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ADP-ribosylation factors are determining monomeric G proteins in actin cytoskeleton reorganization, lipid membrane remodeling, and vesicule formation. Our laboratory has previously shown that ARF1 is overexpressed in highly invasive breast cancer cells and contribute to their enhanced proliferation and migration phenotype. In this study, we propose to define the role of ARF1 on the activation of Rac1, an important member of the Rho family of GTPases implicated in the formation of lamellipodia and the migration process. Overall, we evaluated whether ARF1 activation could affect Rac1 activation and the signaling pathway necessary for cell migration. We first determined that the endogenous inhibition of ARF1 expression, in MDA-MB-231 cells, permitted to alter EGF-dependent Rac1 activation. We next examined Rac1 interaction partners. We showed that ARF1 and Rac1 are constitutively complexed and that ARF1 is necessary for Rac1 associastion with IRSp53, an essential protein for lamellipodia formation. We finally confirmed the role of Rac1 in migration by an RNAi approach that showed the essential contribution of Rac1 in ARF1-dependant migration. In conclusion, this study will contribute to better define the role of ARF1 in the migration process of breast cancer cells and to demonstrate that this GTPase is a potential pharmacological target.
Introduction (Je n'ai pas parlé de l'EGFR…)
Breast cancer is the most common cancer in woman population (1). Although considerable advances has been realized over the past years, patients with triple-negative breast cancer (TNBC), wich represents 15-20% of all breast cancers (2), still suffer from a limited choice of targeted therapies (3,4). Therefore, TNBC remains associated with a poor prognosis (5,6), a phenomenon accentuated the aggressive nature and the high risk of metastasis formation linked to this type of cancer (7,8). In this regard, cell migration has been considered as a promising pharmacological target in the development of new cancer treatment strategies since it is a key step in metastasis.
The Rho GTPase family plays an important role in intracellular actin dynamics processes including cell adhesion, polarity, motility and cell-cycle progression (9-12). Among the family members, Ras related C3 botulinum toxin substrate 1 (Rac1) is known to regulate different signaling pathways to promote cytoskeleton remodeling, lamellipodia formation and cell migration (13). In pathological conditions, Rac1 activation can induce invasion and metastasis of in vitro and in vivo breast cancer cell line models (14). To achieve its functions, Rac1 cooperates with the Wiskott-Aldrich syndrome protein family verprolin homologous protein (WAVE) complex to activate actin-related protein 2/3 (Arp2/3) complex, leading to actin nucleation of a network of branched actin filaments (15). It has also been shown that the insulin receptor tyrosine kinase substrate p53 (IRSp53) acts as the adaptor protein linking Rac1 to WAVE2 to induce actin polymerization (16-18).
ADP-ribosylation factors (ARFs) are determining monomeric G proteins in actin cytoskeleton reorganization, lipid membrane remodeling, and vesicule formation [REFsss]. Our laboratory has previously shown that ARF1 is overexpressed in highly invasive breast cancer cells and contribute to their enhanced proliferation (19) and migration phenotype (20). [Inclure EGFR ici?] In this study, we investigated the molecular mechanisms by which ARF1 regulates Rac1-dependent migration in MDA-MB-231, a TNBC cell line. Here, we show that the active state of ARF1 influence the EGF-dependent activation of the second GTPase. This inhibition was traduced by a reduced migratory phenotype, showing the potential of ARF1 in inhibiting metastasis formation. In this study, ARF1 was found constitutively bound to Rac1 and is thought to act on him by inhibiting is association with IRSp53, blocking the formation of lamellipodia necessary for cell migration. The comprehension by wich ARF1 controls EGF-dependant cell migration is important for the development of future breast cancer therapies.
