Metformin Blocks Melanoma Invasion and Metastasis ... · Cancer Therapeutics Insights Metformin Blocks Melanoma Invasion and Metastasis Development in AMPK/p53-Dependent Manner Micha€el

Cancer Therapeutics Insights

Metformin Blocks Melanoma Invasion and MetastasisDevelopment in AMPK/p53-Dependent Manner

Micha€el Cerezo1,2, M�elanie Tichet3,2, Patricia Abbe1,2, Micka€el Ohanna1,2, Abdelali Lehraiki1,2,Florian Rouaud1,2, Maryline Allegra1,4, Damien Giacchero4, Philippe Bahadoran1,4, Corine Bertolotto1,2,4,Sophie Tartare-Deckert3,2, Robert Ballotti1,2,4, and St�ephane Rocchi1,2,4

AbstractMetformin was reported to inhibit the proliferation of many cancer cells, including melanoma cells. In this

report, we investigated the effect of metformin on melanoma invasion and metastasis development. Using

different in vitro approaches, we found that metformin inhibits cell invasion without affecting cell migration

and independently of antiproliferation action. This inhibition is correlated with modulation of expression of

proteins involved in epithelial–mesenchymal transition such as Slug, Snail, SPARC, fibronectin, and N-

cadherin andwith inhibition ofMMP-2 andMMP-9 activation. Furthermore, our data indicate that this process

is dependent on activation of AMPK and tumor suppressor protein p53. Finally, we showed that metformin

inhibits melanomametastasis development inmice using extravasation andmetastasis models. The presented

data reinforce the fact that metformin might be a good candidate for clinical trial in melanoma treatment.

Mol Cancer Ther; 12(8); 1605–15. �2013 AACR.

IntroductionMetastatic melanoma is one of the most aggressive and

highly proliferative human malignancies with a mediansurvival of only 6 to 9 months once distant sites becomeseeded from skin (1). Typically, primary lesions progressto malignant tumors through a multistep process includ-ing dysplasia, radial growth phase (RGP), invasive verti-cal growth phase (VGP), and metastasis.Melanoma is a neoplasm of neuroectodermal origin;

this melanoma cells may not undergo classic epithelial–mesenchymal transition (EMT)-like changes. However,their ability to invade into the dermis is associatedwith anEMT-like phenotype characterized by changes in expres-sion of cell–cell adhesion molecules as the cadherin fam-ily. During malignant transformation, there is loss ofexpression of E-cadherin in favor of the N-cadherin (2,3). These changes allow the melanocytes to escape kera-tinocytes control, and after crossing the stratum basale, tointeract with new cell types such as fibroblasts or vascular

endothelial cells which promote tumor progression andmetastasis. Melanocytes cross the basal lamina by secret-ing matrix metalloproteinases (MMP) such as collage-nases MMP-2 and MMP-9, which will degrade a majorconstituent of the basal lamina, collagen IV. Several tran-scription factors that belong to the Snail superfamily ofzing finger transcription factors including Snail/SNAI1and Slug/SNAI2 are involved in this mechanism. Forexample, these 2 proteins are central regulators of EMTduring neural crest cell migration and cancer (4, 5). Inaddition, itwas shown inmelanoma that Slug functions asa melanocyte-specific factor required for the strong met-astatic propensity of this tumor (6). More interesting, Slugis a p53 target that antagonizes p53-mediated apoptosis(7) and invasion (8). Elevated mortality caused by mela-noma is attributed to its strong propensity to form distalmetastases in organs, such as lung, liver, brain, and bones,and its notorious resistance to all current therapies (9). Thenew important challenge is thus to discover new thera-peutic drugs that inhibit melanoma cell proliferation butalso exhibit antimetastatic properties.

The oral antidiabetic drug metformin belongs to thefamily of biguanides and is the most widely used antidi-abetic drug in the world. This drug has been shown toinhibit the energy-sensitive AMPK/mTOR signalingpathway that leads to reduced protein synthesis and cellproliferation. Recent studies indicate that metformin canpotentially be used as an efficient anticancer drug invarious neoplasms such as prostate carcinoma, breast,lung, and pancreas cancers (10, 11). These results wereconfirmed by retrospective epidemiologic studies thatreported adecrease in cancer risk in patientswithdiabetestreated with metformin (12). In addition, metformin was

Authors' Affiliations: 1Equipe Biologie et Pathologie des cellulesm�elanocytaire: de la pigmentation cutan�ee au m�elanome, CentreM�editerran�een de M�edecine Mol�eculaire (C3M), INSERM, U1065; 2UFRde M�edecine, Universit�e de Nice Sophia Antipolis; 3INSERM, U1065; and4Service de Dermatologie, Hopital Archet II, CHU Nice, France

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

CorrespondingAuthor:St�ephaneRocchi, INSERMU1065, team1,CentreM�editerran�een deM�edecineMol�eculaire (C3M), Batiment ARCHIMED, 151route de Saint Antoine de Ginesti�ere, 06204 Nice cedex 3, France. Phone:33-4-89-06-43-33; Fax: 33-4-89-06-43-33; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-12-1226-T

�2013 American Association for Cancer Research.

