Cell migration is a critical step in the progression of prostate cancer to the metastatic state, the lethal form of the disease. The antidiabetic drug metformin has been shown to display antitumoral properties in prostate cancer cell and animal models; however, its role in the formation of metastases remains poorly documented. Here, we show that metformin reduces the formation of metastases to fewer solid organs in an orthotopic metastatic prostate cancer cell model established in nude mice. As predicted, metformin hampers cell motility in PC3 and DU145 prostate cancer cells and triggers a radical reorganization of the cell cytoskeleton. The small GTPase Rac1 is a master regulator of cytoskeleton organization and cell migration. We report that metformin leads to a major inhibition of Rac1 GTPase activity by interfering with some of its multiple upstream signaling pathways, namely P-Rex1 (a Guanine nucleotide exchange factor and activator of Rac1), cAMP, and CXCL12/CXCR4, resulting in decreased migration of prostate cancer cells. Importantly, overexpression of a constitutively active form of Rac1, or P-Rex, as well as the inhibition of the adenylate cyclase, was able to reverse the antimigratory effects of metformin. These results establish a novel mechanism of action for metformin and highlight its potential antimetastatic properties in prostate cancer. Mol Cancer Ther; 14(2); 586–96. ©2014 AACR.

Metformin is an antidiabetic drug used by more than 120 million people worldwide. In agreement with retrospective epidemiologic studies in which diabetic patients on metformin display decreased cancer incidence and cancer-related mortality (1–3), metformin has been shown to inhibit cancer cell proliferation and decrease tumor growth in many animal models (4–7). Prostate cancer is the second leading cause of death by cancer in men, and most prostate cancer-related deaths are due to metastasis, a process that requires cancer cell migration. This migration is a complex biologic process orchestrated by environmental factors, signal transduction, and cytoskeletal rearrangement. Several studies demonstrated that metformin exerts an antimigratory effect on cancer cells; however, its mechanism of action remains largely unknown (8–13). In addition, how metformin interferes with the small GTPase Rac1, one of a master regulator of cell migration, is not known.

Rac1 belongs to the family of the Rho GTPases that play a central role in the control of cytoskeleton organization and cell motility. The best characterized family members are: Rho, involved in stress fibers and focal adhesion formation, together with Rac and Cdc42, respectively, involved in lamellipodia and filipodia formation (14). Rho GTPases switch from a GTP-bound active form to a GDP-bound inactive form. The exchange of GDP to GTP is regulated by guanine nucleotide exchange factors (GEF), and the inactivation of Rho GTPases is controlled by GTPase-activating enzymes. The Rac subclass (or subfamily) of RhoGTPases includes Rac1, Rac2, and Rac3. Rac1 is required for lamellipodium extension induced by growth factors, cytokines, and extracellular matrix (ECM) components (15). Rac1 is overexpressed in cancers, including prostate cancer, in which its expression is significantly increased in aggressive tumors (16). The PIP3 phosphatidylinositol (3,4,5)-triphosphate–dependent Rac exchanger 1 (P-Rex1), a Rac-selective GEF, plays an important role in actin remodeling and cell migration. Importantly, upregulation of P-Rex1 promotes metastasis whereas its downregulation inhibits cell migration in prostate cancer cells (17).

Rac1 activity is regulated by numerous biologic signals, such as cAMP and cytokines. Recent studies have highlighted an important role for cAMP metabolism in the migration of carcinoma cells (18) and the regulation of Rac1 activity (19). For example, cAMP-specific phosphodiesterases facilitate cell migration as well as lamellae formation by lowering cAMP levels. In addition, Chen and colleagues (20) have shown that increased cAMP levels correlated with the inhibition of cell migration in both Mouse Embryonic Fibroblasts and 4T1a breast tumors cells by interfering with the formation of lamellipodia.

Chemokines are also important regulators of Rac1, one of them, CXCL12 (also known as SDF-1α) activates Rac1, decreases cAMP levels, and favors prostate cancer cells migration (21–23). In addition, CXCR4, the CXCL12 receptor, is frequently overexpressed in malignant epithelial cells, and the CXCL12/CXCR4 axis plays a pivotal role in directing the metastasis of CXCR4-positive tumor cells to organs that express CXCL12, such as lungs, liver, and bones (24, 25). Here, we investigated the effects of metformin on Rac1 GTPase activity and determined whether it interferes with some of Rac1 multiple upstream signaling pathways, namely P-Rex1, cAMP, and CXCL12/CXCR4.

We demonstrate that metformin inhibits the migration of prostate cancer cells and limits the formation of metastasis to fewer solid organs in an orthotopic xenograft model using PC3 cells. In addition, we show that metformin strongly modifies actin cytoskeletal organization. Reversal of the decreased Rac1GTPase activity through the expression of constitutively active Rac1GTP or P-Rex1, overturned the antimigratory effects of metformin. Similarly, blocking the metformin-induced cAMP increase with an adenylate cyclase inhibitor hampered the effects of metformin on migration. We also show that metformin inhibits CXCL12 chemotactism and counteracts the increase of Rac1GTP by CXCL12. Our study reveals a novel mechanism of action for metformin, in which it targets Rac1GTPase and cytoskeletal organization.

Orthotopic implantation of PC3–GFP prostate cancer cells and analysis of metastasis

Intraprostatic human prostate cancer xenografts were established in nude mice by surgical orthotopic implantation as originally described (26). Briefly, mice were anesthetized by isoflurane inhalation and placed in the supine position. A lower midline abdominal incision was made and a tumor cell suspension (1 × 106 cells/20 μL) was injected into the dorsal lobe of the prostate using a 30-gauge needle and glass syringe (Hamilton). After implantation, the surgical wound was closed in two layers with 4-0 Dexon-interrupted sutures. All procedures were performed with a dissecting microscope. Autopsy and in vivo fluorescence imaging were conducted as previously detailed. The measurements were performed blinded. Animal use and care was approved by the local Animal Care committee according to the European Legislation.

Cell culture and transfection

The human PC3 and DU145 cancer cell lines were obtained from the ATCC and authenticated by the ATCC, the experiments performed in this work were performed during the year and half following the reception of the cells. Cells were grown in DMEM supplemented with 10% FCS, 100 U/mL penicillin, and 50 μg/mL streptomycin. GFP-expressing PC3 cells were generated as described previously (26). Cells were maintained at 37°C in a humidified atmosphere of 5% CO2, and the media were replaced every 2 to 3 days. In all experiments, cells were treated for 4 hours with 5 mmol/L metformin. Cells were transiently transfected with HA–P-rex1–expressing vector (kind gift of Dr. C. Mitchell, Monash University, Victoria, Australia; ref. 27), or the Rac1-mutant expression vectors: RacQ61L and RacV12 expression vector using Lipofectamine 2000 (Invitrogen).

Chemicals

Metformin, the adenylate cyclase inhibitor (SQ22536), dibutyryl-cAMP (dbcAMP), and fibronectin were purchased from Sigma-Aldrich. The Rac inhibitor and AMD3100 were from Merck Chemicals. CXCL12 was purchased from Peprotech.

Boyden chamber assay

Boyden chambers with filter inserts coated with fibronectin (10 μg/mL) and 8-μm pores (BD Biosciences) were used to quantify cell migration. To respond better to the chemoattractant cells were serum starved overnight, 12 × 104 cells were seeded in the upper chamber in serum-free DMEM medium in the presence or absence of 5 mmol/L metformin. The lower chamber contained complete DMEM, 10% FBS, or DMEM with CXCL12 at the indicated concentration. Cell migration was determined after 4 hours by counting all cells in five randomly selected counting areas at the lower surface of the filter. Cells on the upper surface were removed with a cotton swab; filters were fixed and stained with blue toluidin. Each experiment was repeated at least three times. For invasion experiments, the inserts were coated with 25 μg/μL of Matrigel (Beckton Dickinson), and invading cells were counted after a 24-hour incubation with metformin.