Materials and Methods
Reagents and Antibodies
Polyclonal anti-ARF1 (CAT#20226-1-AP) was purchased from Proteintech (Chicago, IL, USA), monoclonal anti-Rac1 (CAT#05-389) from Millipore (Billerica, MA, USA) and polyclonal anti-IRSp53 (CAT#ab37542) from Abcam (Cambridge, MA, USA). EGF (CAT#30R-AE004) was purchased from Fitzgerald (Acton, MA, USA). All others products were from Sigma Aldrich Company (Oakville, ON, Canada).
DNA Plasmids and Small Interfering RNA
Double-stranded small interfering RNA (siRNA) targeting human Rac1 (sequence: 5′-GAGGAAGAGAAAAUGCCUG-3′), human ARF1 siRNA (CAT#J-011580-08-0050) and the non-targeting control (CAT#D-001810-01-50) were purchased from Thermo Fisher Scientific (Nepean, ON, Canada). GST-Rac1(Δ CAAX) was a gift from J. D. Lambeth (Emory University, Atlanta, GA, USA). Rac1-myc, Rac1T17N-myc, Rac1(Q61L)-myc, and GST-PAK(CRIB) were obtained from Dr. N. Lamarche-Vane (McGill University, Montreal, QC, Canada). GST-GGA3 was from Dr. J.-L. Parent (Université de Sherbrooke, Sherbrooke, QC, Canada).
Cell Culture and Transfection
MDA-MB-231 cells were obtained from Dr. Sylvie Mader (Université de Montréal, Montreal, QC, Canada). Cells were maintained at 37°C, 5% CO2, in Dulbecco's modified eagle medium (DMEM) (CAT#319-005-CL) supplemented with 10% fetal bovine serum (CAT#080-150), 10% penicillin/streptomycin (CAT#450-201-EL) and 1X MEM non essential amino acids (CAT#321-011-EL). All cell culture reagents were purchased from Wisent Bioproducts (St-Bruno, QC, Canada). Transfection of DNA plasmids (48h) and siRNAs (72h) were realized using Lipofectamine® 2000 (CAT#11668-019) from Invitrogen (Burlington, ON, Canada) according to the manufacturer's instructions.
Proteins were run on polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes were blotted for relevant proteins using specific antibodies described in the following sections. For Rac1 detection, FITC-conjugated secondary antibody fluorescence was detected using a Typhoon 9410 scanner (Amersham Biosciences, Baie D'Urfé, QC, Canada) while the other proteins where detected by enhanced chemiluminescence of a HRP-conjugated secondary antibody.
GTPase Activation Assay
MDA-MB-231 cells were plated in 10 cm dishes and serum starved overnight. The cells were stimulated with EGF (10 ng/ml) at 37°C for the indicated times. Cells were lysed in 150 μl of ice-cold MLB buffer (pH 7.5, 25 mM HEPES, 150 mM NaCl, 10 mM MgCl2, 1 mM EDTA, 10% glycerol, 1% nonidet P-40, 0.3 mg/ml PMSF, 1.0 mM Na3VO4 and protease inhibitors). Samples were spun for 10 min at 12,000 g (4°C). Glutathione S-transferase-golgi-associated, gamma adaptin ear containing, ARF binding protein 3 (GST-GGA3) or glutathione-S-transferase-p21-activated kinase-Cdc42/Rac interactive binding domain (GST-PAK-CRIB) coupled to glutathione-sepharose 4B was added to each tube. Samples were tumbled at 4 °C for 1 h. Beads were washed three times with lysis buffer and proteins were eluted in 20 μl of SDS-sample buffer by heating to 65°C for 15 min. Detection of GTP-bound GTPases was performed by immunoblot analysis using a specific anti-ARF1 or anti-Rac1 antibody.