MolecularCancer

Therapeutics

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on May 6, 2021. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst June 5, 2013; DOI: 10.1158/1535-7163.MCT-12-1226-T

http://mct.aacrjournals.org/

reported by several groups, including ours, to inhibitmelanoma cell proliferation (13–16). In our previousstudy, we showed that metformin dramatically impairsthe growth of melanoma tumors in vitro and in vivo byinducing autophagic cell death leading to massive apo-ptosis (13). Interestingly, in the present study, we showalso that metformin, independently of its effect on mela-noma cell survival, could display anti-invasive and anti-metastatic properties.

Materials and MethodsReagents and antibodies

Metformin was purchased from Sigma-Aldrich. Dul-becco’s Modified Eagle Medium (DMEM), penicillin/streptomycin, and trypsin were from Invitrogen andfetal calf serum (FCS) from Hyclone. Slug, Snail, p53,HSP90, AMPKa1, and AMPKa2 antibodies were pur-chased from Santa Cruz Biotechnology (TEBU). Anti-AMPKa and phospho-acetyl coA carboxylase (Ser79)antibodies were from Cell Signaling. Antibodies againstfibronectin were from BD Biosciences. Antibody tohuman SPARC was purchased from Hematologic Tech-nologies. Antibody to human N-Cadherin was pur-chased from Invitrogen. Antibody to human S100 waspurchased from Abcam).

Cell linesAllmelanoma cell lineswere purchased fromAmerican

Tissue Culture Collection (ATCC) and used within 6months between resuscitation and experimentation. TheATCC authentication protocols include testing for myco-plasma, bacteria, fungi contamination, confirmation ofspecies identity, and detection of cellular contaminationor misidentification using COI for interspecies identifica-tion and DNA profiling as well as cytogenetic analysis,flow cytometry, and immunocytochemistry with consis-tent refinement of cell growth conditions as well as doc-umentation systems, ensuring traceability. Cells weregrown in RPMI-1640 (A375, WM9, and SKMel28) or inDMEM (1205Lu, Mel501, andMewo) supplemented with10% FCS and penicillin/streptomycin (100 U/mL/50mg/mL) at 37�C and 5% CO2. Patient melanoma cellswere prepared as described (13) after obtaining informedconsent from this patient. Briefly, biopsy was dissectedand digested for 1 to 2 hours with collagenase A (0.33 U/mL), dispase (0.85 U/mL), and DNase I (144 U/mL) withrapid shaking at 37�C. Large debris were removed byfiltration through a 70-mm cell strainer. Viable cells wereobtained by Ficoll gradient centrifugation.

siRNA transfectionTransfection of duplex siRNAs (50nmol/L)was carried

out using Lipofectamine RNAiMAX (Invitrogen) in Opti-MEM (Invitrogen) as described (17). The day after trans-fection,metforminwas added to themediumandproteinswere extracted 24 hours after the addition of metformin.Stealth siRNA targeting AMPKa1, AMPKa2, and p53were purchased from Invitrogen, whereas AMPK siRNA

were from Dharmacon. As nonspecific control, scramblesequence siRNAs were used.

Infection with adenovirusAdenoviruses encoding a dominant-negative form (Ad

AMPK-DN) of subunits a1 and a2 of AMPK were agenerous gift of Dr. Foufelle (INSERM, UMR-S 872, Paris,France). An adenovirus of which the expression cassettecontains the major late promoter with no exogenous genewas used as control (Ad control). Adenoviruses werepropagated in human embryonic kidney 293 cells andstored at �80�C. 1205Lu cells were infected for 24 hourswith the Ad AMPK-DN before the metformin treatment.

Luciferase assaysMelanoma cells were seeded in a 24-well plate, and

transient transfections were conducted the following dayusing 2mLLipofectamine (Gibco-BRL) and 0.3mgof PG13-Luc, a p53-dependent firefly luciferase reporter gene in a200-mL final volume. pCMVbGal plasmid was cotrans-fected to control the variability of transfection efficiency inthe reporter assays. The day after the transfection, met-formin was added to the medium. At 24 hours afterstimulation, cells were harvested in 50 mL of lysis bufferand soluble extracts assayed for luciferase and b-galacto-sidase activities. All transfections were repeated severaltimes using different plasmid preparations. Luciferaseassays were conducted exactly as described (18).

Western blot assaysProtein were extracted in buffer containing 50 mmol/L

Tris HCl, pH 7.5, 15 mmol/L NaCl, 1% Triton X-100, and1� protease and phosphatase inhibitors. Briefly, celllysates (30mg) were separated by SDS-PAGE, transferredonto a polyvinylidene fluoride (PVDF) membrane (Milli-pore), and then exposed to the appropriate antibodies.Proteins were visualized with the ECL System fromAmersham. The Western blot analyses shown are repre-sentative of at least 3 independent experiments. Quanti-fication of Western blot analyses was conducted usingImageJ software.