Spheroid migration assays in three-dimensional Matrigel matrices

Prostate spheroids were generated using the liquid overlay technique. Briefly, 24-well culture plates were coated with 1.5% agarose prepared in sterile water. Cells from a single-cell suspension were added at 10,000 cells per well. The plates were gently swirled and incubated at 37°C in 5% CO2 atmosphere until spheroid aggregates were formed. Then, spheroids were included in a Matrigel matrix and images of invasion were obtained 24 hours later.

Cell migration observation with video microscopy

Cell migration was monitored in duplicate experiments by time-lapse digital microscopy. Cells were seeded on a 6-well plate at low density. Computer-assisted cell tracking of 20 to 30 randomly selected cells was performed. Briefly, the x and y coordinates were collected from the center of the cell with a step interval of 5 minutes and reconstructed either as path at orthotopic position or as migration speed over time.

Immunofluorescence and fluorescence microscopy

Cells grown on coverslips were fixed in 3.7% paraformaldehyde, permeabilized in 0.2% Triton X-100 for 20 minutes, blocked with 2% BSA for 1 hour (all reagents were diluted in PBS), and then incubated with Texas red Phalloidin and anti-HA antibodies (Covance). Cells were simultaneously stained with Hoescht. Images were recorded with a Leica scanning microscopy system DM5500B. Image acquisition and image analysis were performed on the C3M (or MicorBio) Cell Imaging Facility.

Western blot analysis

A total of 40 μg cell lysate protein was separated by SDSPAGE, transferred on a polyvinylidene difluoride membrane (Millipore), and incubated with the antibodies against Rac1 (BD Biosciences); ERK and HSP90 (Santa Cruz Biotechnology); HA (Covance).

Pull-down assay for the measurement of RAC1 GTP activity

The assay was performed as previously described (28). DU145 and PC3 were seeded in DMEM medium with 10% FBS. After a 4-hour treatment with 5 mmol/L metformin, cells were washed once with ice-cold PBS and immediately lysed in 25 mmol/L Tris buffer, pH 7.5, 150 mmol/L NaCl, 5 mmol/L MgCl2, 0.5% Triton X100, 4% glycerol, 10 mmol/L sodium fluoride, 2 mmol/L sodium orthovanadate, 5 mmol/L DTT, 1 mmol/L phenylmethylsulfonylfluoride. Cleared extracts were mixed with 20 μg of GST–PAK in the presence of glutathione–agarose beads (Sigma-Aldrich). After a 40-minute incubation at 4°C, beads were pelleted by centrifugation and washed three times in lysis buffer, and the proteins were eluted in SDSPAGE sample buffer for analysis by Western blot analysis using a monoclonal antibody to Rac1 (BD Biosciences).

cAMP concentration

cAMP levels were assessed using a commercially available fluorimetric kit (Arbor Assays). In brief, DU145 or PC3 were seeded in 6-well plates and half the wells were treated with 5 mmol/L of metformin for 4 hours before cAMP measurement performed according to the manufacturer's protocol.

Flow cytometry

Cells were harvested after 4 hours of metformin treatment (5 mmol/L). Cells were labeled with anti–CRCR4-APC–conjugated antibody (R&D Systems) and fixed in PAF 3.7% for 10 minutes. Labeling was carried out in ice for 2 hours. Cells were then washed in PBS 0.5% BSA at 1,100 rpm for 5 minutes and resuspended in 400 μL of PBS. For each tube, 10,000 events were acquired. Samples were analyzed using FACSCanto II cytometer (Beckton Dickinson).

CRE–luciferase reporter gene assay

PC3 and DU145 cells were transiently transfected using lipofectamine 2000 with 1 μg of a plasmid encoding for the cyclic AMP–responsive element (CRE) coupled to the luciferase gene (CRE–Luc), and 1 μg of pRL Renilla Luciferase Control vector. Two days after transfection, the culture medium was discarded and cells were treated with DMEM supplemented with 10% FCS ± 5 mmol/L metformin. After a 4-hour incubation at 37°C, the stimulation medium was discarded and the Luciferase activity was determined using the Dual Luciferase reporter Assay System (Promega).

Statistical analysis

The statistical significance of differences between the means of two groups was evaluated using the Student t test.

Metformin inhibits tumor growth and reduces metastasis in an orthotopic model of PC3 cells

We first investigated the effects of metformin on the formation of metastases using an orthotopic model of PC3 cells overexpressing GFP. In these experimental conditions, cells grow in their native environment, and the primary tumor forms distant metastasis (26, 29). Tumor growth and metastasis dissemination were analyzed 5 weeks after the injection of PC3–GFP cells into the prostate. Metformin (100 mg/kg/d) was given in drinking water for 5 weeks (Met, 5 wk) starting 3 days after cell injection or only 2 final weeks (Met, 2 wk). A group was injected i.p with docetaxel (20 mg/kg) for the last 2 weeks. Metformin had no toxic effect on mice, and it did not affect animal weight and insulinemia (Supplementary Fig. S1). A whole-body open imaging of the animals revealed the fluorescence of primary tumors and metastases, including periaortic and periadrenal lymph nodes, liver, pancreas, lungs, and mesentery, indicating a disseminating disease as described previously (29). A representative picture of the GFP-positive tumors is shown in Fig. 1A. As expected, the tumors were significantly smaller in the docetaxel-treated group and metformin induced a strong antitumoral effect. Indeed, it significantly reduced by more than 50% the growth of the primary tumor when given for 5 weeks (Fig. 1 A and B). However, when administrated only during the last 2 weeks like docetaxel, metformin did not have any impact on tumor growth (Fig. 1 A and B). Our findings show that metformin has a preventative effect on primary tumor growth, but yet does not manifest a curative effect when the tumor is already established. Interestingly, the dissemination pattern of metastases showed that all mice had metastases regardless of the treatment except 2 mice in the “metformin 5 weeks” group (Table 1). Among the 3 mice without solid metastasis 2 of them had primary tumors bigger than the average tumor volume of the metformin 5 weeks group (162.51 and 295.64 vs. 135.53 mm3 for the average tumor volume), suggesting that the absence of metastasis is not associated with small tumors. Only docetaxel significantly reduced the formation of retroperitoneal lymph nodes as well as liver, pancreas, lung, and mesentary metastases (Table 1). Nevertheless, mice from the “metformin 5 weeks” arm exhibited statistically less metastasis (P = 0.04), suggesting that metformin may hinder the metastatic dissemination.

Figure 1.

Effects of metformin on growth of established human fluorescent PC-3 orthotopic xenografts in nude mice. The day of the orthotopic implantations with 1 × 106 PC-3 cells, mice were randomized into four groups. Animals were given drinking water (control, C) or 100 mg/kg metformin (Met, 5 wk) in drinking water for 5 weeks. Alternatively, 3 weeks after implantation, animals were treated for the final 2 weeks with metformin in drinking water (Met, 2 wk) or weekly i.p. with 20 mg/kg docetaxel A, representative photos of the primary tumors (magnification ×0.8). The tumors are in green and the bladder appears in orange (autofluorescence) on the picture. B, tumor volume calculated as described in Materials and Methods. Columns, mean from 6 to 8 animals; bars, SEM. The statistical analysis was performed using the Student t test; *, P < 0.05.

Figure 1.

Effects of metformin on growth of established human fluorescent PC-3 orthotopic xenografts in nude mice. The day of the orthotopic implantations with 1 × 106 PC-3 cells, mice were randomized into four groups. Animals were given drinking water (control, C) or 100 mg/kg metformin (Met, 5 wk) in drinking water for 5 weeks. Alternatively, 3 weeks after implantation, animals were treated for the final 2 weeks with metformin in drinking water (Met, 2 wk) or weekly i.p. with 20 mg/kg docetaxel A, representative photos of the primary tumors (magnification ×0.8). The tumors are in green and the bladder appears in orange (autofluorescence) on the picture. B, tumor volume calculated as described in Materials and Methods. Columns, mean from 6 to 8 animals; bars, SEM. The statistical analysis was performed using the Student t test; *, P < 0.05.

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Table 1.