MDA-MB-231 cells were transiently transfected with 25 μM of Rac1 siRNA, 50 μM of ARF1, 2 μg of Rac1(Q61L)-myc DNA or the equal quantity of control. 72h (siRNAs) or 48h (plasmids) later, cells were trypsinized and 40,000 cells were seeded into Transwell® permeable support of 8.0 μm polycarbonate membrane (Corning, NY, USA) coated with 6 μg/ml collagen solution from bovine skin (Sigma-Aldrich, Oakville, ON, Canada). One hour later, cells were stimulated with 10 ng/ml EGF and incubated for 6h migration. A paraformaldehyde solution (4% in phosphate buffered saline, 20 min) was used to fix the cells and a violet crystal solution (0.1% in 20% methanol, O/N) for staining. Cells present in the upper chamber were removed with a cotton bud and migrated cells were quantified in the lower chamber.
Wound Healing Assay
MDA-MB-231 cells were transiently transfected into 6-well plates with 25 μM of Rac1 siRNA, 50 μM of ARF1 or 50 μM of non-targeting siRNA. After 72h, confluent cells were serum-starved for 8h. Three scratches per well were then performed using a micropipette tip under an angle of around 30 degrees. Cells were washed twice with serum deprived DMEM, treated with 100 ng/ml EGF, and incubated for 24h migration. A paraformaldehyde solution (4% in phosphate buffered saline, 20 min) was used to fix the cells and a violet crystal solution (0.1% in 20% methanol, O/N) for staining. Scratch images were taken with a MICROSCOPE DU GÉPROM. A violet pseudocolor was applied to the pictures with Adobe Photoshop CS5.1 to facilitate visualization.
GST Pulldown Assay
GST pulldown assays were described previously (21). Briefly, for the fusion protein loading experiments, equal amounts of GST and GST-Rac1(Δ CAAX) were incubated at 30°C with either GDPβS (100 μM) or GTPγS (10 μM) for 30 min with an agitation of 900 rpm. Nucleotide loading was stopped by adding 60 mM MgCl2 at 4°C. Purified nonmyristoylated recombinant ARF1 (gift from Nicolas Vitale) was then added to the mixture and samples were tumbled for 4h at 4°C. For ARF1 nucleotide loading experiments, the purified ARF1 was incubated with either nucleotide before being mixed with GST-Rac1(Δ CAAX) in the inverse similar way described for the fusion protein loading experiments. After tumbling, beads were washed three times with lysis buffer. Proteins were eluted into 20 μl of SDS sample buffer by heating to 65°C for 15 min. Detection of interacting GTPases was performed by immunoblot analysis using a specific anti-ARF1 or anti-Rac1 antibody respectively.
MDA-MB-231 cells were plated in 10 cm dishes and serum starved overnight. The cells were stimulated with EGF (10 ng/ml) at 37°C for the indicated times. Cells were lysed in 150 μl of ice-cold TGH buffer (pH 7.3, 50 mM HEPES, 50 mM NaCl, 5 mM EDTA, 10% glycerol, 1% triton and protease inhibitors). Samples were spun for 10 min at 12,000 g (4°C) and equal amounts of soluble protein were incubated with specific anti-ARF1 or anti-Rac1 antibodies for 1h (4°C). Protein G+ agarose beads (CAT#sc2002, Santa Cruz Biotech Inc, Santa Cruz, CA, USA) were then added for 2h. Beads were washed three times with lysis buffer and proteins were eluted in 20 μl of SDS-sample buffer by heating to 65°C for 15 min. Detection of co-precipitated GTPases was performed by immunoblot analysis using a specific anti-ARF1 or anti-Rac1 antibody.
Quantification of the digital images obtained by immunoblot analyses was performed using ImageJ 1.46o software (National Institutes of Health, USA). Statistical analyses were calculated using a one-way analysis of variance followed by a Bonferroni's multiple comparison tests using GraphPad Prism Software (ver. 5.02; San Diego, CA, USA).
