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Priority Report Adipocyte Exosomes Promote Melanoma Aggressiveness through Fatty Acid Oxidation: A Novel Mechanism Linking Obesity and Cancer Ikrame Lazar 1 , Emily Clement 1 , St ephanie Dauvillier 1 , Delphine Milhas 1 , Manuelle Ducoux-Petit 1 , Sophie LeGonidec 2 ,C edric Moro 2 , Vanessa Soldan 3 , St ephane Dalle 4 , St ephanie Balor 3 , Muriel Golzio 1 , Odile Burlet-Schiltz 1 , Philippe Valet 2 , Catherine Muller 1 , and Laurence Nieto 1 Abstract Malignant progression results from a dynamic cross-talk between stromal and cancer cells. Recent evidence suggests that this cross-talk is mediated to a signicant extent by exosomes, nanovesicles secreted by most cell types and which allow the transfer of proteins, lipids, and nucleic acids between cells. Adi- pocytes are a major component of several tumor microenviron- ments, including that of invasive melanoma, where cells have migrated to the adipocyte-rich hypodermic layer of the skin. We show that adipocytes secrete exosomes in abundance, which are then taken up by tumor cells, leading to increased migration and invasion. Using mass spectrometry, we analyzed the proteome of adipocyte exosomes. Interestingly, these vesicles carry proteins implicated in fatty acid oxidation (FAO), a feature highly specic to adipocyte exosomes. We further show that, in the presence of adipocyte exosomes, FAO is increased in melanoma cells. Inhi- bition of this metabolic pathway completely abrogates the exo- some-mediated increase in migration. Moreover, in obese mice and humans, both the number of exosomes secreted by adipo- cytes as well as their effect on FAO-dependent cell migration are amplied. These observations might in part explain why obese melanoma patients have a poorer prognosis than their nonobese counterparts. Cancer Res; 76(14); 40517. Ó2016 AACR. Introduction Among the cells found in tumor microenvironments, adipo- cytes, the main cellular components of adipose tissue (AT), can promote tumor progression (1, 2). Understanding their role in cancer, particularly in obesity, is of major clinical importance as obesity affects cancer occurrence and prognosis (2). Research into the communication between adipocytes and tumor cells has been limited to soluble factors, such as leptin or proinammatory cytokines (2), although emerging evidence suggests that exosomes also play a role in cellcell communication. Exosomes are nano- vesicles secreted by most cell types. Tumor cellderived exosomes are implicated in tumor progression through various mechanisms, such as immune evasion, proliferation, invasion, or metastatic niche preparation (3). "Normal" cells in the tumor microenvi- ronment also secrete exosomes (4, 5). However, the role of adipocyte exosomes (ad-exos) in tumor progression has not yet been studied. Subcutaneous adipocytes are the main component of the hypodermis, and adipocyte secretions may promote mel- anoma aggressiveness by stimulating cell growth (6) and/or invasion (7). Obesity is associated with an increased risk of developing melanoma as well as malignant progression (8, 9). In obesity, adipose tissue is submitted to stress such as inam- mation that is known, in other cells, to modify exosomes and consequently their activity on recipient cells (10). Few studies have focused on ad-exos in obesity, although their function and content are modied. For instance, they are implicated in insulin resistance (11) and their miRNAs mediate inammation and brosis (12). We show here that ad-exos stimulate melanoma cell migration and invasion. These exosomes, specically enriched in proteins implicated in fatty acid oxidation (FAO), induce a metabolic reprogramming in tumor cells in favor of FAO, promoting aggres- siveness. In obesity, both the number of exosomes secreted as well as their effect on tumor cell migration is increased, thus contrib- uting to the amplication of the deleterious dialog between adipocytes and cancer cells. Materials and Methods Suppliers and antibodies are available in Supplementary Materials and Methods. Cell lines and treatment The murine 3T3-F442A preadipocyte line (obtained and authenticated from ATCC in 2014) was differentiated into 1 Institut de Pharmacologie et de Biologie Structurale, Universit e de Toulouse, CNRS, UPS, Toulouse, France. 2 Institut des Maladies M etaboliques et Cardiovasculaires, Universit e de Toulouse, INSERM U1048, UPS, Toulouse, France. 3 Plateforme de Microscopie Electro- nique Int egrative, CNRS, Universit e de Toulouse, Toulouse, France. 4 Centre de recherche en Canc erologie de Lyon, Universit e Lyon 1, Pierre B enite, France. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). I. Lazar and E. Clement contributed equally to this article. Corresponding Author: Laurence Nieto, IPBS CNRS UMR5089/University of Toulouse, 205 Route de Narbonne, Toulouse 31077, France. Phone: 335-6117- 5509; Fax: 3305-6117-5994; E-mail: [email protected] doi: 10.1158/0008-5472.CAN-16-0651 Ó2016 American Association for Cancer Research. Cancer Research www.aacrjournals.org 4051 on September 28, 2020. © 2016 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst May 23, 2016; DOI: 10.1158/0008-5472.CAN-16-0651

