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Abstract
The endocannabinoid system regulates cell proliferation and migration in human breast cancer cells. In this study, we showed that a metabolically stable analog of anandamide, 2-methyl-2′-F-anandamide (Met-F-AEA), inhibited the RHOA activity and caused a RHOA delocalization from the cell membrane to cytosol determining a decrease in actin stress fibers. Overexpression of a dominant negative of RHOA activity and treatment of these cells with a RHO-associated protein kinase (ROCK) inhibitor, Y 27632, mimicked Met-F-AEA effects on actin organization and cell migration. We suggest that the inhibitory effect of Met-F-AEA on tumor cell migration could be related to RHOA-ROCK-dependent signaling pathway.
Introduction
Metastasis is a multifactorial process that includes the acquisition of a motile and invasive phenotype. During progression to a metastatic phenotype, tumor cells undergo a series of changes that begin with loss of contact inhibition and increased motility, allowing them to migrate from the primary tumor site, invade distant organs, and induce neo-vascularization resulting in metastasis (Chambers et al. 2002). Many of these changes are associated with dynamic actin reorganization and activation of signaling pathways through transmembrane receptors (Oft et al. 1998, Seasholtz et al. 1999, Muller et al. 2001, Roymans & Slegers 2001, Vivanco & Sawyers 2002). Members of the small guanosine triphosphatase (GTPase) family control cell adhesion and motility through reorganization of the actin cytoskeleton and regulation of actomyosin contractility (Yoshioka et al.1998, Etienne-Manneville & Hall 2002). Both RHOA (Fritz et al. 1999) and the related RHOC (Clark et al. 2000) are expressed at a relatively higher level in metastatic tumors, and their expression levels positively correlate with the stage of the tumors. However, mutations in the RHO gene have not yet been found in human tumors; rather, the overexpression of RHOA in the cell facilitates its translocation from the cytosol to the plasma membrane, where its activation results in stimulation of the actomyosin system, followed by cellular invasion both in vitro and in vivo (Yoshioka et al. 1999). One of the target molecules of RHO is the family of RHO-associated serine—threonine protein kinases (ROCK; Leung et al. 1995), which also participates in cell-to-substrate adhesions, stress fiber formation, and stimulation of actomyosin-based cellular contractility (Narumiya et al. 1997). In particular, several breast cancers contain large amounts of RHOA proteins, whereas RHOA was hardly or not detectable in adjacent normal tissue. In addition, the progression of breast tumors from WHO grade I to grade III is accompanied by a significant average increase in RHOA protein levels (Van Aelst & D'Souza-Schorey 1997, Hall 1998, Fritz et al. 1999, Yoshioka et al. 1999, Kamai et al. 2001, 2003, Horiuchi et al. 2003). The endogenous cannabinoid system, consisting of the cannabinoid CNR1 and CNR2 transmembrane receptors G-protein coupled, their endogenous ligands (endocannabinoids), and the proteins that regulate endocannabinoid biosynthesis and degradation, is an almost ubiquitous signaling system involved in the control of cell fate. The endocannabinoids (anandamide, AEA, and 2-arachidonoyglycerol, 2AG) have been shown to inhibit the growth of tumor cells in culture and animal models by modulating key cell signaling pathways (Bifulco et al. 2006, Mackie & Stella 2006). We have also shown that a metabolically stable anandamide analog, 2-methyl-2′-F-anandamide (Met-F-AEA), arrests the growth of K-ras-dependent tumors, induced and/or already established in vivo, and it inhibits the formation of metastatic nodules in the Lewis lung carcinoma model, these effects being largely mediated by cannabinoid CNR1 receptors (De Petrocellis et al. 1998, 2000, Bifulco et al. 2001, Bifulco & Di Marzo 2002, Portella et al. 2003). Moreover, it has been observed that cannabinoid receptors activation is able to inhibit vascular endothelial cell migration and to downregulate the expression and the activity of matrix metalloproteinase-2, a proteolytic enzyme involved in tissue remodeling during angiogenesis and metastasis (Pisanti et al. 2007). Anandamide inhibits the migration of human colon carcinoma cells (SW480) through a CNR1-dependent mechanism (Joseph et al. 2004). Interestingly, it was recently found that increasing endogenous 2-AG, by blocking its metabolism, inhibits invasion of androgen-independent prostate cancer (PC-3 and DU-145) cells. The anti-invasive effect of endogenous 2-AG relies, once again, on CNR1 activation and consequent inhibition of the adenylyl cyclase and protein kinase A (PKA) pathway (Nithipatikom et al. 2004). Nevertheless, the molecular mechanism of the cannabinoids anti-metastatic effect has not been fully explored. Recently, we described that Met-F-AEA treatment is able to inhibit cell migration of the highly invasive and metastatic breast cancer cell lines, MDA-MB-231, as tested in migration assays with type IV collagen, the major component of the basement membrane. This effect is mediated by CNR1 receptor and it is accompanied by a remarkable decrease in phosphorylation of focal adhesion kinase (FAK) and CSK protein, two tyrosine kinases involved in the loss of adhesion and invasion (Grimaldi et al. 2006). In this study, we further investigated the molecular mechanism of anti-metastatic potential of Met-F-AEA by studying the effect of this agonist of CNR1 receptor on RHOA activity and actin organization.
Materials and methods
Drugs and cell culture
Met-F-AEA was purchased from Sigma. The selective CNR1 antagonist, SR141716, was kindly provided by Sanofi-Aventis (Montpellier, France). Toxin B was purchased from Calbiochem (La Jolla, CA, USA), the inhibitor of RHO kinase, Y 27632 from Sigma, GGTI-298 was purchased from Calbiochem. Monoclonal antibody to RHOA and polyclonal antibody to CNR1 receptor were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The human breast carcinoma cell line MDA-MB-231 was maintained in RPMI 1640 culture medium (Gibco BRL Life Technologies) supplemented with 10% inactivated fetal bovine serum (FBS) and 2 mM L-glutamine. Cells were cultured in a humidified environment containing 5% CO2 and held at a constant temperature of 37 °C.
Separation of particulate and cytosolic fractions
Cells were grown to subconfluency (60—70%), then lysed by ice-cold lysis buffer (50 mmol/l HEPES (pH 7.5), 50 mmol/l NaCl, 1 mmol/l MgCl2, 2 mmol/l EDTA, 10 mmol/l NaF, 1 mmol/l dithiothreitol, 1 mmol/l phenylmethylsulfonyl fluoride, 10 mg/ml aprotinin, and 10 mg/ml leupeptin) and centrifuged at 100 000 g for 30 min in micro-ultracentrifuge, and the supernatant was collected as the cytosolic fraction. Pellets were resuspended, and membrane proteins homogenized in 150 μl lysis buffer containing 2% Triton X-114. The homogenate was centrifuged at 800 g for 10 min. The supernatant (particulate fraction) and pellet (detergent insoluble particulate fraction) were collected separately. Whole cell, cytosolic, and particulate fraction proteins were separated by SDS-PAGE.
