Metformin inhibits melanoma development through autophagy and apoptosis mechanisms

Metformin is the most widely used antidiabetic drug because of its proven efficacy and limited secondary effects. Interestingly, recent studies have reported that metformin can block the growth of different tumor types. Here, we show that metformin exerts antiproliferative effects on melanoma cells, whereas normal human melanocytes are resistant to these metformin-induced effects. To better understand the basis of this antiproliferative effect of metformin in melanoma, we characterized the sequence of events underlying metformin action. We showed that 24 h metformin treatment induced a cell cycle arrest in G0/G1 phases, while after 72 h, melanoma cells underwent autophagy as demonstrated by electron microscopy, immunochemistry, and by quantification of the autolysosome-associated LC3 and Beclin1 proteins. In addition, 96 h post metformin treatment we observed robust apoptosis of melanoma cells. Interestingly, inhibition of autophagy by knocking down LC3 or ATG5 decreased the extent of apoptosis, and suppressed the antiproliferative effect of metformin on melanoma cells, suggesting that apoptosis is a consequence of autophagy. The relevance of these observations were confirmed in vivo , as we showed that metformin treatment impaired the melanoma tumor growth in mice, and induced autophagy and apoptosis markers. Taken together, our data suggest that metformin has an important impact on melanoma growth, and may therefore be beneficial in patients with melanoma.

To elucidate whether AMPK activation is modulated in response to metformin, we studied the phosphorylation at Thr-172 of AMPK- α (AMPK T172) as a read-out of its activation state. Figure 2a shows that metformin induced a dose-dependent increase of AMPK T172 phosphorylation in A375 melanoma cell lines. We next examined the effect of metformin on the activity of mTOR, a downstream effector of AMPK. We observed a net decrease in the phosphorylation status of mTOR and S6 Ribosomal protein, suggesting a blockade of the mTOR pathway. As the mTOR pathway is a central regulator of multiple cellular responses to nutrient and growth factor signaling, including autophagy, we analyzed whether metformin might induce characteristic hallmarks of autophagy. During autophagy, LC3-I is converted to LC3-II through the ATG4-dependent insertion of a phosphoethanolamine moiety and recruited into the membrane of the forming phagophore, a double membrane required for the recycling of protein aggregates and organelles. 16 Increased LC3 expression was detected for two doses of metformin. At the same concentrations, metformin also enhanced the expression level of Beclin 1, a protein required for the initiation of the formation of the autophagosome in autophagy. These data collectively strongly argue the induction of macroautophagy by metformin. Accordingly, electron microscopy images of A375 cells treated for 72 h with metformin showed typical images of autophagy including accumulation of numerous vesicles with distinct double membrane ( Figure 2b ). In addition, early autophagosomes sequestering cytosol, mitochondrion, or endoplasmic reticulum membranes were observed. By contrast, control melanoma cells (PBS) showed no autophagic vacuoles. Similar results were obtained with SKMel28 melanoma cells (Supplementary Figure S2). In addition, following transfection of melanoma cells with RFP-LC3 construct, the formation of RFP puncta in cells treated with metformin was observed, whereas the untreated cells exhibited a diffuse distribution of red fluorescence in the cytoplasm and nucleus ( Figure 2c ). The observation that metformin affected the lysosomal compartment was further substantiated by the increase of cathepsin B activity mediated by metformin at 72 h in A375 and SKMel28 cells ( Figure 2d ). Importantly, increased cathepsin B activity has been associated with the late steps of autophagy. 17

The anticancer effects of metformin have been essentially attributed to its ability to activate the AMPK pathway. To evaluate the function of AMPK in the effects of metformin, we knockdown both α 1 and α 2 catalytic subunits of AMPK with previously validated siRNAs. 18 As shown in Figures 5a and b , inhibition of α 1 and α 2 AMPK subunits partially prevented cell death, and PARP cleavage induced by 5 and 10 mM metformin. Further, AMPK siRNA inhibited partially LC3 cleavage at 5 mM metformin but not at 10 mM metformin. These observations suggest that metformin mediates its anti-melanoma effects via both AMPK-dependent and -independent pathways.