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Page 1: Adipocyte Exosomes Promote Melanoma Aggressiveness … · Adipocytes secrete exosomes that promote melanoma migration and invasion. A, left, 3T3-F442A ad-exos viewed by electron microscopy

Priority Report

Adipocyte Exosomes Promote MelanomaAggressiveness through Fatty Acid Oxidation:A Novel Mechanism Linking Obesity and CancerIkrame Lazar1, Emily Clement1, St�ephanie Dauvillier1, Delphine Milhas1,Manuelle Ducoux-Petit1, Sophie LeGonidec2, C�edric Moro2, Vanessa Soldan3,St�ephane Dalle4, St�ephanie Balor3, Muriel Golzio1, Odile Burlet-Schiltz1,Philippe Valet2, Catherine Muller1, and Laurence Nieto1

Abstract

Malignant progression results from a dynamic cross-talkbetween stromal and cancer cells. Recent evidence suggests thatthis cross-talk is mediated to a significant extent by exosomes,nanovesicles secreted by most cell types and which allow thetransfer of proteins, lipids, and nucleic acids between cells. Adi-pocytes are a major component of several tumor microenviron-ments, including that of invasive melanoma, where cells havemigrated to the adipocyte-rich hypodermic layer of the skin. Weshow that adipocytes secrete exosomes in abundance, which arethen taken up by tumor cells, leading to increased migration andinvasion. Using mass spectrometry, we analyzed the proteome of

adipocyte exosomes. Interestingly, these vesicles carry proteinsimplicated in fatty acid oxidation (FAO), a feature highly specificto adipocyte exosomes. We further show that, in the presence ofadipocyte exosomes, FAO is increased in melanoma cells. Inhi-bition of this metabolic pathway completely abrogates the exo-some-mediated increase in migration. Moreover, in obese miceand humans, both the number of exosomes secreted by adipo-cytes as well as their effect on FAO-dependent cell migration areamplified. These observations might in part explain why obesemelanoma patients have a poorer prognosis than their nonobesecounterparts. Cancer Res; 76(14); 4051–7. �2016 AACR.

IntroductionAmong the cells found in tumor microenvironments, adipo-

cytes, the main cellular components of adipose tissue (AT), canpromote tumor progression (1, 2). Understanding their role incancer, particularly in obesity, is of major clinical importance asobesity affects cancer occurrence and prognosis (2). Research intothe communication between adipocytes and tumor cells has beenlimited to soluble factors, such as leptin or proinflammatorycytokines (2), although emerging evidence suggests that exosomesalso play a role in cell–cell communication. Exosomes are nano-vesicles secreted by most cell types. Tumor cell–derived exosomesare implicated in tumor progression through various mechanisms,such as immune evasion, proliferation, invasion, or metastatic

niche preparation (3). "Normal" cells in the tumor microenvi-ronment also secrete exosomes (4, 5). However, the role ofadipocyte exosomes (ad-exos) in tumor progression has not yetbeen studied. Subcutaneous adipocytes are the main componentof the hypodermis, and adipocyte secretions may promote mel-anoma aggressiveness by stimulating cell growth (6) and/orinvasion (7). Obesity is associated with an increased risk ofdeveloping melanoma as well as malignant progression (8, 9).In obesity, adipose tissue is submitted to stress such as inflam-mation that is known, in other cells, to modify exosomes andconsequently their activity on recipient cells (10). Few studies havefocused on ad-exos in obesity, although their function and contentare modified. For instance, they are implicated in insulin resistance(11) and their miRNAs mediate inflammation and fibrosis (12).

We show here that ad-exos stimulate melanoma cell migrationand invasion. These exosomes, specifically enriched in proteinsimplicated in fatty acid oxidation (FAO), induce a metabolicreprogramming in tumor cells in favor of FAO, promoting aggres-siveness. In obesity, both the number of exosomes secreted aswellas their effect on tumor cell migration is increased, thus contrib-uting to the amplification of the deleterious dialog betweenadipocytes and cancer cells.