Western blot analysis
Cells plated in 100 mm dishes in regular medium with serum were washed with ice-cold PBS and scraped into lysis buffer (50 mM Tris—HCl, 150 mM NaCl, 0.5% Triton X100, 0.5% deossicolic acid, 10 mg/ml leupeptin, 2 mM phenylmethylsulfonyl fluoride, and 10 mg/ml aprotinin). After removal of cell debris by centrifugation (14 000 g, 5 min), about 50 μg of proteins were loaded on 12% SDS-polyacrylamide gels under reducing conditions. After SDS-PAGE, proteins were transferred to nitrocellulose membranes that were blocked with 5% milk (Bio-Rad Laboratories, Inc.) and incubated with anti-CNR1 antibody. After three washes, filters were incubated for 1 h at room temperature with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody. The membranes were then stained using a chemiluminescence system (ECL-Amersham Biosciences) and then exposed to X-ray film (Kodak).
Invasion assay
For chemotaxis assays, Boyden chambers (8 Am Transwell polycarbonate membrane, Costar, NY, USA) were coated with 50 μg/ml type IV collagen and blocked with 5 mg/ml BSA. Cells (1×105 cells) treated for 24 h with Met-F-AEA alone or in combination with toxin B (100 ng/ml) or Y 27632 (10 μM) were added to the upper compartment and incubated (at 37 °C for 4 h) in migration media RPMI in the presence or absence of FBS used as a chemotactic stimulus in the lower compartment. Chambers were washed with PBS, and migratory cells on the lower membrane surface were fixed with 3% formaldehyde (10 min). Cells were permeabilized with 0.2% Triton X-100 (5 min) and stained with Hoechst dye (5 min). The number of migrated cells was counted by a light microscope at 20× magnification: ten randomly chosen microscopic fields were counted per well, and the mean number was determined. Background levels of cells migrated in the absence of chemotactic stimuli (chemokinesis) were subtracted from all the experimental points.
RHOA activation assay
The amount of activated Rho(GTP-Rho) was determined using a Rho activation assay kit (Upstate Biotechnology, Inc., Lake Placid, NY, USA). The human MDA-MB-231 cells were grown to confluence and then placed in serum free for 24 h. After treatment, the cells were washed with ice-cold PBS. Cellular lysates were prepared according to the manufacturer's instructions.
Immunofluorescence
Cells plated on cover slips were washed once with PBS and fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.5% Triton X-100 for 5 min, and incubated with PBS containing 4% BSA for 1 h at room temperature. F-actin was stained with tetramethyl rhodamine B isothiocyanate-labeled phalloidin (500 ng/ml; Sigma—Aldrich) to visualize filamentous actin. Cells were plated in 24-well plates on cover slips (Becton—Dickinson Labware, NY, USA). When they were 60±80% confluent, they were treated with Met-F-AEA (10 μM for 15′, 3 and 24 h). After the incubation with various drugs, the cells were washed twice with PBS, fixed in 3.7% paraformaldehyde in PBS for 20 min and followed by two washes in 50 mM NH4Cl for 10 min. Permeabilization was achieved by incubating the fixed cells in 0.1% Triton X-100 in PBS for 5 min at room temperature. The cells were then blocked in final dilution buffer (FDB) (1 mM MgCl2, 1 mM CaCl2, 5% fetal calf serum, and 2% BSA in PBS) for 30 min at room temperature. All primary and secondary antibody incubations were performed in FDB buffer for 1 h at room temperature. Cover slips were mounted on 50% glycerol in PBS and examined by using a Zeiss Laser Scanning Confocal Microscope (410 or 510).
Cell transfection
Plasmids were purified using the endotoxin-free plasmid midi prep kit (Qiagen).
For transient transfections, MDA-MB-231 cells were seeded in 6 cm plates and grown to 60—80% confluence in RPMI with 10% FBS without antibiotics and transfected the next day using FuGene reagent (Promega), according to the manufacturer's protocol, with a total of 2 μg/well of plasmid DNA. The cells were transfected with control vector (pcDNA3) or pcDNA3-N19RHOA provided by Dr Mario Chiariello (IEOS, CNR Napoli). Twenty-four hours after transfection, cells were prepared for immunofluorescence analysis and for wound-healing assay. For knocking down CNR1 protein expression, we used a 25-nucleotide small interfering RNA (siRNA) duplex (Dharmacon Research, Lafayette, CO, USA) designed for specific silencing of CNR1 (siRNA-CNR1) covered the sequence sense 5′-CCCAAGUGACGAAAACAUU-dTdT-3′ gently provided by Dr Patrizia Gazzerro (University of Salerno). We transfected cells using Lipofectamine (Invitrogen) and 30 nM siRNA for each transfection, in accordance with the manufacturer's protocol. An equal concentration of SilencerR Negative Control-1 siRNA (Ambion Inc. NY, USA) was used as negative control. The transfected MDA-MB-231 cells were serum starved and after 24 h assayed for RHOA activity. Specific silencing of targeted genes was confirmed by western blot analysis for CNR1 receptor.
Statistical analysis
All data were presented as means±S.D. Statistical analysis was performed using one-way ANOVA analysis. In the case of a significant result in the ANOVA, Student's t-test was used for dose—response curves and Bonferroni's test for post hoc analysis for all other experiments. A P value less than 0.05 was considered statistically significant.