Pattern of metastatic dissemination

TreatmentsShamMetformin 5 weeksMetformin 2 weeksDocetaxel 2 weeks
Number of mice with metastases/total number of mice 7/7 (100%6/8 (75%6/6 (100%7/7 (100%
Retroperitoneal lymph nodes 
 Periaortic lymph nodes 2, 2, 2, 2, 2, 2, 1 2, 0, 2, 2, 2, 2, 2, 0 2, 2, 2, 2, 2, 2 2, 1, 1, 2, 1, 2, 2 
 Periadrenal 2, 2, 1, 2, 2, 1, 1 0, 0, 2, 2, 2, 2, 1, 0 1, 2, 2, 1, 2, 1 2, 0, 0, 2, 0, 0, 0 
Number of metastases 24/28 (3.5/animal21/32 (2.6/animal)NS 21/24 (3.5/animal)NS 15/28 (2.1/animal)a 
Liver 1, 1, 1, 1, 1, 1, 1 0, 0, 1, 1, 0, 1, 1, 0 0, 1, 1, 1, 0, 1 0, 0, 0, 1, 0, 0, 1 
Pancreas 1, 1, 1, 1, 1, 1, 1 0, 0, 1, 1, 0, 1, 1, 0 1, 1, 1, 1, 0, 1 0, 0, 0, 1, 0, 1, 1 
Lung 1, 1, 1, 1, 1, 1, 0 0, 0, 1, 1, 1, 1, 0, 0 1, 1, 1, 1, 0, 1 1, 0, 0, 1, 0, 1, 0 
Mesentery 1, 1, 0, 1, 1, 1, 1 0, 0, 1, 1, 0, 1, 1, 0 1, 1, 1, 1, 0, 1 0, 0, 0, 1, 0, 0, 1 
Number of metastases 26/28 (3.7/animal16/32 (2.0/animal)b 19/24 (3.2/animal)NS 10/28 (1.4/animal)c 
TreatmentsShamMetformin 5 weeksMetformin 2 weeksDocetaxel 2 weeks
Number of mice with metastases/total number of mice 7/7 (100%6/8 (75%6/6 (100%7/7 (100%
Retroperitoneal lymph nodes 
 Periaortic lymph nodes 2, 2, 2, 2, 2, 2, 1 2, 0, 2, 2, 2, 2, 2, 0 2, 2, 2, 2, 2, 2 2, 1, 1, 2, 1, 2, 2 
 Periadrenal 2, 2, 1, 2, 2, 1, 1 0, 0, 2, 2, 2, 2, 1, 0 1, 2, 2, 1, 2, 1 2, 0, 0, 2, 0, 0, 0 
Number of metastases 24/28 (3.5/animal21/32 (2.6/animal)NS 21/24 (3.5/animal)NS 15/28 (2.1/animal)a 
Liver 1, 1, 1, 1, 1, 1, 1 0, 0, 1, 1, 0, 1, 1, 0 0, 1, 1, 1, 0, 1 0, 0, 0, 1, 0, 0, 1 
Pancreas 1, 1, 1, 1, 1, 1, 1 0, 0, 1, 1, 0, 1, 1, 0 1, 1, 1, 1, 0, 1 0, 0, 0, 1, 0, 1, 1 
Lung 1, 1, 1, 1, 1, 1, 0 0, 0, 1, 1, 1, 1, 0, 0 1, 1, 1, 1, 0, 1 1, 0, 0, 1, 0, 1, 0 
Mesentery 1, 1, 0, 1, 1, 1, 1 0, 0, 1, 1, 0, 1, 1, 0 1, 1, 1, 1, 0, 1 0, 0, 0, 1, 0, 0, 1 
Number of metastases 26/28 (3.7/animal16/32 (2.0/animal)b 19/24 (3.2/animal)NS 10/28 (1.4/animal)c 

NOTE: For retroperitoneal lymph nodes, the numbers 0, 1, or 2 represent the quantity of invaded lymph nodes. For other organs, 0 means no presence of metastasis; 1 means a metastatic organ (regardless of the intensity of metastasis dissemination in this organ).

Abbreviation: NS, not significant.

aP = 0.049, compared with sham-treated animals (t test).

bP = 0.040, compared with sham-treated animals (t test).

cP = 0.002, compared with sham-treated animals (t test).

Metformin inhibits the migration and the invasive properties of PC3 and DU145 prostate cancer cell lines

Because metastasis requires cancer cell migration, we investigated the effects of metformin on human prostate cancer cell migration using Boyden chamber assay. According to our previous studies (4, 30) and a dose response experiment (data not shown), we treated the cells with 5 mmol/L metformin. To exclude any action of metformin on cell proliferation, PC3 and DU145 were treated with metformin for 4 hours during the migration toward culture medium supplemented with FBS (chemoattractant medium). We monitored cell viability and apoptosis in the same conditions. As expected, viabilities in all cell cultures treated for 4 hours with metformin exceeded 95% (data not shown) and markers of apoptosis were negative (Supplementary Fig. S2). Interestingly, a significant inhibitory effect of metformin of 50% on the migration of PC3 and DU145 cells was revealed (Fig. 2A). In contrary, metformin did not alter the migration of normal epithelial prostate cells (P69 cells; Supplementary Fig. S3). Next, we determined the impact of metformin on invasion. Cells were treated 4 hours with metformin before assessing 2D-invasion in Matrigel using Boyden chambers, as described in Materials and Methods section. As shown in Fig. 2B, metformin strongly inhibited the invasive properties of PC3 and DU145 cells. To further explore whether metformin reduces invasion, we performed a spheroid assay with DU145 cells. Untreated DU145 cells were able to invade the adjacent Matrigel matrix in a collective migration/invasion pattern. Spheroids treated with metformin remained compact with almost no cells migrating out (Supplementary Fig. S4). We then tracked individual cell migration over a period of 12 hours using time-lapse video microscopy. Untreated PC3 cells moved in several directions over an extended area compared with those treated with metformin (Supplementary Fig. S5). The total accumulated distance covered by the untreated cells was 1,329.1 ± 369.2 μm versus 9.30 ± 7.74 μm for those treated with metformin, and the mean euclidean distance (shortest linear distance between points A and B) was 89.02 ± 56.73 μm versus 6.47 ± 4.22 μm. Metformin also affected cell velocity because untreated cells migrated at a 1.84 ± 0.51 μm/min versus 0.012 ± 0.01 μm/min for metformin-treated cells. Our results establish that metformin inhibits all movement parameters of prostate cancer cells with a major inhibitory impact on their invasive properties.

Figure 2.

Metformin inhibits prostate cancer cell migration and invasion. A, PC3 and DU145 cells were seeded in Boyden chambers, and metformin (5 mmol/L) was added during the migration for 4 hours. Graphs are expressed as a percentage of cells migrating across the Boyden chamber relative to the control conditions (100%), and the insets represent picture of the counted fields. B, quantification of the invasion assay performed in Boyden chambers during 24 hours in presence of 5 mmol/L metformin. C, immunofluorescence performed with Texas red Phalloidin in PC3 and DU145 treated with 5 mmol/L metformin for 4 hours. The statistical analysis was performed using the Student t test; *, P < 0.05; **, P < 0.01.

Figure 2.

Metformin inhibits prostate cancer cell migration and invasion. A, PC3 and DU145 cells were seeded in Boyden chambers, and metformin (5 mmol/L) was added during the migration for 4 hours. Graphs are expressed as a percentage of cells migrating across the Boyden chamber relative to the control conditions (100%), and the insets represent picture of the counted fields. B, quantification of the invasion assay performed in Boyden chambers during 24 hours in presence of 5 mmol/L metformin. C, immunofluorescence performed with Texas red Phalloidin in PC3 and DU145 treated with 5 mmol/L metformin for 4 hours. The statistical analysis was performed using the Student t test; *, P < 0.05; **, P < 0.01.