Materials and MethodsSuppliers and antibodies are available in Supplementary

Materials and Methods.

Cell lines and treatmentThe murine 3T3-F442A preadipocyte line (obtained and

authenticated from ATCC in 2014) was differentiated into

1Institut de Pharmacologie et de Biologie Structurale, Universit�e deToulouse, CNRS, UPS, Toulouse, France. 2Institut des MaladiesM�etaboliques et Cardiovasculaires, Universit�e de Toulouse, INSERMU1048, UPS, Toulouse, France. 3Plateforme de Microscopie Electro-nique Int�egrative, CNRS, Universit�e de Toulouse, Toulouse, France.4Centre de recherche en Canc�erologie de Lyon, Universit�e Lyon 1,Pierre B�enite, France.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

I. Lazar and E. Clement contributed equally to this article.

Corresponding Author: Laurence Nieto, IPBS CNRS UMR5089/University ofToulouse, 205 Route de Narbonne, Toulouse 31077, France. Phone: 335-6117-5509; Fax: 3305-6117-5994; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-16-0651

�2016 American Association for Cancer Research.

CancerResearch

www.aacrjournals.org 4051

on September 28, 2020. © 2016 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst May 23, 2016; DOI: 10.1158/0008-5472.CAN-16-0651

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adipocytes as described previously (1). Melanoma line char-acteristics and conditions of culture are described in Supple-mentary Materials and Methods. They were obtained andauthenticated from L. Larue (Institut Curie, Orsay, France) in2013 and passaged for fewer than 2 months after receiptor resuscitation. Cells (1 � 105) were treated with exosomes(5 � 1010 per well) for 48 hours. When indicated, etomoxir(50 mmol/L) or trimetazidine (1 mmol/L) were added 24 hoursafter exosomes. Actin was stained with phalloidin as describedpreviously (13).

Animals and primary cell isolationMice were handled in accordance with National Institute

of Medical Research (INSERM) guidelines. Experimentswere approved by the local committee on ethics of animalexperimentation. Eight-week-old C57BL/6J male mice werefed a normal or high fat diet as described in Supplemen-tary Materials and Methods. Adipocytes and SVF cells wereisolated from adipose tissue as described previously (14). Atotal of 1 � 106 cells/mL was incubated to conditionmedium.

For tail vein metastasis assays, female nude athymic micewere intravenously injected with 5 � 105 GFP-expressingSKMEL28 cells treated or not with ad-exos. Seven weeks later,mice were sacrificed and lungs excised, fixed in 4% parafor-maldehyde for 24 hours, dehydrated, and embedded in paraf-fin. Sections (10 mm) were cut and rehydrated before countingGFP-positive micrometastases using an Axio Imager M2 micro-scope (Zeiss).

Preparation of human adipose tissue samplesHuman adipose tissue samples were collected from abdom-

inal dermolipectomies in accordance with the recommenda-tions of the ethics committee of the Toulouse Hospital (Tou-louse, France). Patients gave their consent in accordance withthe Declaration of Helsinki Principles as revised in 2000.Tissues were processed within 30 minutes of surgical resection.Adipose tissue was separated from skin, large blood vessels,glandular tissue and fascia, weighed, cut into 1 cm3 pieces, andincubated to condition medium (4 mL/g of tissue). The age,sex, and body mass index (BMI) were available for all patients.Samples were separated into three groups of 8 patients: lean(BMI < 25 kg/m2; mean BMI ¼ 22.3 � 2.1; mean age ¼ 55.7 �7.5), overweight (BMI, 25–29.9 kg/m2; mean BMI¼ 27.5� 1.6;mean age ¼ 43.9 � 5.8), and obese (BMI � 30 kg/m2; meanBMI ¼ 32.2 � 2.3; mean age ¼ 49.5 � 5.6).

Exosome preparation and analysisCells were incubated in DMEM supplemented with 10% FCS

depleted of vesicles by overnight centrifugation (100,000 � g).Lines and primary cells and tissueswere respectively incubated for48 and 24 hours. Exosome purification, density, number and sizedistribution analysis, and uptake monitoring were performed asdescribed previously (13).

Transmission electron microscopySpecimens were prepared as described previously (13). Mito-

chondria were quantified by counting the number per picture onat least 50pictures per experiment (an average of 20mitochondriaper picture).

Migration and invasion assaysCells were assessed in Boyden chamber assays as described in

Supplementary Materials and Methods.