Results
Met-F-AEA inhibits RHOA activity and induced delocalization of RHOA from cell membrane
Since in a recent report we demonstrated that metabolically stable anandamide analog, Met-F-AEA at 10 μM inhibited the migration of MDA-MB-231 human breast cancer cells (Grimaldi et al. 2006), we hypothesized that this agonist of the CNR1 receptor could affect the activity of RND1/2/3 involved in the cell migration and actin organization of several cancer cells. To examine this hypothesis, we analyzed the level of the active GTP-bound form of RHOA by a pull-down assay with the RHO-binding fragment of rhotekin in cells treated with Met-F-AEA at 10 μM. The cells were developed in medium serum free for 24 h, and after treatment we collected the cells and performed the assay. As shown in Fig. 1A, a large proportion of the RHOA protein detected in MDA-MB-231 cells is present in an active form. Upon the stimulation of CNR1 receptor by Met-F-AEA, the level of GTP-RhoA was decreased after 15 min and strongly reduced after 1 h. The RHOA activity is restored in Met-F-AEA-treated cells in presence of SR141716, antagonist of CNR1 receptor. To evaluate the maximum effect of Met-F-AEA on RHOA activity, MDA-MB-231 cells were treated with increasing concentrations of Met-F-AEA from 2.5 to 10 μM, then they were collected and was performed a pull-down assay. As shown in Fig. 1D, the anandamide analog strongly reduced the RHOA activity in a dose-dependent manner. The reduction was statistically significant at 10 μM concentration compared with the control. Furthermore, in order to confirm that this effect is mediated by CNR1 receptor, we reduced the level of expression of CNR1 receptor by RNA interference. To specifically silence the CNR1 gene, MDA-MB-231 cells were transfected with siRNA targeting CNR1 mRNA. Western blot analysis (Fig. 1C) showed that the protein level of CNR1 was markedly downregulated by CNR1 siRNA at 24 h after administration of the siRNA, with reductions of 80% of protein expression of CNR1 receptor in MDA-MB-231 cell line compared with lysates transfected with no silencing siRNA. Incubation of CNR1-siRNA transfected cells with Met-F-AEA at 10 μM for 1 h did not affect the RHOA activity (Fig. 1B). More, because RHOA protein must be targeted to the plasma membrane for activation and full function, we examined the translocation of this protein from the cytosol to the cell membrane in MDA-MB-231 cells in the presence of Met-F-AEA at 10 μM, after separation of particulate and soluble fractions. In untreated cells without serum, most of RHOA protein was present on the cell membrane (particulate fractions; Fig. 2A, ctr and B), suggesting that in these breast cancer cells the overexpression of RHOA facilitated its translocation from the cytosol to the cell membrane as previously described (Fritz et al. 1999). Treatment of MDA-MB-231 cells with Met-F-AEA clearly decreased the amount of RHOA in the membrane fraction and accumulates in the cytosolic fraction in a time-dependent manner.
Anandamide induces reorganization of the actin cytoskeleton
As RHOA is involved in the regulation and assembly of contractile actin filaments (stress fibers), we studied the effect of Met-F-AEA on actin cytoskeleton in these cells by immunofluorescence confocal microscopy using phalloidin TRITC-conjugated. As shown in Fig. 3A, various stress fibers were detected in untreated cells. The treatment of these cells with Met-F-AEA at 10 μM caused a significant decrease in F-actin containing stress fibers, which was apparent within 15 min (Fig. 3C). After longer incubation times, many cells displayed a dense meshwork of unpolarized actin filaments around the cell periphery (3 and 24-h time point are shown in Fig. 3E and F). We also observed the intracellular distribution of RHOA with a specific monoclonal antibody and a secondary antibody FITC-conjugated. Interestingly, RHOA was present at the membrane periphery in untreated cells (Fig. 3B); while at 15 min of treatment with Met-F-AEA, the RHOA patches to the cell membrane are reduced (Fig. 3D). After 3 h, RHOA remained largely diffused in the cytoplasm mainly in the perinuclear region in confront to control sample and, at 24 h, the RHOA fluorescence-associated is equally widespread into cytosol. To confirm the role of RHOA in the actin organization, we transiently transfected the cells with a vector carrying a dominant negative of RHOA (N19RHOA) which antagonizes the RHO guanine nucleotide exchange factors (ARHGEFs), when compared with cells transfected with empty vector. As shown in Fig. 4, in cell transfected with empty vector (mock), the actin is organized in stress fibers, while in that with vector carrying N19RHOA we observed a significant reduction in actin stress fiber as how in Met-F-AEA-treated cells (Fig. 3E, C and G). In addition, because ROCK, the immediate downstream effector of RHOA, has been clearly implicated in cancer migration, we examined the effect of a selective inhibitor of ROCK I and II, Y 27632 (Ishizaki et al. 2000) on actin organization. Cells treated with this ROCK inhibitor at 10 μM reduced actin stress fibers (Fig. 4A). We previously reported that Met-F-AEA inhibited MDA-MB-231 cell migration on type IV collagen (Grimaldi et al. 2006), in order to verify the involvement of RND1 signaling pathway in the Met-F-AEA effect on cell migration, we used Toxin B of Clostridium difficile, which glycosylates threonine residue of RHOA causing the inactivation of small RND1 (Just et al. 1995), and Y 27632, a highly specific inhibitor of ROCK (also termed RHO-associated kinase, RHO-kinase), major effectors of RHOA (Ishizaki et al. 2000) alone and in combination with Met-F-AEA in chemotaxy assay. Data obtained in the absence of chemotactic-factor gradient (chemokinesis) were shown in Fig. 5. We observed that Y 23762 10 μM and Toxin B (100 ng/ml) inhibited cell invasion and the combination of Met-F-AEA and Y 23762 or toxin B did not show any additivity (Fig. 5).
Mevalonate (MVA) induces stress fibers reversion
In addition to GDP/GTP cycling, RND1/2/3 function is also believed to be critically dependent on post-translational modification by isoprenoid lipids. RND1/2/3 terminate in a COOH-terminal CAAX tetrapeptide sequence motif. This sequence signals for covalent attachment of an isoprenoid lipid group. These modifications (prenylation) promote the association of RND1/2/3 with plasma and intracellular membranes. The MVA pathway produces isoprenoids lipids such as farnesyl pyrophosphate and geranylgeranyl pyrophosphate which in turn post-translationally modify the carboxy termini of many signaling molecules, such as RND1/2/3. Most RND1/2/3 are geranylgeranylated (Adamson et al. 1992, Collisson et al. 2002, Solski et al. 2002, Sebti 2005, McTaggart 2006). In order to ascertain the role of RHOA prenylation for its activity, we examined the ability of the MVA, central precursor of all isoprenoid lipids, to restore cytoskeletal changes in Met-F-AEA-treated cells. Figure 6E illustrates the recovery of actin stress fibers in response to MVA supplementation of Met-F-AEA-treated cells. The MVA supplementation recovered the subcellular distribution of RHOA in comparison with cells treated with the agonist alone, interestingly RHOA fluorescence-associated was mainly distributed in patches in proximity of the plasma membrane (Fig. 6F). Furthermore, we evaluated the hypothesis that the MVA supplementation to Met-F-AEA-treated cells could recover the cell migration by chemotaxis assay. As shown in Fig. 7A, the supplementation of MVA recovered the migration of Met-F-AEA-treated cells. In order to demonstrate the role of geranylgeranylation of RHOA in cell migration of MDA-MB-231 cells, we used GGTI-298 (Kusama et al. 2003, 2006), an inhibitor of geranylgeranyltransferase I, enzyme that catalyzes the RHOA prenylation. GGTI-298 at 10 μM markedly attenuated the in vitro invasive capacity of MDA-MB-231 cells. Moreover, the MVA did not recover the effect of Met-F-AEA on cell migration in presence of GGTI-298 (Fig. 7A), suggesting that the geranylgeranylation of RHOA has an important role in cell migration of these breast cancer cells. In order to confirm the role of RHOA activity in cell migration of MDA-MB-231, we performed a RHOA activity assay for each treatment. As shown in Fig. 7B, the RHOA activity was inhibited in cells treated with Met-F-AEA and GGTI-298.