Depletion of ARF1 inhibits EGF-dependent Rac1 activation
We first examined the profile of ARF1 and Rac1 activation in MDA-MB-231 cells upon EGF stimulation. As shown in Figure 1A, a rapid and transient activation of endogenous ARF1 was observed after 1 min stimulation whereas maximal levels of endogenous Rac1-GTP were detected after 5 min. It was previously reported that the activation of ARF6 by the GEF ARNO leads to the activation of Rac1 (22). To assess whether ARF1 and Rac1 could both act in the same signaling axis, we next investigated whether activation of one GTPase could regulate the function of the other using an RNAi approach. Depletion of ARF1 significantly inhibited EGF-dependent activation of Rac1 (Fig. 1B). However, depletion of Rac1 had no effect on ARF1 activation, suggesting that this ARF isoform acts upstream to control key processes mediating the activation of the Rho GTPase. Altogether, these results suggest that EGF-induced ARF1 activation modulates Rac1 activation in MDA-MB-231 cells.
ARF1 is overexpressed in many cancers and is sign of a poor diagnosis. We first examined the effect of ARF1 state of activation on Rac1 activation.
ARF1 interacts with Rac1
We have previously shown that ARF6 can be found in complex with Rac1 upon Ang II stimulation of HEK 293 cells (21). To further address the role of ARF1 on Rac1, we examined whether these two GTPases could be found in complex in MDA-MB-231 cells. As depicted in Figure 3A, endogenously expressed Rac1 was found in ARF1 co-immunoprecipitates and vice-versa. This association was not modulated by EGF stimulation. We next examined whether this interaction could be direct. Using purified proteins preloaded with either GDPßS or GTPï§S, we showed that ARF1 can directly interact with Rac1 and that the nature of the nucleotide bound to the GTPases does not impact their interaction (Fig. 3B). Altogether, these results suggest that in MDA-MB-231 cells, ARF1 and Rac1 are found constitutively associated whether they are in their active or inactive state. (Manip avec mutants pas finie.)
ARF1 controls Rac1 interaction with IRSp53
In renal cell carcinoma, it was demonstrated that some proteins regulating the actin cytoskeleton like the Kank family could inhibit actin remodeling by preventing the interaction between IRSp53 and Rac1 (23). Since ARF1 also acts has a molecular switch to control cell migration, we investigated if this GTPase could prevent cell motility in a similar way in breast cancer cells. As we can see in Figure 5A, Rac1 and IRSp53 had the ability to form an EGF-dependent complex after 15 min stimulation. This interaction was blocked to basal level by inhibition of endogenous expression of ARF1 and completely lost when depleting Rac1 as a negative control (Figure 5B). In sum, these results suggest that ARF1 is required for Rac1 functions and could therefore be a good candidate for the pharmacological inhibition of cell migration.
Depletion of ARF1 inhibits EGF-induced Rac1 cell migration
Inhibition of Rac GTPases has been shown to block the spread of intact human breast cancers (24). Knowing that ARF1 has the ability to modulate Rac1 activation, we next studied the impact of ARF1 on Rac1-dependent cell migration. Overall, we observed a twofold increase in the motility of MDA-MB-231 cells upon EGF stimulation (Fig. 2). In a wound healing assay, depletion of either isoform markedly impaired the ability of cancer cells to migrate, demonstrating the key roles of these GTPases in the progression of cancer (Fig. 2A). Similarly, depletion of Rac1 (Fig. 2C) or ARF1 (Fig. 2B) completely abrogated EGF-induced cell migration in a collagen-coated Boyden chamber assay. Furthermore, overexpression of a constitutively active mutant form of Rac1, Rac1(Q61L), spontaneously increased the basal level of migration to the level of control stimulated cells while resulting in a threefold increase in EGF-promoted migration. Interestingly, ARF1 depletion failed to alter Rac1(Q61L)-enhanced basal motility but prevented EGF-increased cell migration. Taken together, these results suggest that ARF1 cooperates with Rac1 to modulate EGF-induced migration.
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This work was supported by grants from the Canadian Institutes of Health Research.