Nano-LC/MS-MS analysisProteins (50 mg) were identified using Nano-LC/MS-MS and

analyzed as described in Supplementary Materials and Methods.

Western blotting analysisCells or exosomes were lysed in PBS 1% SDS. Proteins (1 mg)

were electrophoresed on SDS-PAGE. Western blotting analyseswere performed as described previously (13).

Measurement of FAOCells were incubated for 3 hours with [1-14C]palmitate or

[U-14C]D-glucose (1 mCi/mL). Following incubation, 14CO2 wasextracted, measured by liquid scintillation (LS 6500; BeckmanCoulter), and normalized as described previously (15).

Statistical analysisValues are means � SEM. The statistical significance of results

(at least three independent assays) was evaluated using Studentt tests. �, P < 0.05; ��, P < 0.01; and ���, P < 0.001were deemed assignificant (ns, nonsignificant).

ResultsAdipocytes secrete exosomes that are taken up by tumors andpromote aggressiveness

Exosomes were purified from the conditioned medium of3T3-F442A mature adipocytes. The isolated particles display thetypical morphology, density, and size of exosomes and containALIX, TSG101, and flotillin-1 (FLOT1), all of which being hall-marks of exosomes (Fig. 1A; ref. 16). Mature adipocytes exhibithigh rates of exosome shedding. Indeed, they secrete much moreexosomes thanmelanoma cells, which are known to secretemanyvesicles (Fig. 1B; ref. 17). Exposure of melanoma cell lines,SKMEL28 and 1205Lu, to DiD-labeled ad-exos resulted in apunctate fluorescence in cells, indicating exosome internalization(Fig. 1C). Melanoma cells treated with ad-exos exhibited anelongated morphology with actin-rich membrane protrusions,consistent with an increase in cell migration (Fig. 1C). Indeed,ad-exos significantly increased melanoma cell migration andinvasion, whereas proliferation was not affected (Fig. 1D andSupplementary Fig. S1). Moreover, melanoma cells incubatedwith ad-exos induced increased lung colonization in immuno-deficient mice when compared with control cells (Fig. 1D).Interestingly, exosome secretion progressively increased duringadipocyte differentiation (Supplementary Fig. S2A), and preadi-pocyte-derived exosomes failed to promote migration (Supple-mentary Fig. S2B), showing the specific effect of mature ad-exos.The effect of ad-exoswas not restricted tomelanoma, as it was alsoseen in a prostate cancer model (Supplementary Fig. S3). Overall,these results show that ad-exos enhance tumor cell aggressiveness,highlighting a new mode of communication between adipocytesand cancer cells.

Ad-exos promote cancer cell migration through metabolicreprogramming

To decipher the mechanisms responsible for the promigratoryeffect of ad-exos, we analyzed their protein content by mass

Lazar et al.

Cancer Res; 76(14) July 15, 2016 Cancer Research4052

on September 28, 2020. © 2016 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

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spectrometry (Supplementary Fig. S4A; Supplementary Table).Among the proteins identified, all common exosomal markerswere present (Supplementary Fig. S4B).When classified dependingon their function, the most represented cellular process associatedwith ad-exo proteins was "metabolism and transporters" (Fig. 2A).This protein signature is specific to ad-exos as exosomalproteins areusually predominantly associated with "vesicular trafficking" (13).Moreover, 53%of the proteins implicated inmetabolismhadnever

been identified in exosomes fromother cells. In contrast,more than90%ofproteins implicated in "vesicular trafficking" hadpreviouslybeen identified in exosomes (Fig. 2A, top). Themajority of proteinsimplicated in cell metabolism were found to be involved in lipidmetabolism, a trait highly specific to ad-exos (Fig. 2A, bottom).Among them, we identified a large number of proteins involved inFAO (Fig. 2B), including ECHA (a subunit of the trifunctionalenzyme) and HCDH (hydroxyacyl-coenzyme A dehydrogenase).

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Figure 1.

Adipocytes secrete exosomes that promote melanoma migration and invasion. A, left, 3T3-F442A ad-exos viewed by electron microscopy (scale bar, 200 nm);right, ad-exos isolated on a sucrose density gradient analyzed by silver staining or Western blotting. d, density. B, NanoSight analysis of exosomes isolatedfrom 3T3-F442A or melanoma cell–conditioned medium. One representative analysis for each line is shown. C, labeled ad-exos (red) were incubated with theindicated melanoma cells. Actin was stained using phalloidin (green; scale bar, 20 mm). D, left and middle, migration or invasion assays of melanoma linesincubated or not with ad-exos; right, number of lung metastases per mouse intravenously injected with GFP-SKMEL28 cells treated or not with ad-exos(control, n ¼ 9; ad-exos, n ¼ 8). � , P < 0.05; �� , P < 0.01; ��� , P < 0.001.