Discussion
Metastasis is the direct cause of mortality in most cancer patients. Metastatic process requires the abrogation of cell—cell contacts, the remodeling of cell matrix interactions, and finally the movement of the cell mediated by actin cytoskeleton. Many of these components are controlled by members of Ras superfamily of GTP-binding proteins that includes RND1/2/3 subfamily. All aspects of cellular motility and invasion, including cellular polarity, cytoskeleton organization, and transduction of signals from the outside environment are controlled through an interplay between the RND1/2/3. Once activated, RHOA triggers a complex set of signal transduction pathways that include both the RHO-associated coiled-coil containing protein kinase (ROCK) activation pathway, which is responsible for actin polymerization required for cell locomotion (Zhong et al. 2005). In the past, we proved that CNR1 receptor mediates the inhibition of migration of MDA-MB-231, a highly invasive human breast cancer cell line, in response to Met-F-AEA, through a reduction of tyrosine phosphorylation of FAK and CSK protein. Because this pathway interconnected with RHOA/RHO kinase pathway for the regulation of cell migration (Frame 2004), we supposed that this agonist by CNR1 receptor could affect the RND1/2/3 activity and the downstream signaling. In the present study, we show that Met-F-AEA could alter endogenous RHO signaling in MDA-MB-231 cells and this alteration might decrease metastatic potential in vitro of these cells. We observed that CNR1 stimulation by Met-F-AEA affects the distribution and activation state of RHOA and consequently actin organization. We described that Met-F-AEA at 10 μM caused an inhibition in the GTPase activity of RHOA that it is recovered in the presence of antagonist of CNR1 receptor suggesting that this effect is CNR1-receptor mediated. Moreover, in order to confirm the receptor-dependent mechanism, we inhibited specifically the CNR1 receptor signaling using chemically synthesized siRNAs to achieve direct gene silencing for CNR1 receptor. After verifying the specificity and efficacy of CNR1-siRNA for knocking down CNR1 protein expression, we tested the RHOA activity upon the CNR1 stimulation by Met-F-AEA. We reported that the silencing of CNR1 receptor reversed the Met-F-AEA effect on RHOA activity. Consequently, treatment of MDA-MB-231 cells with Met-F-AEA attenuated the translocation from the cytosol (inactive form) to the plasma membrane (active form) in a time-dependent manner (Fig. 2). This event could cause a significant reduction of stress fibers formation determining the loss of traction forces dependent on actin cytoskeleton required for cell motility. Indeed, we showed a reduction of the stress fibers after treatment of MDA-MB-231 cells with Met-F-AEA at 10 μM The RHOA distribution also changed upon the CNR1 stimulation by Met-F-AEA; in untreated cells, fluorescence RHOA-associated was in proximity of the plasma membrane, while in treated cells after 3 h RHOA was in perinulclear region and afterwards widespread into cytosol, suggesting that Met-F-AEA induced a delocalization of RHOA from cell membrane to the cytoplasm and this effect led to the disruption of skeleton actin stress fibers, as already described (Vincent et al. 2001). To gain further insight into the role of RHOA in the migration of breast cancer cell, we examined the effect of the overexpression of dominant negative RHOA (N19RHOA) on actin organization and cell migration. The overexpression of dominant negative of RHOA markedly inhibited both stress fibers organization and the migration cell (Figs 4C and 5B). Moreover, the treatment of these cells with Toxin B from C. difficile, that inactivates the RHO family members by glycosylation of threonine residue of these proteins, caused the similar effects on cell migration (Fig. 5A). In addition, we demonstrated that the inhibition of ROCK by Y 23762 inhibited the transmigration of MDA-MB-231 cancer cells through Matrigel-coated membranes (Fig. 6) and it reduced stress fibers formation (Fig. 4C). Taken together, data showed in this study suggest that RHOA/ROCK signaling could be involved in the maintenance of actin organization and induction of migration in the MDA-MB-231 cells and that this CNR1 agonist could affect this pathway. Although various RND family members (such as RHOA, RHOB, and RHOC) are highly homologous and could be affected by Met-FAEA, our present data suggest that the inhibition of RHOA/ROCK signaling is critical for suppressing metastatic activity in the treated cells. These data support results by Pillè who used anti RHOA siRNA to inhibit the RHOA synthesis in MDA-MB-231 cells. In vitro, these siRNAs inhibited cell proliferation and invasion more effectively than conventional blockers of RHO cell signaling (Pillé et al. 2005). In conclusion, RND1/2/3 are well-characterized proteins whose activity depends on post-translational modification by isoprenoids compounds. The pharmacologic inhibition of RHO geranylgeranylation produced similar inhibition of RHO localization and signaling that include the reorganization of the actin cytoskeleton, and focal adhesion assembly suggesting that the prenylation of RHOA is critical event for cancer cell invasion (Denoyelle et al. 2001, Schmidmaier et al. 2004, Zhong et al. 2005). Here, we observed that the supplementation of MVA, central precursor of isoprenoid compounds, to cell treated with Met-F-AEA restored F-actin stress fibers, intracellular distribution of RHOA, and cell migration. These results suggest that the stimulation of CNR1 receptor by Met-F-AEA could affect the pathway of isoprenoid synthesis. To confirm this hypothesis, we showed that in vitro invasion of breast cancer cells was inhibited potently by GGTI-298, inhibitor of geranyl transferase I GGTase-I, enzyme of pathway of isoprenoids that it catalyzes the geranylgeranylation of RHOA. Moreover, we observed that the RHOA activity was inhibited in cells treated with GGTI-298 while was recovered in cells treated with Met-F-AEA in presence of MVA. These data strongly suggest that the inhibition of the geranylgeranylation of RHOA is critical for the RHOA activity and consequently for cancer cell invasion. Finally, the crucial point of the anti-metastatic action of Met-F-AEA could be related to the inhibition of the RHOA prenylation, causing the disruption of RHOA localization to the membrane, required to modulate its interaction with upstream and downstream signaling components, thereby attenuating RHO's ability to promote invasion. This report represents the first study to disclose a unique underlying mechanism of Met-F-AEA against tumor metastasis.