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Metformin induces the reorganization of actin cytoskeleton

Because cells coordinate their migration through the regulation of actin dynamics (31), we studied the effect of metformin on α-actin, β-actin, and fascin expression, three important proteins implicated in cell migration. We did not observe any change in the expression these proteins except a slight decrease of fascin expression in PC3 cells only (Supplementary Fig. S6). More importantly, we analyzed actin cytoskeleton organization, PC3 and DU145 cells were seeded on fibronectin-coated wells and fluorescence microscopy was used to analyze F-actin. In the control (untreated) conditions, elongated cells forming lamellipodia extensions rich in F-actin and stress fibers and ruffle formations were visible (Fig. 2C). Four hours of treatment with 5 mmol/L metformin induced a drastic change of cell morphology, with cells reorganizing their actin cytoskeleton, becoming circular, displaying less lamellipodia (Fig. 2C). The shape of the PC3 and DU145 cells confirmed that metformin treatment significantly decreased invasive morphology (Fig. 2C).

Metformin decreases Rac1 GTPase activity

The known role of the small GTPase Rac1 as a major driver of cell motility (32, 33) prompted us to assess Rac1 activity, using GST-Pak pull-down assay, as described previously (34). Interestingly, this series of measurements revealed a significant decrease in Rac1–GTP levels in PC3 and DU145 cells treated with 5 mmol/L metformin for 4 hours (Fig. 3A). Rho activity was not affected by metformin, thereby pointing to a specific decrease in Rac1 activity (Supplementary Fig. S7). To establish the link between the inhibition of migration and Rac1GTPase activity triggered by metformin treatment, we used a Rac1 inhibitor that specifically and reversibly inhibits Rac1 GDP/GTP exchange activity, while exhibiting no effect on Cdc42 or RhoA (35). We found that treatment of PC3 and DU145 cells phenocopied the effects of metformin on cell migration (Fig. 3B) and induced a circular cell morphology (Supplementary Fig. S8). To further gain insight in the relationship between metformin and Rac1GTPase, constitutively active forms of Rac1 (HA–Rac1–Q61L or HA–Rac1–V12) were overexpressed in PC3 and DU145 cells. In the presence of metformin, cells expressing the active form of Rac1 no longer displayed the “rounded shape” phenotype that could be observed in nontransfected cells (Fig. 3C). Furthermore, we found that the expression of the constitutive forms of Rac1 slightly but significantly inhibits basal cell migration (Fig 3D). Importantly, the inhibitory effect of metformin on control PC3 and DU145 cell migration was abolished in cells expressing the constitutive forms of Rac1: Rac1–Q61L or Rac1–V12 (Fig. 3D). This reveals that constitutive activation of Rac1 overrides the effects of metformin on actin cytoskeleton reorganization and cancer cell migration.

Figure 3.

Metformin inhibits Rac 1 activity and constitutively active Rac 1 restores cell migration. A, immunoblot analysis of Rac1 performed with DU145 and PC3 prostate cancer cells treated with 5 mmol/L metformin for 4 hours as described in Materials and Methods. The graph represents the quantification of the ratio of Rac1 GTP/Total Rac1. B, quantification of the migration assay in Boyden chambers of cells treated for 4 hours with the Rac inhibitor (50 μmol/L). C, DU145 transfected with HA-RacQ61L and treated with 5 mmol/L metformin was analyzed by immunofluorescence using Texas red Phalloidin (red) and HA (green). D, PC3 and DU145 were transfected with empty vector, active forms of Rac1:RacQ61L or RacV12 and treated with 5 mmol/L metformin for 4 hours during the migration assay. Columns are the mean of five independent experiments; bars are SEM. The statistical analysis was performed using the Student t test; *, P < 0.05; **, P < 0.01.

Figure 3.

Metformin inhibits Rac 1 activity and constitutively active Rac 1 restores cell migration. A, immunoblot analysis of Rac1 performed with DU145 and PC3 prostate cancer cells treated with 5 mmol/L metformin for 4 hours as described in Materials and Methods. The graph represents the quantification of the ratio of Rac1 GTP/Total Rac1. B, quantification of the migration assay in Boyden chambers of cells treated for 4 hours with the Rac inhibitor (50 μmol/L). C, DU145 transfected with HA-RacQ61L and treated with 5 mmol/L metformin was analyzed by immunofluorescence using Texas red Phalloidin (red) and HA (green). D, PC3 and DU145 were transfected with empty vector, active forms of Rac1:RacQ61L or RacV12 and treated with 5 mmol/L metformin for 4 hours during the migration assay. Columns are the mean of five independent experiments; bars are SEM. The statistical analysis was performed using the Student t test; *, P < 0.05; **, P < 0.01.

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P-Rex1 overexpression reverses the antimigratory action of metformin

P-Rex1 is a GEF that modulates cellular Rac1–GTP levels. It is implicated in cytoskeleton remodeling (36) and facilitates prostate cancer metastasis (17). We asked whether P-Rex1 overexpression (HA–P-Rex1 wt) reversed metformin effects on cell migration. HA–P-Rex1 expression did not affect basal Rac1 GTP levels, but restored Rac1 GTP levels in cancer cells treated with metformin (Fig. 4A). Accordingly, the overexpression of wild-type P-Rex1 does not affect cell migration but reversed the antimigratory effects of metformin (Fig. 4B). Altogether, our results support the idea that the forced activation of Rac1 alleviates the metformin-mediated inhibition of cancer cell migration.

Figure 4.

Overexpression of P-Rex1 counteracts the antimigratory effects of metformin. A, immunoblot analysis of Rac1GTP and total Rac1 in DU145 cells transfected with the indicated vectors and treated for 4 hours with 5 mmol/L metformin. Expression of the HA-tagged proteins in the cell lysates was revealed by an anti-HA immunoblot. Similar results were obtained in three independent experiments. B, DU145 transfected with control vector (empty vector) or P-Rex1 expression vector (HA–P-rex1 wt) were assayed for migration during 4 hours in the presence or absence of 5 mmol/L metformin. The graph represents the average of three independent migration assays. The statistical analysis was performed using the Student t test; *, P < 0.05.

Figure 4.

Overexpression of P-Rex1 counteracts the antimigratory effects of metformin. A, immunoblot analysis of Rac1GTP and total Rac1 in DU145 cells transfected with the indicated vectors and treated for 4 hours with 5 mmol/L metformin. Expression of the HA-tagged proteins in the cell lysates was revealed by an anti-HA immunoblot. Similar results were obtained in three independent experiments. B, DU145 transfected with control vector (empty vector) or P-Rex1 expression vector (HA–P-rex1 wt) were assayed for migration during 4 hours in the presence or absence of 5 mmol/L metformin. The graph represents the average of three independent migration assays. The statistical analysis was performed using the Student t test; *, P < 0.05.

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Metformin increases cAMP levels in prostate cancer cells

Because cyclic AMP inhibits Rac1 activity (37), we investigated whether cAMP acts as a potential mediator by which metformin modulates migration of prostate tumor cells. Accordingly, we measured cAMP content in cells treated with metformin. We did not detect any change in cAMP concentration after 4 hours of treatment with metformin in PC3 cells (Supplementary Fig. S9). On contrary, metformin induced a slight but significant increase in cAMP levels in DU145 (Fig. 5A), which was associated with the augmentation of luciferase activity in cells transfected with the CRE–Luc construct (to monitor cAMP increase through the activation of CREB, the cAMP response element–binding protein; Fig. 5B) and increased CREB phosphorylation (Supplementary Fig. S10).

Figure 5.

Metformin increases cAMP levels and inhibition of adenylate cyclase reverses the antimigratory effects of metformin. A, cAMP concentration in DU145 cells treated with 5 mmol/L metformin for 4 hours in the presence or absence of 100 μmol/L SQ22536 (Adenylate cyclase inhibitor). B, luciferase activity of the CRE promoter element in DU145 cells transfected with CRE–Luc vector and treated with 5 mmol/L metformin for 4 hours. C, quantification of a migration assay performed in Boyden chambers with DU145 cells treated with both 5 mmol/L metformin and 100 μmol/L SQ22536 (Adenylate cyclase inhibitor) for 4 hours. D, quantification of a migration assay performed in Boyden chambers with DU145 cells treated with 5 mmol/L metformin (M) or 100 μmol/L of the cell-permeant cAMP analogue dbcAMP for 4 hours. E, DU145 cells were transfected with empty vector, active forms of Rac1:RacQ61L or RacV12 and treated with dbcAMP for 4 hours during the migration assay. The graphs represent the quantification of at least three experiments performed independently. The statistical analysis was performed using the Student t test. The differences are significant; *, P < 0.05; **, P < 0.01.