Coimmunoprecipitation assaysFor the co-immunoprecipitation experiments, 1205Lu

melanoma cells were treated for 24 hours with metformin10mmol/L and lysed in Fischer buffer. Fiftymicroliters ofprotein G agarose (Invitrogen) was mixed with 2 mg ofmonoclonal anti-p53 antibody for 2 hours at 4�C. Thenlysates were added and mixed at 4�C overnight. Theimmunoprecipitated complexes were analyzed by 10%SDS-PAGE and immunoblotted using anti-AMPKa anti-body and anti-p53 antibody.

Invasion assaysBoyden chambers (8.0-mm pores, Transwell; Corning,

Inc.) were coated with 1 mg/mL Matrigel (BD Bios-ciences) and were placed into 24-well chambers contain-ing medium supplemented with 10% FCS. The cells were

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Mol Cancer Ther; 12(8) August 2013 Molecular Cancer Therapeutics1606




resuspended in FCS-starved medium, loaded into the topchamber. Five hours later, adherent cells to the undersideof the filters were fixed with 4% paraformaldehyde (PFA)and stained with 0.4% crystal violet, and 5 random fieldsat� 20magnification were counted. Results represent theaverage of triplicate samples from 3 independentexperiments.

Three-dimensional spheroid growthMelanoma spheroids were prepared using the liquid

overlay method. Briefly, 500 mL of melanoma cells(20,000/mL) were added to a 24-well plate coated with1.5% agar (Difco). Plateswere left to incubate for 72 hours;by this time, cells had organized into 3-dimensional (3D)spheroids. Spheroids were then harvested using a P1000pipette. The medium was removed and the spheroidswere implanted into a gel of bovine collagen I containingMEM (Invitrogen). Normalmelanomamediumwas over-laid on top of the solidified collagen. After different times,pictures of the invading spheroids were taken using aZeiss microscope.

MMP activity measurementThe culturemedia from stimulated cellswere harvested

and incubated in a 96-well platewith 0.2mmol/L ofNH2-RA-Dpa-LGLP-AMC as a substrate for various times at37�C. MMP activity was measured in quadruplicate byquantifying the emission at 460 nm (excitation at 390 nm)in the presence or absence of 10 mmol/L CP471474. Theenzyme activities were expressed in arbitrary units permg of protein.

Substrate zymographyThe culture media from 1205Lu melanoma cells were

concentrated in centrifugal filter unit and loaded on 10%SDS-PAGE containing 1 mg/mL type I collagen (BDBiosciences). To estimate the protein concentration,1205Lu melanoma cells were lysed in a buffer containing1% Triton X-100, 150 mmol/L NaCl, and 20mmol/L Tris,pH 7.4, supplemented with a protease inhibitor mixture(Complete EDTA-free, Roche Molecular Biochemicals) at4�C under agitation for 30 minutes. Lysates were clarifiedby brief spinning, and protein concentration was evalu-ated by bicinchoninic acid technique (BCA protein assaykit, Pierce).Following electrophoresis, proteins were renatured by

incubating gels in 2.5% Triton X-100 for 2 hours at 37 �C.Gels were then washed 3 times in distilled water andincubated in substrate buffer (50mmol/LTris, pH7.4, and1mmol/LCaCl2) at 37

�C for 24 hourswith gentle shaking.Gels were stained with 0.1% Coomassie Blue R-250 (Sig-ma) and destained in 7% acetic acid. Enzymatic activitiesappear as cleared bands in a dark background.

In vivo studies1205Lu cells stably transfected with a vector encoding

luciferase come fromDr. Tartare-Deckert team.A total of 1� 106/150 mL PBS 1205Lu-Luc cells were injected via the

tail vein of nude mice (Harlan Laboratories). The micewere treated with or without intraperitoneal injection of60 mg/kg metformin every day. Melanoma cells werevisualized in the animal after intraperitoneal injection of50 mg/kg luciferin (Caliper Life Sciences) by biolumines-cence imaging using a Photon Imager (Biospace Lab).Mice were sacrificed and the lungs were excised, fixed,and serially sectioned. S100 (1:100) andSlug (1:100) immu-nostaining was conducted.

To conduct pulmonary extravasation analysis, 1.5� 106

1205Lu cells were labeled for 1 hour with CellTrackerGreen (Invitrogen) and injected via the tail vein of nudemice. After 24 hours, mice were sacrificed, and the lungswere harvested for analysis with a Zeiss Inverted scope.

Statistical analysisResults are presented as mean � SE with experiment

numbers indicated in the figure legends. Statistical sig-nificance was assessed using the Student t test. P � 0.05was accepted as statistically significant.