Source, Graphs and Figures: The anandamide analog, Met-F-AEA, controls human breast cancer cell migration via the RHOA/RHO kinase signaling pathway
The endocannabinoid system regulates cell proliferation and migration in human breast cancer cells. In this study, we showed that a metabolically stable analog of anandamide, 2-methyl-2′-F-anandamide (Met-F-AEA), inhibited the RHOA activity and caused a RHOA delocalization from the cell membrane to cytosol determining a decrease in actin stress fibers. Overexpression of a dominant negative of RHOA activity and treatment of these cells with a RHO-associated protein kinase (ROCK) inhibitor, Y 27632, mimicked Met-F-AEA effects on actin organization and cell migration. We suggest that the inhibitory effect of Met-F-AEA on tumor cell migration could be related to RHOA-ROCK-dependent signaling pathway.
Introduction
Metastasis is a multifactorial process that includes the acquisition of a motile and invasive phenotype. During progression to a metastatic phenotype, tumor cells undergo a series of changes that begin with loss of contact inhibition and increased motility, allowing them to migrate from the primary tumor site, invade distant organs, and induce neo-vascularization resulting in metastasis (Chambers et al. 2002). Many of these changes are associated with dynamic actin reorganization and activation of signaling pathways through transmembrane receptors (Oft et al. 1998, Seasholtz et al. 1999, Muller et al. 2001, Roymans & Slegers 2001, Vivanco & Sawyers 2002). Members of the small guanosine triphosphatase (GTPase) family control cell adhesion and motility through reorganization of the actin cytoskeleton and regulation of actomyosin contractility (Yoshioka et al.1998, Etienne-Manneville & Hall 2002). Both RHOA (Fritz et al. 1999) and the related RHOC (Clark et al. 2000) are expressed at a relatively higher level in metastatic tumors, and their expression levels positively correlate with the stage of the tumors. However, mutations in the RHO gene have not yet been found in human tumors; rather, the overexpression of RHOA in the cell facilitates its translocation from the cytosol to the plasma membrane, where its activation results in stimulation of the actomyosin system, followed by cellular invasion both in vitro and in vivo (Yoshioka et al. 1999). One of the target molecules of RHO is the family of RHO-associated serine—threonine protein kinases (ROCK; Leung et al. 1995), which also participates in cell-to-substrate adhesions, stress fiber formation, and stimulation of actomyosin-based cellular contractility (Narumiya et al. 1997). In particular, several breast cancers contain large amounts of RHOA proteins, whereas RHOA was hardly or not detectable in adjacent normal tissue. In addition, the progression of breast tumors from WHO grade I to grade III is accompanied by a significant average increase in RHOA protein levels (Van Aelst & D'Souza-Schorey 1997, Hall 1998, Fritz et al. 1999, Yoshioka et al. 1999, Kamai et al. 2001, 2003, Horiuchi et al. 2003). The endogenous cannabinoid system, consisting of the cannabinoid CNR1 and CNR2 transmembrane receptors G-protein coupled, their endogenous ligands (endocannabinoids), and the proteins that regulate endocannabinoid biosynthesis and degradation, is an almost ubiquitous signaling system involved in the control of cell fate. The endocannabinoids (anandamide, AEA, and 2-arachidonoyglycerol, 2AG) have been shown to inhibit the growth of tumor cells in culture and animal models by modulating key cell signaling pathways (Bifulco et al. 2006, Mackie & Stella 2006). We have also shown that a metabolically stable anandamide analog, 2-methyl-2′-F-anandamide (Met-F-AEA), arrests the growth of K-ras-dependent tumors, induced and/or already established in vivo, and it inhibits the formation of metastatic nodules in the Lewis lung carcinoma model, these effects being largely mediated by cannabinoid CNR1 receptors (De Petrocellis et al. 1998, 2000, Bifulco et al. 2001, Bifulco & Di Marzo 2002, Portella et al. 2003). Moreover, it has been observed that cannabinoid receptors activation is able to inhibit vascular endothelial cell migration and to downregulate the expression and the activity of matrix metalloproteinase-2, a proteolytic enzyme involved in tissue remodeling during angiogenesis and metastasis (Pisanti et al. 2007). Anandamide inhibits the migration of human colon carcinoma cells (SW480) through a CNR1-dependent mechanism (Joseph et al. 2004). Interestingly, it was recently found that increasing endogenous 2-AG, by blocking its metabolism, inhibits invasion of androgen-independent prostate cancer (PC-3 and DU-145) cells. The anti-invasive effect of endogenous 2-AG relies, once again, on CNR1 activation and consequent inhibition of the adenylyl cyclase and protein kinase A (PKA) pathway (Nithipatikom et al. 2004). Nevertheless, the molecular mechanism of the cannabinoids anti-metastatic effect has not been fully explored. Recently, we described that Met-F-AEA treatment is able to inhibit cell migration of the highly invasive and metastatic breast cancer cell lines, MDA-MB-231, as tested in migration assays with type IV collagen, the major component of the basement membrane. This effect is mediated by CNR1 receptor and it is accompanied by a remarkable decrease in phosphorylation of focal adhesion kinase (FAK) and CSK protein, two tyrosine kinases involved in the loss of adhesion and invasion (Grimaldi et al. 2006). In this study, we further investigated the molecular mechanism of anti-metastatic potential of Met-F-AEA by studying the effect of this agonist of CNR1 receptor on RHOA activity and actin organization.
Materials and methods
Drugs and cell culture
Met-F-AEA was purchased from Sigma. The selective CNR1 antagonist, SR141716, was kindly provided by Sanofi-Aventis (Montpellier, France). Toxin B was purchased from Calbiochem (La Jolla, CA, USA), the inhibitor of RHO kinase, Y 27632 from Sigma, GGTI-298 was purchased from Calbiochem. Monoclonal antibody to RHOA and polyclonal antibody to CNR1 receptor were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The human breast carcinoma cell line MDA-MB-231 was maintained in RPMI 1640 culture medium (Gibco BRL Life Technologies) supplemented with 10% inactivated fetal bovine serum (FBS) and 2 mM L-glutamine. Cells were cultured in a humidified environment containing 5% CO2 and held at a constant temperature of 37 °C.
Separation of particulate and cytosolic fractions
Cells were grown to subconfluency (60—70%), then lysed by ice-cold lysis buffer (50 mmol/l HEPES (pH 7.5), 50 mmol/l NaCl, 1 mmol/l MgCl2, 2 mmol/l EDTA, 10 mmol/l NaF, 1 mmol/l dithiothreitol, 1 mmol/l phenylmethylsulfonyl fluoride, 10 mg/ml aprotinin, and 10 mg/ml leupeptin) and centrifuged at 100 000 g for 30 min in micro-ultracentrifuge, and the supernatant was collected as the cytosolic fraction. Pellets were resuspended, and membrane proteins homogenized in 150 μl lysis buffer containing 2% Triton X-114. The homogenate was centrifuged at 800 g for 10 min. The supernatant (particulate fraction) and pellet (detergent insoluble particulate fraction) were collected separately. Whole cell, cytosolic, and particulate fraction proteins were separated by SDS-PAGE.