Figure 5.

Metformin increases cAMP levels and inhibition of adenylate cyclase reverses the antimigratory effects of metformin. A, cAMP concentration in DU145 cells treated with 5 mmol/L metformin for 4 hours in the presence or absence of 100 μmol/L SQ22536 (Adenylate cyclase inhibitor). B, luciferase activity of the CRE promoter element in DU145 cells transfected with CRE–Luc vector and treated with 5 mmol/L metformin for 4 hours. C, quantification of a migration assay performed in Boyden chambers with DU145 cells treated with both 5 mmol/L metformin and 100 μmol/L SQ22536 (Adenylate cyclase inhibitor) for 4 hours. D, quantification of a migration assay performed in Boyden chambers with DU145 cells treated with 5 mmol/L metformin (M) or 100 μmol/L of the cell-permeant cAMP analogue dbcAMP for 4 hours. E, DU145 cells were transfected with empty vector, active forms of Rac1:RacQ61L or RacV12 and treated with dbcAMP for 4 hours during the migration assay. The graphs represent the quantification of at least three experiments performed independently. The statistical analysis was performed using the Student t test. The differences are significant; *, P < 0.05; **, P < 0.01.

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To firmly establish that increased cAMP is directly implicated in the antimigratory effects of metformin, we treated DU145 cells with SQ22536, an inhibitor of adenylate cyclase. Treatment with 100 μmol/L SQ22536 prevented the increase of cAMP (Fig. 5A) as well as the decrease in cell migration (Fig. 5C) induced by metformin, while leaving basal cAMP concentration and basal cell migration unaffected (Fig. 5A and C). To directly observe the effects of elevated cAMP on cell migration, we treated DU145 cells with 500 μmol/L of dbcAMP, a cell-permeable cAMP analogue. A 4 hours treatment with dbcAMP inhibited the migration of DU145 cells (Fig. 5D) and decreased Rac1 activity (Supplementary Fig. S11). Importantly, the overexpression of a constitutively active Rac1 in DU145 cells overcame the antimigratory effects of dbcAMP (Fig. 5E). These results suggest that the antimigratory effect of metformin requires increased cAMP levels.

Metformin inhibits CXCL12 chemotactism in prostate cancer cells

Regardless of its chemoattractive properties, CXCL12 was recently shown to regulate Rac1 (38). Therefore, CXCL12 was used as a chemoattractant in a cell migration assay, in which it significantly promoted DU145 migration (Fig. 6A). Importantly, we found that addition of metformin prevented CXCL12 promigratory effects (Fig. 6A). CXCL12 binds to the chemokine receptor 4 (CXCR4) to affect cell migration. To validate the role of CXCL-12/CXCR4 signaling in prostate cancer cell migration, we treated cells with AMD3100, a well-characterized and specific antagonist of CXCR4, which inhibits the binding and function of CXCL12 (39). In the presence of CXCL12, AMD3100 significantly inhibited DU145 cell migration showing that the CXCL12/CXCR4 axis plays an important role in the migration of prostate cancer cells (Fig. 6B). Flow-cytometry analysis to monitor expression of CXCR4 at the cell surface revealed a decrease upon metformin treatment (Fig. 6C and Supplementary Fig. S12). We measured Rac1–GTP levels and found that CXCL12 increased Rac1 activity in a metformin-sensitive manner (Fig. 6D). In conclusion, our results show that metformin interferes with CXCL12 signaling through the regulation of CXCR4 and Rac1 to inhibit prostate cancer cell migration.

Figure 6.

Metformin inhibits the promigratory effects of CXCL12. A, quantification of a migration assay performed with DU145 cells treated with 250 ng/mL of CXCL12 in the absence (C) or presence of 5 mmol/L metformin for 4 hours. 0, the untreated condition. B, quantification of migration assay with DU145 cells incubated with 250 ng/mL CXCL12 for 4 hours in the presence or absence of 25 μg/mL AMD3100 (a CXCR4 antagonist). C, relative expression of CXCR4, determined by flow-cytometry analysis, in DU145 cells treated with 5 mmol/L metformin for 4 hours. D, immunoblot analysis of Rac1GTP in DU145 cells treated with CXCL12 in presence of 5 mmol/L metformin for 4 hours. The graphs represent the quantification of at least three independent experiments performed independently. The statistical analysis was performed using the Student t test. The differences are significant; *, P < 0.05; **, P < 0.01.

Figure 6.

Metformin inhibits the promigratory effects of CXCL12. A, quantification of a migration assay performed with DU145 cells treated with 250 ng/mL of CXCL12 in the absence (C) or presence of 5 mmol/L metformin for 4 hours. 0, the untreated condition. B, quantification of migration assay with DU145 cells incubated with 250 ng/mL CXCL12 for 4 hours in the presence or absence of 25 μg/mL AMD3100 (a CXCR4 antagonist). C, relative expression of CXCR4, determined by flow-cytometry analysis, in DU145 cells treated with 5 mmol/L metformin for 4 hours. D, immunoblot analysis of Rac1GTP in DU145 cells treated with CXCL12 in presence of 5 mmol/L metformin for 4 hours. The graphs represent the quantification of at least three independent experiments performed independently. The statistical analysis was performed using the Student t test. The differences are significant; *, P < 0.05; **, P < 0.01.

Close modal

Prostate cancer can be very aggressive in advanced stages and commonly metastasizes to bone and lymph nodes, more rarely to the liver and lung and cell migration, which is required for metastasis, is a complex biologic process regulated by environmental factors, signaling pathways and cytoskeletal rearrangement. Here, we report that the antidiabetic drug metformin reduces the formation of metastasis to fewer solid organs in an orthotopic mouse model and affects cell cytoskeleton organization, which drastically inhibits prostate cancer cell migration through decreased Rac1 activity. Because our previous studies showed that metformin inhibits cancer cell proliferation and blocks cell cycle in G0–G1 within 24 hours (4), all cell migration assays were performed within 4 hours of treatment to exclude any effects due to cell-cycle arrest.

Metformin inhibits the migration of glioblastoma, ovarian, and pancreatic cancer cells (8, 12, 13). However, the cellular and molecular mechanisms responsible for this inhibition are poorly documented. In melanoma, metformin does not affect cell migration, but inhibits invasion by reducing the activity of matrix metalloproteinases (MMP; ref. 9). Similarly, two studies reported that metformin inhibits the activity of MMP-9, and therefore blocks cancer cell invasion in endothelial and fibrosarcoma cells (10, 40). Bao and colleagues (8) correlated the antimigratory effects of metformin with the decreased expression of let-7b, miR-26a, and miR-200b. In glioma cell lines, metformin suppresses MMP-2 expression and affects cell adhesion through the diminution of fibulin-3, a secreted glycoprotein that associates to the ECM (41). Here, we show that metformin induces drastic changes in cell morphology with a marked reduction of lamellipodia. These modifications are not associated with changes in α-actin or β-actin expressions (Supplementary Fig. S6). However, we observed a slight decrease of fascin upon metformin treatment. Fascin downstream of Rac contributes to cancer cell migration and the formation of metastasis (42–44). Further investigations are required to determine how metformin interferes with lamellipodia formation, and whether fascin is implicated in its antimigratory effect. The drastic change in prostate cancer cell morphology is associated with a decrease in the active form of Rac1, a master regulator of actin polymerization (14). Expression of a constitutively active form of Rac1 inhibited the antimigratory effects of metformin and restored the formation of lamellipodia in cancer cells. Conflicting reports have been published regarding the role Rac1 in cell migration (45–49). For instance, the RacGEF Tiam1 inhibits cell migration of melanoma cells (48), in accordance, we also observe a slight inhibition of cell migration when we express Rac61L and RacV12 in DU145. On the other hand, the GEF P-Rex1 promotes cell migration and its downregulation with siRNA inhibits PC3 cell migration (17). In the present study, the expression of P-Rex1 reversed the antimigratory effects of metformin, supporting the notion that metformin acts in a Rac1-dependent manner. Metformin could, therefore, act as a GEF inhibitor. Indeed, P-Rex1 activity is enhanced by PIP3 and Gβγ proteins, which is inhibited by cAMP through the phosphorylation of P-Rex1 by the protein kinase A (PKA; ref. 50). As a result, the increased cAMP levels induced by metformin could inhibit P-Rex1 through PKA and downregulate Rac1. In line with this hypothesis, increased intracellular cAMP levels and PKA activity following morphine treatment lead to inhibition of Rac1GTPase and p38 MAPK, cause attenuation of actin polymerization, and decrease bacterial phagocytosis (51).