ResultsMetformin inhibits cell invasion but not cellmigration

We previously showed that the antidiabetic drug met-formin (Fig. 1A) induced melanoma cell death after long-term treatment of 96 hours (13). We now wanted todetermine whether metformin was able to inhibit migra-tion and invasion properties of melanoma cells at earlytimes. As presented in cell migration assay using Boydenchambers (Supplementary Fig. S1A and S1B), metformindid not inhibit migration of 1205Lu and A375 melanomacell lines after 24 hours. Results were confirmed usingwound healing assay (Supplementary Fig. S1C). We nextdetermined themetformin capacity to inhibit cell invasionusing Boyden chambers coated with Matrigel (Fig. 1B).Metformin decreases cell invasion in a dose-dependentmanner in melanoma cell lines 1205Lu, A375, and WM9.At concentration of 10 mmol/L, metformin inhibits by95%, 90%, and 60% cell invasion in 1205Lu, A375, andWM9 cells, respectively. Similar results were obtainedwith cells freshly isolated from patients (Fig. 1B).

To determine whether these anti-invasive effects ofmetformin are apoptosis mediated, we carried out anexperiment in which we analyzed apoptosis in our con-ditions. As presented in Supplementary Fig. S2, weobserved in all tested cell lines, absence of PARP cleavageand decrease of procaspase-3 expression in response tometformin. As expected, apoptosis control staurosporineinduces cleavage of PARP and decrease of procaspase-3expression. These results indicated that the anti-invasiveeffect of metformin is not mediated by apoptosis.

Tumor invasion was then analyzed in a more physio-logic context; WM9 melanoma cells were grown asspheroids embedded in collagen. Metformin significantlyreduced cell invasion into collagen (Fig. 1C). To confirmthat invasion inhibition is not due to apoptosis inducedby metformin, we performed the same experiment in

Metformin Inhibits Melanoma Invasion

www.aacrjournals.org Mol Cancer Ther; 12(8) August 2013 1607




presence of apoptosis inhibitor, QVD. As expected, con-trary to QVD alone, association of QVD with metforminblocks invasion, indicating that apoptosis does notaccount for the inhibitory effects on cell invasion medi-ated by metformin.

Metformin decreases expression of proteins involvedin EMT

To determine proteins involved in the inhibition ofinvasionmediated bymetformin, we checked byWesternblot analysis expression of proteins involved in EMT.

Figure 2A indicates that metformin inhibited in a dose-dependentmanner expression of key proteins involved inthis process such as fibronectin, N-cadherin, or SPARC in1205Lu melanoma cells (Fig. 2A). In contrast, vimentinexpressionwas notmodified bymetformin. Levels of both

transcription factors Slug and Snail that initiate EMTwerealso decreased.

Similar results were found in A375 melanoma cellsand in patient-isolated melanoma cells (Fig. 2B and C,respectively).

Metformin inhibits activation of matrix MMPs inmelanoma cells

We next examined MMP activities in melanoma cellstreated with metformin. Total MMP activity level wasassessed using a broad-spectrum fluorogenic MMP sub-strate on 1205Lu melanoma cells treated by metformin(Fig. 3A). Metformin (10 mmol/L) induced a slight butsignificant decrease of approximately 30% of total MMPactivities. In addition, cell-associated metalloproteinaseactivities were assessed by type I collagen substrate gel

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Figure 1. Effects of metformin onmelanoma invasion. A, structure of metformin. B, invasion assay in coated Boyden chambers with 1205Lu, A375, WM9, andfreshly isolated from patient melanoma cells treated for 24 hours with metformin (Metf; at the indicated concentrations) or PBS was conducted. The resultsare expressed as percentages of the control. The bars indicate the mean � SD of triplicate samples. �, P < 0.05; ��, P < 0.01; ��, P < 0.001. C, 3D spheroidgrowth assay using WM9 melanoma cells treated with 10 mmol/L of metformin or PBS for 4 days was conducted. QVD was used at 10 mmol/L.

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zymography (Fig. 3B). Collagen zymography allowed thedetection of enzymatic activities at 82 and 62 kDa that areconsistent with active forms of MMP-9 and MMP-2,respectively. In basal condition (PBS), activities were highfor both MMPs. Quantification shows an important dim-inution in response to 5mmol/Lmetformin for bothMMPto reach 80% and 45% of decrease at 10 mmol/L metfor-min for MMP-2 and MMP-9, respectively (Fig. 3C).

AMPK is involved in inhibition of invasion mediatedby metforminTo determine whether AMPK activation plays a role in

inhibition of invasion bymetformin,we abrogatedAMPKactivation by metformin using infection of dominant-negative forms of AMPK adenoviruses (AdAMPK DN)in 1205Lu melanoma cells (Fig. 4). As expected, infectionof AdAMPK DN a1 and a2 increases the expression ofAMPK a1 and a2 indicating that dominant-negativeforms of AMPK are expressed in the cells. Furthermore,basal and metformin-stimulated phosphorylation ofdirect AMPK substrate, acetyl coA carboxylase is abol-ished in cells infected by AdAMPK DN a1 and a2 show-ing that AMPK activation is inhibited.In parallel, we observed that Slug and SPARC are

inhibited in response to metformin in cells infected withAd control. In contrast, expression of AMPK DN con-structs abrogates these inhibitory effects.Finally, we tested metformin capacity to inhibit inva-

sion using Boyden chambers in presence (Ad control) or

absence (AdAMPK DN a1 and a2) of active AMPK.Interestingly, metformin-induced inhibition of invasionwas abolished in presence of dominant-negative forms ofAMPK. Taken together, these results suggest an implica-tion of AMPK in the inhibitory effects of metformin ininvasion.

