERK kinase (MEK) inhibitor U0126 was obtained from Cell Signaling Technology, Inc. (Beverly, MA).
Generation of doxycycline-responsive gene inducible cell line. Human glioblastoma cell line U373 was transfected with pTet-On regulator plasmid (Clontech) using Lipofectamine 2000 (Invitrogen). After selection by G418 (500 mg/mL) for about 3 weeks, a stable U373 Tet-On cell line was generated. This stable cell line was then transfected with either pTRE2hyg-VSVG-EFA6A or pTRE2hyg alone. After 2 days, the transfected cells were selected with 200 mg/mL hygromycin B (Invitrogen) in the presence of 500 mg/mL G418. Twenty stable hygromycin-resistant cell lines were screened for the expression of EFA6A on addition of doxycycline (1 mg/mL) by RT-PCR and Western blotting. Three stable hygromycin-resistant cell lines, U373-EFA6A, which expressed EFA6A on doxycycline induction, were chosen for subsequent experiments. Induction of luciferase activity in the control pTRE2-Luc transfectants was also confirmed by a Luciferase assay kit (Promega, Madison, WI).
Western blot analysis. Cells were washed twice with PBS and solubilized in radioimmunoprecipitation assay lysis buffer [50 mmol/L Tris-HCl (pH 7.4), 1% NP40, 0.25% Na-deoxycholate, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mg/mL each of aprotinin, leupeptin, and pepstatin, 1 mmol/L Na3VO4, 1 mmol/L NaF]. The supernatants, which contained the whole-cell protein extracts, were obtained after centrifugation of the cell lysates at 10,000 x g for 10 minutes at 4°C. The protein concentration was determined by bicinchoninic acid protein assay kit (Pierce, Rockford, IL). Twenty-microgram protein samples were loaded on an SDS-PAGE gel and analyzed by Western blotting.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was done to assess the effect of EFA6A expression on cell proliferation. U373-pTRE2hyg or U373-EFA6A cells (5.0 x 103) were plated in each well of a 96-well plate. The cells were cultured in growth medium with or without 1 µg/mL doxycycline in a total volume of 100 µL. At various times after doxycycline treatment, 25 µL of sterile MTT dye (5 mg/mL; Sigma, St. Louis, MO) were added and the cells were then incubated for 4 hours at 37°C. After incubation, the MTT solution was removed and 200 µL of DMSO were added and thoroughly mixed for 30 minutes. Spectrometric absorbance was measured on a microplate reader at a wavelength of 570 nm with background subtraction at 660 nm (Spectra Max 340, Molecular Devices, Sunnyvale, CA).
Cell cycle analysis. U373-pTRE2hyg and U373-EFA6A Tet-On cell lines were cultured in medium with or without doxycycline for 2 days, harvested, and washed with ice-cold PBS containing 0.1% glucose. The cells were then fixed with 70% ethanol for 1 hour and incubated in 1 mL of PBS containing 50 mL/mL of propidium iodide (Sigma) and 66 units/mL RNase (Invitrogen) on ice for 30 minutes. DNA content analysis was done by FACScan with CellQuest software (Becton Dickinson, San Jose, CA).
Wound healing assay. In vitro wound healing assay was carried out to investigate the formation of membrane protrusion and cell migration. Equal numbers of U373-pTRE2hyg or U373-EFA6A cells (1.0 x 105) were seeded into six-well tissue culture plates. When the confluence reached 90%, a single wound was created in the center of the cell monolayer by gently removing the attached cells with a sterile plastic pipette tip. The debris was removed by washing the cells with serum-free medium. Migration of the cells into the wound was then observed at different time points. Cells that migrated into the wounded area or cells with extended protrusion from the border of the wound were visualized and photographed under an inverted microscope. A total of nine areas were selected randomly from each well under a 40x objective and the cells in three wells of either group were quantified in each experiment.
In vitro invasion assay. Cell invasiveness in vitro was reflected by the ability of the cell to transmigrate a layer of extracellular matrix in Biocoat Matrigel Invasion Chambers (Becton Dickinson Labware, Bedford, MA). U373-pTRE2hyg or U373-EFA6A cells were plated at a density of 3.0 x 104 per insert. Medium with 10% FBS was added to the lower chamber as a chemoattracctant. For induction of EFA6A expression, doxycycline (1 mg/mL) was added to the lower chamber. After incubation for 22 hours, cells on the upper surface of the membrane were removed. Invasive cells which had the ability to push themselves through the 8-µm pores and grow on the lower surface were fixed with 100% methanol and stained with 1% toluidine blue (Sigma) before counting under an inverted microscope (Leica, Solms, Germany). In all experiments, data were collected from triplicate chambers.