Western blot analysis
Cells plated in 100 mm dishes in regular medium with serum were washed with ice-cold PBS and scraped into lysis buffer (50 mM Tris—HCl, 150 mM NaCl, 0.5% Triton X100, 0.5% deossicolic acid, 10 mg/ml leupeptin, 2 mM phenylmethylsulfonyl fluoride, and 10 mg/ml aprotinin). After removal of cell debris by centrifugation (14 000 g, 5 min), about 50 μg of proteins were loaded on 12% SDS-polyacrylamide gels under reducing conditions. After SDS-PAGE, proteins were transferred to nitrocellulose membranes that were blocked with 5% milk (Bio-Rad Laboratories, Inc.) and incubated with anti-CNR1 antibody. After three washes, filters were incubated for 1 h at room temperature with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody. The membranes were then stained using a chemiluminescence system (ECL-Amersham Biosciences) and then exposed to X-ray film (Kodak).
Invasion assay
For chemotaxis assays, Boyden chambers (8 Am Transwell polycarbonate membrane, Costar, NY, USA) were coated with 50 μg/ml type IV collagen and blocked with 5 mg/ml BSA. Cells (1×105 cells) treated for 24 h with Met-F-AEA alone or in combination with toxin B (100 ng/ml) or Y 27632 (10 μM) were added to the upper compartment and incubated (at 37 °C for 4 h) in migration media RPMI in the presence or absence of FBS used as a chemotactic stimulus in the lower compartment. Chambers were washed with PBS, and migratory cells on the lower membrane surface were fixed with 3% formaldehyde (10 min). Cells were permeabilized with 0.2% Triton X-100 (5 min) and stained with Hoechst dye (5 min). The number of migrated cells was counted by a light microscope at 20× magnification: ten randomly chosen microscopic fields were counted per well, and the mean number was determined. Background levels of cells migrated in the absence of chemotactic stimuli (chemokinesis) were subtracted from all the experimental points.
RHOA activation assay
The amount of activated Rho(GTP-Rho) was determined using a Rho activation assay kit (Upstate Biotechnology, Inc., Lake Placid, NY, USA). The human MDA-MB-231 cells were grown to confluence and then placed in serum free for 24 h. After treatment, the cells were washed with ice-cold PBS. Cellular lysates were prepared according to the manufacturer's instructions.
Immunofluorescence
Cells plated on cover slips were washed once with PBS and fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.5% Triton X-100 for 5 min, and incubated with PBS containing 4% BSA for 1 h at room temperature. F-actin was stained with tetramethyl rhodamine B isothiocyanate-labeled phalloidin (500 ng/ml; Sigma—Aldrich) to visualize filamentous actin. Cells were plated in 24-well plates on cover slips (Becton—Dickinson Labware, NY, USA). When they were 60±80% confluent, they were treated with Met-F-AEA (10 μM for 15′, 3 and 24 h). After the incubation with various drugs, the cells were washed twice with PBS, fixed in 3.7% paraformaldehyde in PBS for 20 min and followed by two washes in 50 mM NH4Cl for 10 min. Permeabilization was achieved by incubating the fixed cells in 0.1% Triton X-100 in PBS for 5 min at room temperature. The cells were then blocked in final dilution buffer (FDB) (1 mM MgCl2, 1 mM CaCl2, 5% fetal calf serum, and 2% BSA in PBS) for 30 min at room temperature. All primary and secondary antibody incubations were performed in FDB buffer for 1 h at room temperature. Cover slips were mounted on 50% glycerol in PBS and examined by using a Zeiss Laser Scanning Confocal Microscope (410 or 510).
Cell transfection
Plasmids were purified using the endotoxin-free plasmid midi prep kit (Qiagen).
For transient transfections, MDA-MB-231 cells were seeded in 6 cm plates and grown to 60—80% confluence in RPMI with 10% FBS without antibiotics and transfected the next day using FuGene reagent (Promega), according to the manufacturer's protocol, with a total of 2 μg/well of plasmid DNA. The cells were transfected with control vector (pcDNA3) or pcDNA3-N19RHOA provided by Dr Mario Chiariello (IEOS, CNR Napoli). Twenty-four hours after transfection, cells were prepared for immunofluorescence analysis and for wound-healing assay. For knocking down CNR1 protein expression, we used a 25-nucleotide small interfering RNA (siRNA) duplex (Dharmacon Research, Lafayette, CO, USA) designed for specific silencing of CNR1 (siRNA-CNR1) covered the sequence sense 5′-CCCAAGUGACGAAAACAUU-dTdT-3′ gently provided by Dr Patrizia Gazzerro (University of Salerno). We transfected cells using Lipofectamine (Invitrogen) and 30 nM siRNA for each transfection, in accordance with the manufacturer's protocol. An equal concentration of SilencerR Negative Control-1 siRNA (Ambion Inc. NY, USA) was used as negative control. The transfected MDA-MB-231 cells were serum starved and after 24 h assayed for RHOA activity. Specific silencing of targeted genes was confirmed by western blot analysis for CNR1 receptor.
Statistical analysis
All data were presented as means±S.D. Statistical analysis was performed using one-way ANOVA analysis. In the case of a significant result in the ANOVA, Student's t-test was used for dose—response curves and Bonferroni's test for post hoc analysis for all other experiments. A P value less than 0.05 was considered statistically significant.