cAMP plays an important and sometimes controversial role in apoptosis (52, 53), but cAMP is also a well-established inhibitor of cell migration (20) and a regulator of cytoskeleton organization (54). It was demonstrated that cAMP inhibits the Rho family small GTPases via PKA. For example, prostaglandin E2 inhibits insulin-like growth factor-I–induced cell migration and inhibited Rac1 activity through a mechanism involving cAMP. In addition, cAMP has been shown to regulate Rac1 and breast cancer cell migration via PKA. We showed that metformin increases cAMP levels in DU145, but not in PC3. The cellular cAMP level depends on the activity of two enzymes, the adenylyl cyclases that produce cAMP and the phosphodiesterases that hydrolyze cAMP. This discrepancy between the cell lines may be related to a different action of metformin on adenylyl cyclase or/and phosphodiesterase depending on the cell lines. Indeed, a study demonstrates that metformin decreases phopshodiesterase 3B mRNA levels in breast cancer biopsies after the treatment (55). More recently, a work performed in primary hepatocytes showed that a pretreatment with metformin inhibited glucagon-induced accumulation of cAMP, but did not affect basal cAMP levels (56). Indeed, metformin induced an increase in the AMP levels, possibly due to the decreased ATP concentration, which inhibits the activity of adenylate cyclase. We and others have shown that metformin inhibits the activity of the mitochondrial complex 1, and decreases the intracellular concentration of ATP, resulting in the increase of AMP within 8 hours (30, 57).

We established that CXCL12 increases Rac1 activity as previously shown in endothelial cells (58), and that metformin inhibits CXCL12-induced Rac1 activation. Our work suggests that metformin hampers the promigratory effects of CXCL12 by affecting Rac1 GTPase activity. Interestingly, the CXCL12/CXCR4 pathway was recently associated with Rac activation and metastasis (38). Therapeutic approaches target this pathway by either blocking CXCL12 with antibodies or acting on CXCR4 by preventing CXCL12 binding. We anticipate that metformin may represent a novel and alternative way of inhibiting this pathway known to play a major role in prostate cancer metastasis.

Regarding prostate cancer therapy, we demonstrated in an orthotopic metastatic model that metformin reduces the formation of metastasis to fewer organs in addition to its inhibitory effect on the growth of primary tumors. Several studies have shown in different mouse xenograft models and transgenic mice that metformin inhibits tumor growth (reviewed in ref. 59), but few works analyzed metastasis dissemination. Our data are encouraging for a potential use of metformin in the treatment of advanced metastatic prostate cancer. However, one of the limitation of our in vivo model is the injection of exogenous cancer cells in the mouse prostate. Therefore, we are aware that we need to confirm the effect of metformin on the formation of metastasis in another mouse model. Thus, it would be interesting to test the effects of metformin in the “RapidCaP” model recently described by Cho and colleagues (60). In this new model, unlike our study, mice develop metastasis from mouse prostate tumors. Rattan and colleagues (11) demonstrated that metformin significantly reduces the growth of metastatic nodules of ovarian cancer cells in nude mice. They also indicated that metformin potentiates cisplatin-induced toxicity. To this regard, it would be interesting to determine whether metformin can improve the efficiency of docetaxel, the standard treatment for patients with prostate cancer who are refractory to hormonal manipulations.

Collectively, our results shed light on a new mechanism of action of metformin and novel properties of this drug in prostate cancer.

No potential conflicts of interest were disclosed.

Conception and design: B. Dirat, I. Ader, O. Cuvillier, F. Bost

Development of methodology: B. Dirat, I. Ader, M. Golzio, B. Malavaud, F. Bost

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): B. Dirat, I. Ader, M. Golzio, A. Mettouchi, F. Larbret, B. Malavaud, E. Lemichez, F. Bost

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): B. Dirat, I. Ader, M. Golzio, K. Laurent, O. Cuvillier, F. Bost

Writing, review, and/or revision of the manuscript: B. Dirat, I. Ader, M. Golzio, F. Massa, B. Malavaud, M. Cormont, O. Cuvillier, J.F. Tanti, F. Bost

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): B. Dirat, F. Larbret, F. Bost

Study supervision: B. Dirat, O. Cuvillier, F. Bost

The authors thank Anne Doye and Rachel Paul-Bellon for technical assistance, Pr. Mitchell and Dr. Becanovic for The HA–PREX plasmid, Issam Ben-Sahra, Stéphane Ricoult, Jérôme Gilleron, Sophie Giorgetti-Peraldi, and Yannick Le Marchand Brustel for their help and the critical reading of the article. The authors greatly acknowledge Damien Alcor of the C3M (or MicorBio) Cell Imaging Facility.

This study was supported by The European Foundation for the Study of Diabetes (EFSD), INCA (grants 2010-219 and 2010-214) and the “Fondation ARC.” B. Dirat was supported by INCA grant 2010-219, the Cancerople PACA, and the Région PACA. F. Bost, J.F. Tanti, and A. Mettouchi are investigators of the Centre National de la Recherche Scientifique (CNRS). F. Massa is supported by ITMO-Cancer. This work was supported by the French Government (National Research Agency, ANR) through the “Investments for the Future” LABEX SIGNALIFE (grant ANR-11-0028-01).

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.