Glutathione S-transferase pull-down assay. The glutathione S-transferase (GST) pull-down assay was done as described by Niedergang et al. (9) by using a bait a fragment (1-226) of the Golgi-localized 
ear–containing ARF-binding protein 3 (GGA3) fused to GST. After washing thrice in ice-cold PBS, the cells were lysed in 50 mmol/L Tris-HCl (pH 8.0), 100 mmol/L NaCl, 10 mmol/L MgCl2, 1% Triton X-100, 0.05% sodium cholate, 0.005% SDS, 10% glycerol, 2 mmol/L DTT, and protease inhibitors (Sigma). Lysates were incubated with 20 µg of GST or 40 µg of GST-GGA3(1-226) bound to glutathione-sepharose beads (Amersham Pharmacia Biotech, Uppsala, Sweden). After 1 hour, the beads were washed thrice in lysis buffer and the proteins were eluted by boiling in 30 µL of sample buffer. Equal amounts of proteins of each sample were analyzed by immunoblot by using an anti-ARF6 antibody.
Statistical analysis. Results are expressed as the mean ± SD. Statistical analyses were done by Student's t test. P < 0.05 was considered statistically significant.
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Expression of EFA6A enhances U373 cell invasion and migration. During metastasis, invasion of basement membrane by tumor cells is thought to be a critical event (10). To study whether expression of EFA6A is associated with cell migration, wound healing assay was done. After doxycycline induction (1 µg/mL) for 22 hours, the migration of U373-EFA6A cells was markedly increased whereas the control U373-pTRE2hyg cells did not show any significant changes (Fig. 3A and B).
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Inhibition of ERK signaling blocks U373 cell invasion induced by EFA6A. We also investigated whether inhibiting ERK1/2 phosphorylation can prevent EFA6A-induced glioma cell invasion. Because MEK1/2 is an upstream activator of ERK1/2 (15), inhibition of MEK1/2 will also block ERK1/2 activation and hence the cell invasion induced by EFA6A. To test this hypothesis, we pretreated U373-EFA6A cells with a specific MEK inhibitor (U0126; 20 µmol/L) for 1 hour, and then exposed the cells to doxycycline (1 µg/mL) in the presence of U0126 for 24 hours. Inactivation of MEK abolished EFA6A-induced ERK1/2 activation (Fig. 5D, left). Importantly, MEK inhibitor U0126 also blocked U373 cell invasion induced by EFA6A expression (Fig. 5D, right). Thus, our data show that EFA6A regulates the invasion of U373 cells through ARF6/MEK/ERK signaling.
The GEF-defective mutant EFA6(E242K) inhibited U373 invasion. Finally, we determined whether the dominant negative mutant EFA6A(E242K), which loses GEF activity for ARF6 (2), could inhibit glioma cell invasion. The pcDNA3.1 expression plasmid alone or pcDNA3.1 carrying the GEF-defective mutant EFA6(E242K) was transfected into U373 cells. After selection with G418 (500 µg/mL) for about 2 weeks, expression of EFA6A(E242K) was verified by Western blotting. To check whether the expression of EFA6A(E242K) can lead to the loss of GEF activity in U373 cells, we also assessed the activation level of endogenous ARF6 by using a pull-down assay based on the association of GTP-bound ARF6 and the GGA3 (9). In U373 cells expressing VSVG-EFA6A(E242K), the level of ARF6-GTP was significantly lower than that of the control cells (Fig. 6A). Consistently, the expression of EFA6A(E242K) also reduced the phosphorylated level of ERK1/2 (Fig. 6B). The invasive ability of the pooled clones was also analyzed by the in vitro cell invasion assay. As shown in Fig. 6C, EFA6A(E242K) markedly inhibited U373 cell invasion by >4-fold. These results further verify that ARF6 is a downstream effector of EFA6A and the GEF activity of EFA6A is essential for the U373 cell invasion.
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Discussion |
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Tumor cell invasion involves complex interactions between the normal and malignant cells. It has been well established that this dynamic process requires the concerted effects of various molecules including proteolytic enzymes, growth factors, adhesion molecules, and extracellular matrix molecules (16, 17). However, the mechanisms of glioma cell motility remain unclear. Studies in other cell types, notably fibroblasts, have revealed that cell motility is dependent on the dynamic remodeling of the actin cytoskeleton (18). To this end, GTPases of the Rho family have been implicated as regulators for cell motility (19). More recently, it has become clear that members of the ARF GTPase family also regulate cytoskeletal assembly and may integrate with Rho proteins in this process (20, 21).
EFA6A is mainly expressed in the brain and elevated expression can be detected in 
50% of the human glioma tissues tested. We also found that 5 of 7 (71.4%) glioma cell lines examined have EFA6A expression. These findings led us to hypothesize that EFA6A may play a role in glioma carcinogenesis. Our data showed that overexpression of EFA6A can promote glioma cell invasion and it seems that EFA6A exerts its effect by activating the ARF6/ERK pathway because blocking this pathway by a dominant negative mutant ARF6(T27N), MEK inhibitor U0126, or EFA6A(E242K) can abolish the cell invasiveness induced by EFA6A.