Adipocyte Exosomes: A New Link between Obesity and Cancer

www.aacrjournals.org Cancer Res; 76(14) July 15, 2016 4053

on September 28, 2020. © 2016 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

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We verified by Western blot analysis the presence of these proteinsin exosomes from 3T3-F442A adipocytes, whereas they were unde-tectable in those frompreadipocytes. Interestingly, theywere barelydetectable in the exosomes secreted by melanoma cells, althoughthese cells expressed them at levels comparable with adipocytes(Fig. 2C), highlighting their specific addressing to ad-exos. ECHAand HCDH were also present in murine and human primary ad-exos (Fig. 2C, bottom).

We hypothesized that, through the transfer of functionalenzymes, ad-exos might induce a metabolic reprogramming infavor of FAO in recipient cells. In favor of this hypothesis,SKMEL28 cells treated with ad-exos presented increased levels ofECHA andHCDHproteins, with no difference in the correspond-ing mRNA levels (Supplementary Fig. S5). Moreover, FAO wasincreased in SKMEL28 cells incubatedwith ad-exos, while glucoseoxidation and lactate release were unchanged, indicating thatglycolysis was not impacted by this treatment (Fig. 3A andSupplementary Fig. S6). This metabolic reprogramming was alsoassociated with an increase in mitochondria number and densityin melanoma (Fig. 3B) and prostate tumor cells (Supplemen-

tary Fig. S7A) cells, suggesting enhanced respiratory chainactivity (18). To evaluate the impact of FAO on ad-exos–induced cell migration, two FAO inhibitors, etomoxir andtrimetazidine, were used (targets shown in Fig. 2B). Althoughneither inhibitor affected basal tumor cell migration, a com-plete reversion of the promigratory effect of ad-exos wasobserved in recipient tumor cells (Fig. 3C and SupplementaryFig. S7B), demonstrating the implication of FAO in this process.FAO inhibitors induced a reversion of the ad-exos–mediatedactin cytoskeleton remodeling (Fig. 3D), further supporting thelink between FAO and migration.

In obesity, the effect of ad-exos is increasedWe therefore tested whether ad-exos from obese individuals

exhibit an increased activity on tumor cells. Exosomes secretedby subcutaneous or visceral adipocytes from lean and obesemice were purified (Supplementary Fig. S8A). Interestingly, thenumber of exosomes shed by adipocytes from obese mice washigher than that from lean animals (Fig. 4A, left; SupplementaryFig. S8B). However, this increase was not observed for other cells

CPT1

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Figure 2.

Ad-exos specifically carry proteins implicated inFAO. A, top, distribution of ad-exo proteins intofunctional groups; bottom, classification ofproteins implicated in "metabolism andtransporters" into subcategories. Hatchingindicates proteins specific to ad-exos. ECM,extracellular matrix; TCA, tricarboxylic acid.B, schematic representation of FAO. Ad-exoproteins are framed in green. Etomoxir andtrimetazidine targets are indicated. FA, fattyacid; CoA, coenzyme A. C, Western blot analysisof whole-cell lysate (C) and exosomes (E). FLOT1was used as an exosomal marker. PreAd,preadipocytes; Ad, 3T3-F442A adipocytes; SK,SKMEL28; LU, 1205Lu; mAd/hAd, murine/humanprimary subcutaneous adipocytes.

Lazar et al.

Cancer Res; 76(14) July 15, 2016 Cancer Research4054

on September 28, 2020. © 2016 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

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found in adipose tissue (referred to as "stromal vascular fraction"or SVF). Remarkably, cancer cell migration was increased afterincubation with equal concentrations of ad-exos isolated fromobesemice comparedwith those isolated from leanmice,whereasthis effect was not observed for the SVF (Fig. 4A and Supplemen-tary Fig. S8C). We confirmed these findings using human adiposetissue from individuals with varying BMIs. Indeed, there was apositive correlation between adipose tissue exosome sheddingand BMI (Fig. 4B, left). Moreover, when used at equal concentra-tions, exosomes fromoverweight and obese individuals graduallyincreased melanoma migration compared with exosomes fromlean individuals (Fig. 4B, right). Finally, in both lean and obeseconditions, inhibition of FAO reversed the effect of ad-exos,

showing that FAO is involved in the obesity-associated exacer-bation of this deleterious metabolic reprogramming (Fig. 4C).Overall, these findings show that ad-exos are modified in aquantitative and qualitative manner in obesity. Indeed, not onlyis the number of exosomes secreted per cell increased, but so istheir effect on cell migration when equal concentrations wereused. These modifications might well work in synergy in obesepatients, favoring tumor cell migration to an even higher extent.