1.
Currie
CJ
,
Poole
CD
,
Jenkins-Jones
S
,
Gale
EA
,
Johnson
JA
,
Morgan
CL
. 
Mortality after incident cancer in people with and without type 2 diabetes: impact of metformin on survival
.
Diabetes Care
2012
;
35
:
299
304
.
2.
Evans
JM
,
Donnelly
LA
,
Emslie-Smith
AM
,
Alessi
DR
,
Morris
AD
. 
Metformin and reduced risk of cancer in diabetic patients
.
BMJ
2005
;
330
:
1304
5
.
3.
Margel
D
,
Urbach
DR
,
Lipscombe
LL
,
Bell
CM
,
Kulkarni
G
,
Austin
PC
, et al
Metformin use and all-cause and prostate cancer-specific mortality among men with diabetes
.
J Clin Oncol
2013
;
31
:
3069
75
.
4.
Ben Sahra
I
,
Laurent
K
,
Loubat
A
,
Giorgetti-Peraldi
S
,
Colosetti
P
,
Auberger
P
, et al
The antidiabetic drug metformin exerts an antitumoral effect in vitro and in vivo through a decrease of cyclin D1 level
.
Oncogene
2008
;
27
:
3576
86
.
5.
Huang
J
,
Manning
BD
. 
The TSC1–TSC2 complex: a molecular switchboard controlling cell growth
.
Biochem J
2008
;
412
:
179
90
.
6.
Memmott
RM
,
Mercado
JR
,
Maier
CR
,
Kawabata
S
,
Fox
SD
,
Dennis
PA
. 
Metformin prevents tobacco carcinogen–induced lung tumorigenesis
.
Cancer Prev Res
2010
;
3
:
1066
76
.
7.
Zakikhani
M
,
Dowling
R
,
Fantus
IG
,
Sonenberg
N
,
Pollak
M
. 
Metformin is an AMP kinase-dependent growth inhibitor for breast cancer cells
.
Cancer Res
2006
;
66
:
10269
73
.
8.
Bao
B
,
Wang
Z
,
Ali
S
,
Ahmad
A
,
Azmi
AS
,
Sarkar
SH
, et al
Metformin inhibits cell proliferation, migration, and invasion by attenuating CSC function mediated by deregulating miRNAs in pancreatic cancer cells
.
Cancer Prev Res
2012
;
5
:
355
64
.
9.
Cerezo
M
,
Tichet
M
,
Abbe
P
,
Ohanna
M
,
Lehraiki
A
,
Rouaud
F
, et al
Metformin blocks melanoma invasion and metastasis development in a AMPK/p53-dependent manner
.
Mol Cancer Ther
2013
;
12
:
1605
15
.
10.
Hwang
YP
,
Jeong
HG
. 
Metformin blocks migration and invasion of tumour cells by inhibition of matrix metalloproteinase-9 activation through a calcium and protein kinase Calpha-dependent pathway: phorbol-12-myristate-13-acetate-induced/extracellular signal-regulated kinase/activator protein-1
.
Br J Pharmacol
2010
;
160
:
1195
211
.
11.
Rattan
R
,
Graham
RP
,
Maguire
JL
,
Giri
S
,
Shridhar
V
. 
Metformin suppresses ovarian cancer growth and metastasis with enhancement of Cisplatin cytotoxicity in vivo
.
Neoplasia
2010
;
13
:
483
91
.
12.
Tan
BK
,
Adya
R
,
Chen
J
,
Lehnert
H
,
Sant Cassia
LJ
,
Randeva
HS
. 
Metformin treatment exerts antiinvasive and antimetastatic effects in human endometrial carcinoma cells
.
J Clin Endocrinol Metab
2011
;
96
:
808
16
.
13.
Wu
B
,
Li
S
,
Sheng
L
,
Zhu
J
,
Gu
L
,
Shen
H
, et al
Metformin inhibits the development and metastasis of ovarian cancer
.
Oncol Rep
2012
;
28
:
903
8
.
14.
Parri
M
,
Chiarugi
P
. 
Rac and Rho GTPases in cancer cell motility control
.
Cell Commun Signal
2010
;
8
:
23
.
15.
Nobes
CD
,
Hall
A
. 
Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia
.
Cell
1995
;
81
:
53
62
.
16.
Engers
R
,
Ziegler
S
,
Mueller
M
,
Walter
A
,
Willers
R
,
Gabbert
HE
. 
Prognostic relevance of increased Rac GTPase expression in prostate carcinomas
.
Endocr Relat Cancer
2007
;
14
:
245
56
.
17.
Qin
J
,
Xie
Y
,
Wang
B
,
Hoshino
M
,
Wolff
DW
,
Zhao
J
, et al
Upregulation of PIP3-dependent Rac exchanger 1 (P-Rex1) promotes prostate cancer metastasis
.
Oncogene
2009
;
28
:
1853
63
.
18.
O'Connor
KL
,
Shaw
LM
,
Mercurio
AM
. 
Release of cAMP gating by the alpha6beta4 integrin stimulates lamellae formation and the chemotactic migration of invasive carcinoma cells
.
J Cell Biol
1998
;
143
:
1749
60
.
19.
Bond
M
,
Wu
YJ
,
Sala-Newby
GB
,
Newby
AC
. 
Rho GTPase, Rac1, regulates Skp2 levels, vascular smooth muscle cell proliferation, and intima formation in vitro and in vivo
.
Cardiovasc Res
2008
;
80
:
290
8
.
20.
Chen
L
,
Zhang
JJ
,
Huang
XY
. 
cAMP inhibits cell migration by interfering with Rac-induced lamellipodium formation
.
J Biol Chem
2008
;
283
:
13799
805
.
21.
Dwinell
MB
,
Ogawa
H
,
Barrett
KE
,
Kagnoff
MF
. 
SDF-1/CXCL12 regulates cAMP production and ion transport in intestinal epithelial cells via CXCR4
.
Am J Physiol Gastrointest Liver Physiol
2004
;
286
:
G844
50
.
22.
Ghosh
MC
,
Makena
PS
,
Gorantla
V
,
Sinclair
SE
,
Waters
CM
. 
CXCR4 regulates migration of lung alveolar epithelial cells through activation of Rac1 and matrix metalloproteinase-2
.
Am J Physiol Lung Cell Mol Physiol
2012
;
302
:
L846
56
.
23.
Singh
S
,
Singh
UP
,
Grizzle
WE
,
Lillard
JW
 Jr
. 
CXCL12-CXCR4 interactions modulate prostate cancer cell migration, metalloproteinase expression, and invasion
.
Lab Invest
2004
;
84
:
1666
76
.
24.
Taichman
RS
,
Cooper
C
,
Keller
ET
,
Pienta
KJ
,
Taichman
NS
,
McCauley
LK
. 
Use of the stromal cell-derived factor-1/CXCR4 pathway in prostate cancer metastasis to bone
.
Cancer Res
2002
;
62
:
1832
7
.
25.
Teicher
BA
,
Fricker
SP
. 
CXCL12 (SDF-1)/CXCR4 pathway in cancer
.
Clin Cancer Res
2010
;
16
:
2927
31
.
26.
Pchejetski
D
,
Golzio
M
,
Bonhoure
E
,
Calvet
C
,
Doumerc
N
,
Garcia
V
, et al
Sphingosine kinase-1 as a chemotherapy sensor in prostate adenocarcinoma cell and mouse models
.
Cancer Res
2005
;
65
:
11667
75
.
27.
Waters
JE
,
Astle
MV
,
Ooms
LM
,
Balamatsias
D
,
Gurung
R
,
Mitchell
CA
. 
P-Rex1—a multidomain protein that regulates neurite differentiation
.
J Cell Sci
2008
;
121
:
2892
903
.
28.
Boyer
L
,
Turchi
L
,
Desnues
B
,
Doye
A
,
Ponzio
G
,
Mege
JL
, et al
CNF1-induced ubiquitylation and proteasome destruction of activated RhoA is impaired in Smurf1-/- cells
.
Mol Biol Cell
2006
;
17
:
2489
97
.
29.
Brizuela
L
,
Dayon
A
,
Doumerc
N
,
Ader
I
,
Golzio
M
,
Izard
JC
, et al
The sphingosine kinase-1 survival pathway is a molecular target for the tumor-suppressive tea and wine polyphenols in prostate cancer
.
FASEB J
2010
;
24
:
3882
94
.
30.
Ben Sahra
I
,
Laurent
K
,
Giuliano
S
,
Larbret
F
,
Ponzio
G
,
Gounon
P
, et al
Targeting cancer cell metabolism: the combination of metformin and 2-deoxyglucose induces p53-dependent apoptosis in prostate cancer cells
.
Cancer Res
2010
;
70
:
2465
75
.
31.
Yamazaki
D
,
Kurisu
S
,
Takenawa
T
. 
Regulation of cancer cell motility through actin reorganization
.
Cancer Sci
2005
;
96
:
379
86
.
32.
Sanz-Moreno
V
,
Gadea
G
,
Ahn
J
,
Paterson
H
,
Marra
P
,
Pinner
S
, et al
Rac activation and inactivation control plasticity of tumor cell movement