Transcription factor p53 is involved in inhibition ofinvasion mediated by metformin

As AMPK is involved in p53 activation, we won-dered whether this transcription factor could play arole in inhibition of invasion mediated by metformin.First, we verified that in our system, metformin is ableto activate p53. For this, reporter assay using a pro-moter–luciferase construct that contains p53-bindingsites revealed that treatment with 5 and 10 mmol/Lmetformin led to approximately 10-fold and 20-foldinduction of p53 promoter activity, respectively (Fig.5A). As expected, actinomycin D (ActD) was used aspositive control of p53 activation leads to increase ofp53 promoter activity comparable with 10 mmol/Lmetformin.

We nextwanted to determinewhether uponmetforminstimulation of melanoma cells, AMPKa could associatewith p53 to induce p53 activation. We immunoprecipi-tated p53 from 1205Lu cells stimulated or not with met-formin (10 mmol/L) for 24 hours. Proteins were thenblotted with antibodies to either p53 or AMPKa (Fig.5B). In unstimulated conditions, p53 is poorly associated

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Figure 2. Effects of metformin on EMT markers. 1205Lu (A), A375 (B), and freshly isolated from patient melanoma cells (C) were treated for 24 hourswith metformin (at the indicated concentrations) or PBS, lysed, and analyzed by Western blotting with fibronectin, N-cadherin, vimentin, Sparc, Slug, andSnail antibodies. HSP90 was used for load control.






with AMPKa, but after metformin treatment, a largeincreased amount of p53 is co-immunoprecipitated withAMPKa. As control, total blots were presented andshowed no major modification of AMPKa level and adecrease in Slug, N-cadherin, and fibronectin expressionsin response to metformin. We conclude that in intact1205Lu melanoma cells, p53 associates with AMPKa ina metformin-dependent fashion.

Furthermore, we asked whether decreasing levels ofp53 could prevent inhibition of invasion induced bymetformin (Fig. 5C). We observed that siRNA-mediateddownregulation of p53 prevents downregulation of Slug(bottom) and inhibition of invasion (top) induced bymetformin. Similar results were obtained in another mel-anoma cell line, Mel501 stably transfected with shp53RNA (Supplementary Fig. S3).

Regarding a functional role for p53 in mediating anti-invasion properties of metformin, melanoma cells har-boring amutated TP53 gene (Mewo, SKmel28, andHMV2cells) exhibited resistance to metformin-mediated inhibi-tion of invasion using Western blot analyses and Boydenchamber assays (Fig. 5D and Supplementary Fig. S4A andS4B). Interestingly, re-expression of wild-type (WT) p53expression by adenoviruses infection inMewo cells resen-sitizes cells to metformin (Fig. 5D) and restores the inhi-bition of invasion and thedecrease in Slug andN-cadherinexpressions in response to metformin.

Our results show that inhibition of invasion induced bymetformin occurs through an AMPKa/p53-dependentmechanism.

Metformin inhibits melanoma metastasisdevelopment in mice using extravasation andmetastasis models

Finally, to assess a potential antimetastatic effect ofmetformin in vivo, extravasation and lung metastasismodels were conducted in immunodeficient nude mice.

Green-labeled human melanoma cells 1205Lu treatedor not 24 hours by metformin were injected in the caudalvein of 6-week-old female athymic nude mice, and theirability to extravasate through the pulmonaryparenchymawas evaluated (Fig. 6A). As shown in figure, the control1205Lu cells treated with PBS give much more microme-tastases in the lungs than 1205Lu cells treated by metfor-min. Quantification of experiments by counting extrava-sated cells using inverted microscope confirmed thisresult. In addition, similar experiment carried out withMewo cells harboring p53 mutation shows the incapacityof metformin to inhibit extravasation in lungs (Supple-mentary Fig. S5A), confirming the implication of p53 inthis process in vivo.

In another experiment, 1205Lu melanoma cells (1.5 �106) stably expressing luciferase were injected in caudalvein of 6-week-old female athymic nude mice and werethen treated daily with an intraperitoneal injection ofvehicle or metformin (2 mg/mouse/d) over a period of39 days (Fig. 6B). Seven days after cell injection, biolumi-nescence was detected in the lungs of all mice. Impor-tantly, a 3-fold increase in bioluminescence intensity wasobserved in the lungs of mice treated with vehicle com-paredwith lungs ofmice treated bymetformin. This result

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Figure 3. Effects of metformin on MMP. A, MMP activity was measured on the culture media of 1205Lu melanoma cells treated 24 hours with 10 mmol/L ofmetformin or PBS. The results are expressed in arbitrary units. The bars indicate themean�SDof triplicate samples. �,P<0.05. B,MMP-2 andMMP-9 activitywere evaluated by substrate zymographywith 1205Lumelanoma cells treated for 24 hours withmetformin (at the indicated concentrations). Cell lysates werenext loaded on SDS-PAGE containing type I collagen and stained with Coomassie blue. C, ImageJ quantifications of 3 independent experiment of substratezymography are shown. The results are expressed as percentages of the control. The bars indicate the mean � SD of triplicate samples. ��, P < 0.01;��, P < 0.001.