The discovery of new therapeutic compounds is a very important challenge to treat advanced melanomas that are resistant to existing therapies. To this purpose, using in vitro and in vivo approaches, we demonstrate the potent anti-melanoma activity of the antidiabetic metformin. Interestingly, contrary to normal human melanocytes, drastic inhibition of cell viability mediated by metformin is observed in four different melanoma cell lines independent of the mutational status or melanoma development stage, and in two primary melanoma cell cultures freshly isolated from patients. These findings are consistent with results of Woodard et al. 19 demonstrating AMPK activators AICAR and metformin reduced the proliferation of SKMel2 and SKMel28 melanoma cells. Metformin action is mainly mediated by AMPK activation; however, several recent reports indicate that some effects of metformin in cancer cell lines could be mediated by an AMPK-independent pathway. 20 , 21 , 22 , 23 In our model, AMPK silencing by siRNA ( Figure 5 ) or inhibition of AMPK activation by pharmacological inhibitor (data not shown) inhibits only partially the effect of metformin on melanoma cell death, suggesting that the effects of this drug are mediated, at least in part, through an AMPK-independent mechanism. In addition, AMPK knockdown protects partially the cleavage of PARP but fails to block the induction of LC3-b at 10 mM metformin. This result indicates that AMPK-dependent component of anti-melanoma action of metformin could be autophagy-independent. However, we cannot exclude that the low residual AMPK expression is sufficient to mediate metformin effect on cell death. Noteworthy, treatment of normal human melanocytes that express endogenous AMPK with metformin did not affect cell viability ( Figure 1a ), suggesting that metformin action is restrained to transformed cell lines and likely reflects a tumor-specific regulation. The AMPK/mTOR pathway is under the control of LKB1, a serine-threonine kinase acting as a tumor suppressor. A recent report demonstrates that oncogenic V600E B-RAF, a common somatic mutation found in malignant melanoma (50–70%), negatively regulates LKB1 to promote melanoma cell proliferation. 24 In our model, LKB1 seems not to be involved in metformin's anti-melanoma effect as G361 cells, which do not express LKB1, are also sensitive to the drug ( Figure 1a ). Accordingly, overexpression of a dominant negative form of LKB1 in melanoma cell lines did not prevent metformin sensitivity (data not shown). Another protein that can mediate metformin effect is the tumor suppressor p53. Indeed, p53 functions as a transcriptional regulator of genes involved in cell cycle arrest, autophagy and apoptosis pathways. 25 In addition, the AMPK-p53 pathway may represent a cell cycle checkpoint in response to energy limitation. AMPK has been shown to phosphorylate p53 on Ser-15 to induce G0/G1 phase arrest. 26 In our melanoma cell lines, p53 is not phosphorylated on Ser-15 in response to metformin (data not shown). Further melanoma cells harboring a mutated TP53 gene (SKMel28 cells) exhibited the same sensitivity to metformin as other melanoma cell lines harboring wild-type p53 ( Figure 1a ), indicating that p53 is not involved in metformin effects in our cellular model. Several recent reports have highlighted that metformin induces cell cycle arrest of cancer cell lines. 7 We observed here that the proportion of cells in G0/G1 was significantly increased in melanoma cells treated for 24 h with metformin, while apoptotic cells with fragmented DNA (sub G1) could not be observed at 24 h (Supplementary Figures S1a and b). Cell cycle arrest in G0/G1 phase is associated at molecular level with a dramatic reduction of cyclin D1 expression, a marked increase in the expression of cyclin-dependent kinase inhibitor p21Cip1/Waf1, and hypophosphorylation of pRb (Supplementary Figure S1c). However, the antiproliferative effect of metformin cannot be exclusively explained by cell cycle arrest as a strong increase in cell death was detected for longer time of drug treatment (>48 h; Figure 3a ). Autophagy is a catabolic process for the degradation and recycling of macromolecules and organelles, which is activated during stress conditions. 27 , 28 Autophagy is initiated by the formation of double-membraned vesicles called autophagosomes, which fuse with lysosomes to form autophagolysosomes in which lysosomal hydrolases digest the vesicle contents for recycling. 29 Autophagy is considered as a survival mechanism induced in adverse conditions to maintain cell integrity, but paradoxically, it is also involved in a particular mode of death called autophagic cell death or type II cell death. 30 In our study, several compelling evidences indicate that metformin induces autophagy in melanoma cells ( Figure 2 ). This finding is particularly interesting as until now, only one study has reported that metformin is able to induce autophagy in colon cancer. 14 In

Normal human melanocytes were prepared and maintained as described. 36 Different melanoma cell lines were purchased from American Tissue Culture Collection (Molsheim, France). Cells were grown in RPMI 1640 (A375, WM9 and SKMel28) or in DMEM medium (G361 and B16) supplemented with 10% FCS and penicillin/streptomycin (100 U/ml/50 μ g/ml) at 37°C and 5% CO 2 . Patient melanoma cells were prepared as described. 37 Briefly, biopsy was dissected and digested for 1–2 h with collagenase A (0.33 U/ml), dispase (0.85 U/ml) and Dnase I (144 U/ml) with rapid shaking at 37°C. Large debris were removed by filtration through a 70- μ m cell strainer. Viable cells were obtained by Ficoll gradient centrifugation. For each experiment, cells were starved with or without 1% SVF in appropriate medium during 14 h before drug stimulation.

Electron microscopy experiments were performed as described. 39

All flow cytometry analyses were performed using the FL2 channels of a FACScan (Becton Dickinson, Cowley, UK) and data were analyzed with CellQuest software (Becton Dickinson, Cowley, UK) as previously described. 38 PI staining was performed using Staining Kit (Roche Diagnostics, Meylan, France) according to the manufacturer's protocol.

Cathepsin B activity was determined as described. 41

Data presented are mean ±S.D. of three independent experiments performed in triplicate. Statistical significance was assessed using the Student's t -test except for in vivo experiments in which statistical significance was assessed using two-tailed Wilcoxon rank sum test. A value of P <0.05 was accepted as statistically significant.