Structurally, EFA6A comprises a catalytic Sec7 domain, a PH domain, and a COOH-terminal coiled-coil motif. Each conserved domain has its own function. Among the three conserved domains, the Sec7 domain bears the GDP-GTP exchange activity for ARF6 (3), which is a member of the ARF family comprising the Ras-related, low molecular weight (20 kDa) GTP-binding proteins expressed in all eukaryotes (22, 23). In mammals, there are six ARFs and many more ARF-like proteins. Like most GTPases, ARF6 alternates between its active GTP-bound and inactive GDP-bound conformations via activation by GEFs (24). This ARF6 small G-protein cycle has been shown to be important for processes controlling cell shapes and invasion, including endosome membrane trafficking, exocytosis, and actin rearrangements at the cell surface (20, 25, 26).
The molecular mechanism by which EFA6A promotes cell invasion is not fully understood. Previous studies in fibroblastic cells have shown that activation of ARF6 by ARF nucleotide binding site opener, another GEF for ARF6 and ARF1 (27), stimulates epithelial cell migration through downstream activation of both Rac1 and phospholipase D (21, 28). In the case of Madin-Darby canine kidney cells, this transformation requires the dual activation of both Rac1 and phospholipase D, which presumably function together to regulate actin structure and cell motility (21). In this study, we showed that EFA6A-mediated activation of ARF6/ERK is responsible, at least in part, for the migration and invasion of glioma cells. The existence of other mechanisms and the potential cross-talks between various pathways in the precious control of glioma cell migration and invasion remain to be determined.
Activation of ERK1/2 has been shown in other systems to be the mechanism for promoting the production of matrix metalloproteinases (MMP), such as MMP1 and MMP3 (29), which are important for cell proliferation, invasion, and neovascularization. With reference to our data in glioma U373 cells, blocking of the ERK1/2 pathways by MEK1/2 inhibitor U0126 prevents cell invasion induced by EFA6A/ARF6 signaling. This observation suggests that activation of ERK1/2 is also responsible for glioma cell invasiveness. It has been reported that ERK1/2 directly regulates the transcriptional activation of MMP1 and MMP3 (30). Supporting this notion, Westermarck et al. (31) showed that the activation of ERK1/2 induces activator protein-1 transcription, which in turn stimulates MMP1 promoter activity. Thus, it is possible that EFA6A may enhance glioma cell invasion by stimulating the production of MMPs via activation of ERK1/2.
Because ARF6 is a downstream effector for EFA6A and the number of genes coding for ARF GEFs and ARF GTPase-activating proteins in humans is significantly greater than that of genes coding for the ARF isoforms and ARF-like factors (23, 32), ARF6 might be a bona fide factor for tumor invasion. In agreement with this, Hashimoto et al. (33) recently reported that ARF6 is required for breast cancer invasion and there is a direct correlation between the level of ARF6 protein (but not mRNA level) and the invasive capacity of the breast cancer cell lines. A more recent article also reported that ARF6 enhances melanoma cell invasion through the activation of the MEK/ERK signaling pathway and that the ARF6 GTPase cycle regulates ERK1/2 activation (14). Our data showing that the EFA6A-stimulated glioma cell invasion requires the activation of ARF6 and ERK1/2 further support the role of EFA6A/ARF6/ERK signaling pathway in cancer cell invasion. In this regard, EFA6A, as well as its downstream effectors including ARF6, may be considered as a potential therapeutic target for preventing tumor invasion and metastasis.
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Acknowledgments |
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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 Drs. J.G. Donaldson and P. Chavrier for generously providing the cDNAs, and Dr. M.E. Hegi for the kind gift of LN-308 cell line.
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Footnotes |
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Received 7/11/05. Revised 10/27/05. Accepted 12/ 1/05.
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MAPK enhances collagenase-1 (matrix metalloproteinase (MMP)-1) and stromelysin-1 (MMP-3) expression by mRNA stabilization. J Biol Chem 2002;277:32360–8.This article has been cited by other articles:
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  R. Poincloux, F. Lizarraga, and P. Chavrier Matrix invasion by tumour cells: a focus on MT1-MMP trafficking to invadopodia J. Cell Sci., September 1, 2009; 122(17): 3015 - 3024. [Abstract] [Full Text] [PDF]
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B. Hu, B. Shi, M. J. Jarzynka, J.-J. Yiin, C. D'Souza-Schorey, and S.-Y. Cheng ADP-Ribosylation Factor 6 Regulates Glioma Cell Invasion through the IQ-Domain GTPase-Activating Protein 1-Rac1-Mediated Pathway Cancer Res., February 1, 2009; 69(3): 794 - 801. [Abstract] [Full Text] [PDF]
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and 
, U373-pTRE2hyg cells treated with (+) or without (–) doxycycline, respectively. 
and 
, U373-EFA6A cells treated with (+) or without (–) doxycycline, respectively (n = 6). D, cell cycle analysis by propidium iodide staining assay. a and b, U373-pTRE2hyg cells cultured in the absence (–) or presence (+) of doxycycline, respectively. c and d, U373-EFA6A cells cultured in the absence (–) or presence (+) of doxycycline, respectively (n = 6).
3). *, P < 0.05, statistically significant difference between U373-EFA6A cells and the control U373-pTRE2hyg cells. 
(–), Dox; 
(+), Dox.