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reflects the decreased capacity of cells in mice treated bymetformin to metastase in lung in vivo. After 39 days,bioluminescence intensitywas veryweak in lungs ofmicetreated by metformin in comparison of lungs of controlmice. This inhibition was not found when we usedMewocells with inactive p53 (Supplementary Fig. S5B).To confirm the molecular mechanisms involved in the

antimetastatic effects of metformin in vivo, Slug expres-sion was studied by immunofluorescent staining ontumor sections from mice treated with vehicle or metfor-min (Fig. 6C). S100 staining was used to detect melanomacells in lungs. Sections of lung tumors from mice treatedwith metformin showed a significant decrease in Slugstaining compared with sections of tumors from controlmice injected with vehicle. Quantification of ratio Slug:S100 confirms this observation. Thus, the reduction ofmetastases observed in metformin-treated mice seems tobe, at least in part, related to the inhibition of the expres-sion of Slug protein.

DiscussionThe discovery of new therapeutic compounds is a very

important challenge to treat advancedmelanomas that areresistant to existing therapies. For this purpose, using invitro and in vivo approaches, we and others previouslyshowed the potent effects of the antidiabetic drug met-

formin on reduction of melanoma cells growth and xeno-graft development (13–16). One crucial step of melanomadevelopment appears to be promotion of an EMT-liketransition, a process having central role during RGP andVGP progression, melanoma invasion, and metastaticdissemination of melanoma cells (19, 20). In this study,we show that metformin inhibits melanoma invasion byregulating the EMT-like regulatory factors and showthe critical role of the AMPK/p53 axis in this process.The mode of action and the biologic consequences of theantidiabetic drug metformin in cancer cell migration andinvasion are poorly understood. Indeed, only few studiesdone in fibrosarcoma, carcinoma, and ovarian carcinomacells evoke the role of metformin in this process (21–23).The current study was designed to evaluate the anti-invasive potential of metformin in melanoma and toanalyze the molecular mechanisms involved in thisprocess.

Using different in vitro approaches, we found thatmetformin inhibits cell invasion without affecting cellmigration. The fact that migration was not affected bymetformin indicates that cells are functional and showsthat inhibition of invasion mediated by metforminobserved after 24 hours of treatment is not due to induc-tion of apoptosis observed in cells treated by drug after alonger period of time (96 hours; ref. 13).

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1205Lu Cells 1205Lu Cells

Figure 4. Implication of AMPKa in the metformin effects. A, 1205Lu melanoma cells were infected with adenovirus encoding a dominant negative form of a1and a2 subunits of AMPK (Ad AMPK-DN) or an adenovirus control (Ad control). Twenty-four hours after infection, cells were treated with metformin(at the indicated concentrations) or PBS for 24 hours, lysed, and analyzed by Western blotting with phospho-acetyl coA carboxylase (Ser79), acetyl coAcarboxylase, a1 and a2 AMPK subunits, and Slug antibodies. HSP90 was used for loading control. B, invasion assays in coated Boyden chamberswere conducted on 1205Lumelanoma cells infectedwith adenovirus encoding a dominant-negative form of AMPKa (Ad AMPK-DN) or an adenovirus control(Ad control), treated or not for 24 hours with metformin. The results are expressed as percentages of the control. The bars indicate themean�SD of triplicatesamples. �, P < 0.05.






More interesting, we found the same results usingmorephysiologic tumor invasiveness in 3D collagen matrix, anassay that mimics 3D invasion by melanoma cells. Todefinitively confirm that invasion inhibition is not due toapoptosis induced bymetformin, we carried out the sameexperiment in presence of an apoptosis inhibitor and weobserved that metformin inhibited invasion in the samemanner in presence or not of inhibitor.