Results
Met-F-AEA inhibits RHOA activity and induced delocalization of RHOA from cell membrane
Since in a recent report we demonstrated that metabolically stable anandamide analog, Met-F-AEA at 10 μM inhibited the migration of MDA-MB-231 human breast cancer cells (Grimaldi et al. 2006), we hypothesized that this agonist of the CNR1 receptor could affect the activity of RND1/2/3 involved in the cell migration and actin organization of several cancer cells. To examine this hypothesis, we analyzed the level of the active GTP-bound form of RHOA by a pull-down assay with the RHO-binding fragment of rhotekin in cells treated with Met-F-AEA at 10 μM. The cells were developed in medium serum free for 24 h, and after treatment we collected the cells and performed the assay. As shown in Fig. 1A, a large proportion of the RHOA protein detected in MDA-MB-231 cells is present in an active form. Upon the stimulation of CNR1 receptor by Met-F-AEA, the level of GTP-RhoA was decreased after 15 min and strongly reduced after 1 h. The RHOA activity is restored in Met-F-AEA-treated cells in presence of SR141716, antagonist of CNR1 receptor. To evaluate the maximum effect of Met-F-AEA on RHOA activity, MDA-MB-231 cells were treated with increasing concentrations of Met-F-AEA from 2.5 to 10 μM, then they were collected and was performed a pull-down assay. As shown in Fig. 1D, the anandamide analog strongly reduced the RHOA activity in a dose-dependent manner. The reduction was statistically significant at 10 μM concentration compared with the control. Furthermore, in order to confirm that this effect is mediated by CNR1 receptor, we reduced the level of expression of CNR1 receptor by RNA interference. To specifically silence the CNR1 gene, MDA-MB-231 cells were transfected with siRNA targeting CNR1 mRNA. Western blot analysis (Fig. 1C) showed that the protein level of CNR1 was markedly downregulated by CNR1 siRNA at 24 h after administration of the siRNA, with reductions of 80% of protein expression of CNR1 receptor in MDA-MB-231 cell line compared with lysates transfected with no silencing siRNA. Incubation of CNR1-siRNA transfected cells with Met-F-AEA at 10 μM for 1 h did not affect the RHOA activity (Fig. 1B). More, because RHOA protein must be targeted to the plasma membrane for activation and full function, we examined the translocation of this protein from the cytosol to the cell membrane in MDA-MB-231 cells in the presence of Met-F-AEA at 10 μM, after separation of particulate and soluble fractions. In untreated cells without serum, most of RHOA protein was present on the cell membrane (particulate fractions; Fig. 2A, ctr and B), suggesting that in these breast cancer cells the overexpression of RHOA facilitated its translocation from the cytosol to the cell membrane as previously described (Fritz et al. 1999). Treatment of MDA-MB-231 cells with Met-F-AEA clearly decreased the amount of RHOA in the membrane fraction and accumulates in the cytosolic fraction in a time-dependent manner.
Anandamide induces reorganization of the actin cytoskeleton
As RHOA is involved in the regulation and assembly of contractile actin filaments (stress fibers), we studied the effect of Met-F-AEA on actin cytoskeleton in these cells by immunofluorescence confocal microscopy using phalloidin TRITC-conjugated. As shown in Fig. 3A, various stress fibers were detected in untreated cells. The treatment of these cells with Met-F-AEA at 10 μM caused a significant decrease in F-actin containing stress fibers, which was apparent within 15 min (Fig. 3C). After longer incubation times, many cells displayed a dense meshwork of unpolarized actin filaments around the cell periphery (3 and 24-h time point are shown in Fig. 3E and F). We also observed the intracellular distribution of RHOA with a specific monoclonal antibody and a secondary antibody FITC-conjugated. Interestingly, RHOA was present at the membrane periphery in untreated cells (Fig. 3B); while at 15 min of treatment with Met-F-AEA, the RHOA patches to the cell membrane are reduced (Fig. 3D). After 3 h, RHOA remained largely diffused in the cytoplasm mainly in the perinuclear region in confront to control sample and, at 24 h, the RHOA fluorescence-associated is equally widespread into cytosol. To confirm the role of RHOA in the actin organization, we transiently transfected the cells with a vector carrying a dominant negative of RHOA (N19RHOA) which antagonizes the RHO guanine nucleotide exchange factors (ARHGEFs), when compared with cells transfected with empty vector. As shown in Fig. 4, in cell transfected with empty vector (mock), the actin is organized in stress fibers, while in that with vector carrying N19RHOA we observed a significant reduction in actin stress fiber as how in Met-F-AEA-treated cells (Fig. 3E, C and G). In addition, because ROCK, the immediate downstream effector of RHOA, has been clearly implicated in cancer migration, we examined the effect of a selective inhibitor of ROCK I and II, Y 27632 (Ishizaki et al. 2000) on actin organization. Cells treated with this ROCK inhibitor at 10 μM reduced actin stress fibers (Fig. 4A). We previously reported that Met-F-AEA inhibited MDA-MB-231 cell migration on type IV collagen (Grimaldi et al. 2006), in order to verify the involvement of RND1 signaling pathway in the Met-F-AEA effect on cell migration, we used Toxin B of Clostridium difficile, which glycosylates threonine residue of RHOA causing the inactivation of small RND1 (Just et al. 1995), and Y 27632, a highly specific inhibitor of ROCK (also termed RHO-associated kinase, RHO-kinase), major effectors of RHOA (Ishizaki et al. 2000) alone and in combination with Met-F-AEA in chemotaxy assay. Data obtained in the absence of chemotactic-factor gradient (chemokinesis) were shown in Fig. 5. We observed that Y 23762 10 μM and Toxin B (100 ng/ml) inhibited cell invasion and the combination of Met-F-AEA and Y 23762 or toxin B did not show any additivity (Fig. 5).
Mevalonate (MVA) induces stress fibers reversion
In addition to GDP/GTP cycling, RND1/2/3 function is also believed to be critically dependent on post-translational modification by isoprenoid lipids. RND1/2/3 terminate in a COOH-terminal CAAX tetrapeptide sequence motif. This sequence signals for covalent attachment of an isoprenoid lipid group. These modifications (prenylation) promote the association of RND1/2/3 with plasma and intracellular membranes. The MVA pathway produces isoprenoids lipids such as farnesyl pyrophosphate and geranylgeranyl pyrophosphate which in turn post-translationally modify the carboxy termini of many signaling molecules, such as RND1/2/3. Most RND1/2/3 are geranylgeranylated (Adamson et al. 1992, Collisson et al. 2002, Solski et al. 2002, Sebti 2005, McTaggart 2006). In order to ascertain the role of RHOA prenylation for its activity, we examined the ability of the MVA, central precursor of all isoprenoid lipids, to restore cytoskeletal changes in Met-F-AEA-treated cells. Figure 6E illustrates the recovery of actin stress fibers in response to MVA supplementation of Met-F-AEA-treated cells. The MVA supplementation recovered the subcellular distribution of RHOA in comparison with cells treated with the agonist alone, interestingly RHOA fluorescence-associated was mainly distributed in patches in proximity of the plasma membrane (Fig. 6F). Furthermore, we evaluated the hypothesis that the MVA supplementation to Met-F-AEA-treated cells could recover the cell migration by chemotaxis assay. As shown in Fig. 7A, the supplementation of MVA recovered the migration of Met-F-AEA-treated cells. In order to demonstrate the role of geranylgeranylation of RHOA in cell migration of MDA-MB-231 cells, we used GGTI-298 (Kusama et al. 2003, 2006), an inhibitor of geranylgeranyltransferase I, enzyme that catalyzes the RHOA prenylation. GGTI-298 at 10 μM markedly attenuated the in vitro invasive capacity of MDA-MB-231 cells. Moreover, the MVA did not recover the effect of Met-F-AEA on cell migration in presence of GGTI-298 (Fig. 7A), suggesting that the geranylgeranylation of RHOA has an important role in cell migration of these breast cancer cells. In order to confirm the role of RHOA activity in cell migration of MDA-MB-231, we performed a RHOA activity assay for each treatment. As shown in Fig. 7B, the RHOA activity was inhibited in cells treated with Met-F-AEA and GGTI-298.