.
Cell
2008
;
135
:
510
23
.
33.
Schmitz
AA
,
Govek
EE
,
Bottner
B
,
Van Aelst
L
. 
Rho GTPases: signaling, migration, and invasion
.
Exp Cell Res
2000
;
261
:
1
12
.
34.
Manser
E
,
Loo
TH
,
Koh
CG
,
Zhao
ZS
,
Chen
XQ
,
Tan
L
, et al
PAK kinases are directly coupled to the PIX family of nucleotide exchange factors
.
Mol Cell
1998
;
1
:
183
92
.
35.
Dwivedi
S
,
Pandey
D
,
Khandoga
AL
,
Brandl
R
,
Siess
W
. 
Rac1-mediated signaling plays a central role in secretion-dependent platelet aggregation in human blood stimulated by atherosclerotic plaque
.
J Transl Med
2010
;
8
:
128
.
36.
Balamatsias
D
,
Kong
AM
,
Waters
JE
,
Sriratana
A
,
Gurung
R
,
Bailey
CG
, et al
Identification of P-Rex1 as a novel Rac1-guanine nucleotide exchange factor (GEF) that promotes actin remodeling and GLUT4 protein trafficking in adipocytes
.
J Biol Chem
2011
;
286
:
43229
40
.
37.
Nagasawa
SY
,
Takuwa
N
,
Sugimoto
N
,
Mabuchi
H
,
Takuwa
Y
. 
Inhibition of Rac activation as a mechanism for negative regulation of actin cytoskeletal reorganization and cell motility by cAMP
.
Biochem J
2005
;
385
:
737
44
.
38.
Li
H
,
Yang
L
,
Fu
H
,
Yan
J
,
Wang
Y
,
Guo
H
, et al
Association between Galphai2 and ELMO1/Dock180 connects chemokine signalling with Rac activation and metastasis
.
Nat Commun
2013
;
4
:
1706
.
39.
Rosenkilde
MM
,
Gerlach
LO
,
Jakobsen
JS
,
Skerlj
RT
,
Bridger
GJ
,
Schwartz
TW
. 
Molecular mechanism of AMD3100 antagonism in the CXCR4 receptor: transfer of binding site to the CXCR3 receptor
.
J Biol Chem
2004
;
279
:
3033
41
.
40.
Esfahanian
N
,
Shakiba
Y
,
Nikbin
B
,
Soraya
H
,
Maleki-Dizaji
N
,
Ghazi-Khansari
M
, et al
Effect of metformin on the proliferation, migration, and MMP-2 and -9 expression of human umbilical vein endothelial cells
.
Mol Med Report
2012
;
5
:
1068
74
.
41.
Gao
LB
,
Tian
S
,
Gao
HH
,
Xu
YY
. 
Metformin inhibits glioma cell U251 invasion by downregulation of fibulin-3
.
Neuroreport
2013
;
24
:
504
8
.
42.
Hashimoto
Y
,
Parsons
M
,
Adams
JC
. 
Dual actin-bundling and protein kinase C-binding activities of fascin regulate carcinoma cell migration downstream of Rac and contribute to metastasis
.
Mol Biol Cell
2007
;
18
:
4591
602
.
43.
Li
A
,
Dawson
JC
,
Forero-Vargas
M
,
Spence
HJ
,
Yu
X
,
Konig
I
, et al
The actin-bundling protein fascin stabilizes actin in invadopodia and potentiates protrusive invasion
.
Curr Biol
2010
;
20
:
339
45
.
44.
Schoumacher
M
,
Goldman
RD
,
Louvard
D
,
Vignjevic
DM
. 
Actin, microtubules, and vimentin intermediate filaments cooperate for elongation of invadopodia
.
J Cell Biol
2010
;
189
:
541
56
.
45.
Engers
R
,
Springer
E
,
Michiels
F
,
Collard
JG
,
Gabbert
HE
. 
Rac affects invasion of human renal cell carcinomas by upregulating tissue inhibitor of metalloproteinases (TIMP)-1 and TIMP-2 expression
.
J Biol Chem
2001
;
276
:
41889
97
.
46.
Feng
H
,
Hu
B
,
Liu
KW
,
Li
Y
,
Lu
X
,
Cheng
T
, et al
Activation of Rac1 by Src-dependent phosphorylation of Dock180(Y1811) mediates PDGFRalpha-stimulated glioma tumorigenesis in mice and humans
.
J Clin Invest
2011
;
121
:
4670
84
.
47.
Hwang
SY
,
Jung
JW
,
Jeong
JS
,
Kim
YJ
,
Oh
ES
,
Kim
TH
, et al
Dominant-negative Rac increases both inherent and ionizing radiation-induced cell migration in C6 rat glioma cells
.
Int J Cancer
2006
;
118
:
2056
63
.
48.
Uhlenbrock
K
,
Eberth
A
,
Herbrand
U
,
Daryab
N
,
Stege
P
,
Meier
F
, et al
The RacGEF Tiam1 inhibits migration and invasion of metastatic melanoma via a novel adhesive mechanism
.
J Cell Sci
2004
;
117
:
4863
71
.
49.
Zhang
JY
,
Zhang
D
,
Wang
EH
. 
Overexpression of small GTPases directly correlates with expression of delta-catenin and their coexpression predicts a poor clinical outcome in nonsmall cell lung cancer
.
Mol Carcinog
2013
;
52
:
338
47
.
50.
Mayeenuddin
LH
,
Garrison
JC
. 
Phosphorylation of P-Rex1 by the cyclic AMP-dependent protein kinase inhibits the phosphatidylinositiol (3,4,5)-trisphosphate and Gbetagamma-mediated regulation of its activity
.
J Biol Chem
2006
;
281
:
1921
8
.
51.
Ninkovic
J
,
Roy
S
. 
Morphine decreases bacterial phagocytosis by inhibiting actin polymerization through cAMP-, Rac-1-, and p38 MAPK-dependent mechanisms
.
Am J Pathol
2012
;
180
:
1068
79
.
52.
Kizaki
H
,
Nakada
S
,
Ohnishi
Y
,
Azuma
Y
,
Mizuno
Y
,
Tadakuma
T
. 
Tumour necrosis factor-alpha enhances cAMP-induced programmed cell death in mouse thymocytes
.
Cytokine
1993
;
5
:
342
7
.
53.
Naderi
EH
,
Ugland
HK
,
Diep
PP
,
Josefsen
D
,
Ruud
E
,
Naderi
S
, et al
Selective inhibition of cell death in malignant vs. normal B-cell precursors: implications for cAMP in development and treatment of BCP-ALL
.
Blood
2013
;
121
:
1805
13
.
54.
Han
JD
,
Rubin
CS
. 
Regulation of cytoskeleton organization and paxillin dephosphorylation by cAMP. Studies on murine Y1 adrenal cells
.
J Biol Chem
1996
;
271
:
29211
5
.
55.
Hadad
S
,
Iwamoto
T
,
Jordan
L
,
Purdie
C
,
Bray
S
,
Baker
L
, et al
Evidence for biological effects of metformin in operable breast cancer: a pre-operative, window-of-opportunity, randomized trial
.
Breast Cancer Res Treat
2011
;
128
:
783
94
.
56.
Miller
RA
,
Chu
Q
,
Xie
J
,
Foretz
M
,
Viollet
B
,
Birnbaum
MJ
. 
Biguanides suppress hepatic glucagon signalling by decreasing production of cyclic AMP
.
Nature
2013
;
494
:
256
60
.
57.
El-Mir
MY
,
Nogueira
V
,
Fontaine
E
,
Averet
N
,
Rigoulet
M
,
Leverve
X
. 
Dimethylbiguanide inhibits cell respiration via an indirect effect targeted on the respiratory chain complex I
.
J Biol Chem
2000
;
275
:
223
8
.
58.
Carretero-Ortega
J
,
Walsh
CT
,
Hernandez-Garcia
R
,
Reyes-Cruz
G
,
Brown
JH
,
Vazquez-Prado
J
. 
Phosphatidylinositol 3,4,5-triphosphate-dependent Rac exchanger 1 (P-Rex-1), a guanine nucleotide exchange factor for Rac, mediates angiogenic responses to stromal cell-derived factor-1/chemokine stromal cell derived factor-1 (SDF-1/CXCL-12) linked to Rac activation, endothelial cell migration, and in vitro angiogenesis
.
Mol Pharmacol
2010
;
77
:
435
42
.
59.
Bost
F
,
Ben Sahra
I
,
Le Marchand-Brustel
Y
,
Tanti
JF
. 
Metformin and cancer therapy
.
Curr Opin Oncol
2012
;
24
:
103
8
.
60.
Cho
H
,
Herzka
T
,
Zheng
W
,
Qi
J
,
Wilkinson
JE
,
Bradner
JE
, et al
RapidCaP, a novel GEM model for metastatic prostate cancer analysis and therapy, reveals myc as a driver of Pten-mutant metastasis
.
Cancer Discov
2014
;
4
:
318
33
.