To elucidate themechanismbywhichmetformin inhib-ited melanoma cell invasion, we focused our interest onproteins involved in EMT transition. In melanoma, thetranscriptional factors of the Snail family Snail and Slugare required for the control of the expression of genesinvolved in EMT such as N-cadherin, SPARC, or fibro-nectin and for the development of local tumor invasionandpromotion of distantmetastasis (24, 25). Interestingly,

we found that metformin inhibited in a dose-dependentmanner the expression of transcription factors Slug andSnail in association with inhibition of EMT proteinsN-cadherin, SPARC, and fibronectin. Furthermore, it iswell-established that melanoma cell invasion dependson MMP expression and activity especially MMP-2 andMMP-9 (26). Both MMP are highly expressed in mela-noma cells, and a direct relationship between melanomaprogression and MMP expression and activity hasbeen well-established in many studies (26). Moreover,inhibition of MMP activity has been previously inves-tigated as a new therapeutic strategy to control meta-static spreading. As presented, metformin decreasesglobal MMP activity and, more specifically, expressionand activity of MMP-2 and MMP-9. Taken togetherthese results indicate that decrease in EMT protein

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Figure 5. Implication of p53 in the antimelanoma effects of metformin. A, 1205Lu melanoma cells were transfected with PG13-Luc, a p53-dependent fireflyluciferase reporter gene, and treated 24 hours with metformin (at the indicated concentrations), actinomycin D, or PBS. The results are expressed aspercentages of the control. The bars indicate the mean � SD of triplicate samples. ��, P < 0.001. B, 1205Lu melanoma cells were treated for 24 hourswith 10mmol/L of metformin or PBS and p53was immunoprecipitated from lysates and immunoblotted with AMPKa and p53 antibodies (bottom). In parallel,lysateswere analyzedbyWesternblottingwith fibronectin,N-cadherin, AMPKa, andSlug antibodies.HSP90wasused for load control (top). C, invasion assayin coated Boyden chambers were conducted on 1205Lumelanoma cells transfectedwith siRNAs against p53 (sip53) or a siRNA control (siCtl) and treated for24 hours with metformin (at the indicated concentrations) or PBS. In parallel, lysates were analyzed by Western blotting with p53 and Slug antibodies.HSP90was used for load control. The results are expressed as percentages of the control. The bars indicate themean�SD of triplicate samples. ��,P < 0.01;��, P < 0.001. D, invasion assay in coated Boyden chambers was conducted on Mewo melanoma cells (mutated for p53) infected with adenovirusencoding a WT form of p53 (Adp53) or a control adenovirus (AdCtl) and treated with metformin (at the indicated concentrations) or PBS for 24 hours.The results are expressed as percentages of the control. The bars indicate themean� SD of triplicate samples. ��, P < 0.01. In parallel, lysates were analyzedby Western blotting with N-cadherin, p53, and Slug antibodies. HSP90 was used for load control.

Cerezo et al.





expressions and MMP inhibitions are, at least in part,mechanisms by which metformin negatively regulatesmelanoma invasiveness.Metformin action is mainly mediated by AMPK acti-

vation; however, several recent reports indicate thatsome effects of metformin in cancer cell lines could bemediated by an AMPK-independent pathway (13, 27–29). In our model, AMPK inactivation by AMPK dom-inant-negative construct (30) or inhibition of AMPKexpression by siRNA (data not shown) inhibits the effectof metformin on melanoma invasion, suggesting thatthe effects of this drug are mediated through an AMPK-dependent mechanism.Several recent studies indicate that AMPK activation is

able to activate tumor suppressor gene p53 and thatanticancer effects of metformin could be mediated, inpart, by the activation of this transcription factor (10,11, 31). Indeed, this transcription factor is well-known asa potent suppressor of tumor development by inhibitingcell-cycle progression and promoting senescence, apopto-sis, or autophagy (32, 33). More recently, accumulatingevidence has pointed an alternative role of p53 by nega-tively regulating invasion and tumor cell metastasis(8, 34, 35). In melanoma cells, metformin promotes theinteraction between AMPKa and p53 and strongly stimu-

lates p53 activity. Furthermore, p53 was shown to pro-mote Slug degradation and inhibition of invasion in lungcarcinoma cells by mechanism involving MDM2 degra-dation pathway (8).We therefore examined the regulationof SLUG and invasion by p53 in p53-knockdown cells andfound that Slug and invasion regulation by metformin isdependent of both expression and mutational status ofp53. Accordingly, we observed that reversion of the EMTprocess, including modification of N-cadherin, SPARC,and fibronectin expression requires activation of p53.However, the mechanism by which p53 induces Slugdegradation remains unclear and needs further investiga-tions. Indeed, our experiments show that MDM2 is notinvolved in thismechanism (data not shown) as describedin lung carcinoma cells (8).

Finally, we have evaluated the antimelanoma activityof metformin in mouse models of extravasation andmelanoma metastasis of lungs.

One of the key events in cancer metastasis is extrava-sation. Extravasation of cancer cells is a complicatedprocess which involves adhesive interactions, chemotax-is, and invasion. Our in vivo experiments indicate thatshort-term treatment of melanoma cells (24 hours) withmetformin inhibited the capacity of tumor cells to extrav-asate in lungs to create micrometastasis. Similar results

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Figure 6. Effects ofmetformin onmelanoma invasion in vivo.1205Lumelanomacellswere treated for 24 hourswith 10mmol/L ofmetformin or PBSand labeledwith Green Cell Tracker and then injected via the tail vein in nude mice. After 24 hours, the lungs of the mice were imaged (left) and the number ofmicrometastasis were counted (right). B and C, after injection of 1 � 106 1205Lu melanoma cells expressing luciferase into the tail vein, the nude micewere treated or not with metformin (60 mg/kg) for 39 days. The bioluminescence resulting from the presence of lung metastasis was quantified with aPhoton Imager. The results after 7 days were quantified and presented in B. A characteristic image of 3 mice per group and quantification after39 days were presented in C. The bars indicate the mean � SD of triplicate samples. �, P < 0.05; ��, P < 0.001. D, immunofluorescence on sectionsof lungs was conducted with Slug and S100 antibodies. Hoechst was used to labeled nucleus (left). Quantification of the ratio Slug/S100 was evaluated withImageJ (right).






were observed when mice were pretreated with metfor-min instead of the injected cells (data not shown).