Discussion
Metastasis is the direct cause of mortality in most cancer patients. Metastatic process requires the abrogation of cell—cell contacts, the remodeling of cell matrix interactions, and finally the movement of the cell mediated by actin cytoskeleton. Many of these components are controlled by members of Ras superfamily of GTP-binding proteins that includes RND1/2/3 subfamily. All aspects of cellular motility and invasion, including cellular polarity, cytoskeleton organization, and transduction of signals from the outside environment are controlled through an interplay between the RND1/2/3. Once activated, RHOA triggers a complex set of signal transduction pathways that include both the RHO-associated coiled-coil containing protein kinase (ROCK) activation pathway, which is responsible for actin polymerization required for cell locomotion (Zhong et al. 2005). In the past, we proved that CNR1 receptor mediates the inhibition of migration of MDA-MB-231, a highly invasive human breast cancer cell line, in response to Met-F-AEA, through a reduction of tyrosine phosphorylation of FAK and CSK protein. Because this pathway interconnected with RHOA/RHO kinase pathway for the regulation of cell migration (Frame 2004), we supposed that this agonist by CNR1 receptor could affect the RND1/2/3 activity and the downstream signaling. In the present study, we show that Met-F-AEA could alter endogenous RHO signaling in MDA-MB-231 cells and this alteration might decrease metastatic potential in vitro of these cells. We observed that CNR1 stimulation by Met-F-AEA affects the distribution and activation state of RHOA and consequently actin organization. We described that Met-F-AEA at 10 μM caused an inhibition in the GTPase activity of RHOA that it is recovered in the presence of antagonist of CNR1 receptor suggesting that this effect is CNR1-receptor mediated. Moreover, in order to confirm the receptor-dependent mechanism, we inhibited specifically the CNR1 receptor signaling using chemically synthesized siRNAs to achieve direct gene silencing for CNR1 receptor. After verifying the specificity and efficacy of CNR1-siRNA for knocking down CNR1 protein expression, we tested the RHOA activity upon the CNR1 stimulation by Met-F-AEA. We reported that the silencing of CNR1 receptor reversed the Met-F-AEA effect on RHOA activity. Consequently, treatment of MDA-MB-231 cells with Met-F-AEA attenuated the translocation from the cytosol (inactive form) to the plasma membrane (active form) in a time-dependent manner (Fig. 2). This event could cause a significant reduction of stress fibers formation determining the loss of traction forces dependent on actin cytoskeleton required for cell motility. Indeed, we showed a reduction of the stress fibers after treatment of MDA-MB-231 cells with Met-F-AEA at 10 μM The RHOA distribution also changed upon the CNR1 stimulation by Met-F-AEA; in untreated cells, fluorescence RHOA-associated was in proximity of the plasma membrane, while in treated cells after 3 h RHOA was in perinulclear region and afterwards widespread into cytosol, suggesting that Met-F-AEA induced a delocalization of RHOA from cell membrane to the cytoplasm and this effect led to the disruption of skeleton actin stress fibers, as already described (Vincent et al. 2001). To gain further insight into the role of RHOA in the migration of breast cancer cell, we examined the effect of the overexpression of dominant negative RHOA (N19RHOA) on actin organization and cell migration. The overexpression of dominant negative of RHOA markedly inhibited both stress fibers organization and the migration cell (Figs 4C and 5B). Moreover, the treatment of these cells with Toxin B from C. difficile, that inactivates the RHO family members by glycosylation of threonine residue of these proteins, caused the similar effects on cell migration (Fig. 5A). In addition, we demonstrated that the inhibition of ROCK by Y 23762 inhibited the transmigration of MDA-MB-231 cancer cells through Matrigel-coated membranes (Fig. 6) and it reduced stress fibers formation (Fig. 4C). Taken together, data showed in this study suggest that RHOA/ROCK signaling could be involved in the maintenance of actin organization and induction of migration in the MDA-MB-231 cells and that this CNR1 agonist could affect this pathway. Although various RND family members (such as RHOA, RHOB, and RHOC) are highly homologous and could be affected by Met-FAEA, our present data suggest that the inhibition of RHOA/ROCK signaling is critical for suppressing metastatic activity in the treated cells. These data support results by Pillè who used anti RHOA siRNA to inhibit the RHOA synthesis in MDA-MB-231 cells. In vitro, these siRNAs inhibited cell proliferation and invasion more effectively than conventional blockers of RHO cell signaling (Pillé et al. 2005). In conclusion, RND1/2/3 are well-characterized proteins whose activity depends on post-translational modification by isoprenoids compounds. The pharmacologic inhibition of RHO geranylgeranylation produced similar inhibition of RHO localization and signaling that include the reorganization of the actin cytoskeleton, and focal adhesion assembly suggesting that the prenylation of RHOA is critical event for cancer cell invasion (Denoyelle et al. 2001, Schmidmaier et al. 2004, Zhong et al. 2005). Here, we observed that the supplementation of MVA, central precursor of isoprenoid compounds, to cell treated with Met-F-AEA restored F-actin stress fibers, intracellular distribution of RHOA, and cell migration. These results suggest that the stimulation of CNR1 receptor by Met-F-AEA could affect the pathway of isoprenoid synthesis. To confirm this hypothesis, we showed that in vitro invasion of breast cancer cells was inhibited potently by GGTI-298, inhibitor of geranyl transferase I GGTase-I, enzyme of pathway of isoprenoids that it catalyzes the geranylgeranylation of RHOA. Moreover, we observed that the RHOA activity was inhibited in cells treated with GGTI-298 while was recovered in cells treated with Met-F-AEA in presence of MVA. These data strongly suggest that the inhibition of the geranylgeranylation of RHOA is critical for the RHOA activity and consequently for cancer cell invasion. Finally, the crucial point of the anti-metastatic action of Met-F-AEA could be related to the inhibition of the RHOA prenylation, causing the disruption of RHOA localization to the membrane, required to modulate its interaction with upstream and downstream signaling components, thereby attenuating RHO's ability to promote invasion. This report represents the first study to disclose a unique underlying mechanism of Met-F-AEA against tumor metastasis.
Source, Graphs and Figures: The anandamide analog, Met-F-AEA, controls human breast cancer cell migration via the RHOA/RHO kinase signaling pathway