Importantly, in models of lung metastasis, the doses ofmetformin administered to the mice (2 mg/25 g/d) are inthe same range to those administered to patients withdiabetes (3 g/75 kg/d). We found that short-term admin-istration of metformin (7 days) reduces markedly thedevelopment of melanoma metastasis in lungs mice andalmost abolished metastasis after 39 days. The dramaticeffect of the metformin after 39 days on the inhibition ofmetastases in lungs is probablydue to a combined effect ofthe drug on the induction of the cell death and an inhi-bition of the invasion. Experiments are ongoing in ourlaboratory to confirm this hypothesis.

Todeterminewhether themolecular events observed incell lines were also found in mice, we conducted immu-nohistologic staining of mouse tumor sections. Experi-ments showed a decrease of Slug expression in the tumorsof metformin-treated mice. Furthermore, we observedthat metformin was inactive for inhibition of extravasa-tion and metastasis when melanoma harbors p53 muta-tion. These results suggest that in vivo, p53 activationinduced by metformin is required for Slug inhibition andthe decrease in melanoma metastasis dissemination inlung.

More recently, it was reported that metformin couldincrease melanoma growth in xenograft mice models ofmelanoma A375 cells harboring BRaf V600E mutationthrough increase of angiogenesis (36). In our experi-ments, we did not observe such effects in all the testedmelanoma cell lines (possessing or not BRaf V600Emutation). This discrepancy could be due to high levelof metformin used by Martin and colleagues and couldresult in off-target effects due to lactic acidosisdescribed when concentrations of drug are elevated inserum (37, 38).

In summary, we show for the first time that metformininhibits melanoma invasion and metastasis developmentthrough AMPK/p53 axis activation. This finding bringsnew clues to the understanding of metformin action in

melanoma development. Finally, taking into account thedrastic effects of metformin on melanoma cell growth,survival, invasion, andmetastasis development inmice, itmight be worth evaluating metformin treatment inpatients suffering from metastatic melanoma. However,our data indicate that mutational status of p53 must beconsidered before providing a patient with themetformintherapy.

Disclosure of Potential Conflicts of InterestR. Ballotti has honoraria from speakers bureau from Roche. No poten-

tial conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: M. Cerezo, S. Tartare-Deckert, S. RocchiDevelopment ofmethodology:M.Cerezo,M.Tichet, P.Abbe,M.Ohanna,C. Bertolotto, S. Tartare-Deckert, S. RocchiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.):M.Cerezo, A. Lehraiki, F. Rouaud, D. Giacchero,P. BahadoranAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): M. Cerezo, R. Ballotti, S. RocchiWriting, review, and/or revision of the manuscript: D. Giacchero, R.Ballotti, S. RocchiAdministrative, technical, or material support (i.e., reporting or orga-nizingdata, constructingdatabases):P.Abbe,M.Allegra,D.Giacchero, P.BahadoranStudy supervision: S. Rocchi

AcknowledgmentsThe authors sincerely thank F. Foufelle (INSERM, UMR-S 872, Paris,

France) for AMPK adenoviruses.

Grant SupportThis research was supported by the INSERM, University of Nice

Sophia-Antipolis, ARC contract n� SFI 20121205378, the "INCA RechercheTranslationnelle 2011" and "Soci�et�e Francaise de Dermatologie, grant2011".M. Cerezo is a recipient of a doctoral fellowship from the «Minist�erede l’Enseignement Sup�erieur et de la Recherche ». S. Rocchi is a recipient of« Contrat Hospitalier de Recherche Translationnelle du CHU de Nice ».INSERM U1065, team 1 is « �equipe labelis�ee ligue 2010 ».

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

ReceivedDecember 28, 2012; revisedMay7, 2013; acceptedMay24, 2013;published OnlineFirst June 5, 2013.

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2013;12:1605-1615. Published OnlineFirst June 5, 2013.Mol Cancer Ther Michaël Cerezo, Mélanie Tichet, Patricia Abbe, et al. in AMPK/p53-Dependent MannerMetformin Blocks Melanoma Invasion and Metastasis Development

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Metformin Blocks Melanoma Invasion and Metastasis ... · Cancer Therapeutics Insights Metformin Blocks Melanoma Invasion and Metastasis Development in AMPK/p53-Dependent Manner Micha€el