DiscussionEmerging studies indicate that a cross-talk exists between

adipocytes and cancer cells (1). This dialog entails a vicious circle,

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Figure 3.

Ad-exos promote melanoma migrationthrough metabolic reprogramming.Melanoma lines were incubated or notwith ad-exos. A, 14C palmitate (left) and14C glucose (right) oxidation analysis inSKMEL28 cells. ns, nonsignificant. B, left,representative transmission electronmicrograph of mitochondria (�) inmelanoma cells (scale bar, 500 nm);right, quantification of these organelles.C and D, when indicated, etomoxir andtrimetazidine were added. C, migrationassays. D, actin staining (green) usingphalloidin (scale bar, 20 mm). � , P < 0.05;�� , P < 0.01; ��� , P < 0.001. ns,nonsignificant.

Adipocyte Exosomes: A New Link between Obesity and Cancer

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where cancer cells activate adipocytes, which, in turn, promotetumor progression. The study of this dialog has, until now,been limited to soluble factors, despite increasing evidence foran important role for stromal cell exosomes on tumor progres-sion. For example, it has been shown that exosomes fromcancer-associated fibroblasts promote breast cancer cell motil-ity through Wnt/planar cell polarity signaling (19). Here, weshow the importance of ad-exos in malignant progression.Indeed, ad-exos are key players in a metabolic dialog betweenadipocytes and cancer cells, not only in melanoma but also inprostate cancer, suggesting that their effect on tumor progres-sion is more general. Recent results have shown that exosomesshed by pancreatic cancer cells induce lipolysis in subcutaneousadipose tissue (20), indicating a role for exosomes in bothdirections of the cross-talk between adipocytes and cancer cells.Our study reveals the involvement of FAO in the increasedtumor cell migration induced by ad-exos. In accordance with

our proteomic analysis, previous studies have identified reg-ulators of lipid metabolism, including FAO enzymes, in ad-exos, although none have addressed their functional impact inrecipient cells (21, 22). The importance of FAO in tumorprogression has recently been highlighted (23). Advanced mel-anomas show upregulation of FAO genes, highlighting that thispathway could be key to melanoma progression (24). Finally,ad-exos secretion and activity is amplified in obesity, whichcould explain, at least in part, the poor prognosis observed forthis subset of cancer patients (2). Pharmacologic inhibition ofFAO totally reverses the effect of ad-exos on tumor cell migra-tion in obesity (Fig. 4C). However, the actors responsible forthis effect remain unidentified. Preliminary experiments showthat ECHA and HCDH are not enriched in ad-exos from obesemice (data not shown). In addition to the increased level ofother FAO proteins, the role of fatty acids is an interestingalternative hypothesis since, as ad-exos contain free fatty acids

A

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tion

(vs.

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)

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1205

Lu0.0

0.5

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* **

SKMEL28Rel

ativ

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tion

(vs.

con

trol

)

0

1

2

3

4

ns

** **

***

1205LuRel

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(vs.

con

trol

)

0.0

0.5

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1.5

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ns

** **

*****

ControlEtomoxirAd NDAd ND + EtomoxirAd HFDAd HFD + Etomoxir

Rel

ativ

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SVF

ND

)

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1205

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SVF NDSVF HFD

ns ns

Con

cent

ratio

n (p

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les/

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ells

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0.0

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Num

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ssue

0 10 20 30 400

R2 = 0.7583

25E+11

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Rel

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Ctrl LOW OB

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2

3

4

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***

Figure 4.

The effect of ad-exos is amplified inobesity. A, left, NanoSight analysis ofexosomes secreted by adipocytes orSVF from the subcutaneous adiposetissue of lean [normal diet (ND)] orobese [high fat diet (HFD)] mice;middle and right, migration assays ofmelanoma cells incubated or not withthe exosomes secreted by theindicated cells. ns, nonsignificant.B, left, correlation between humanadipose tissue exosome secretion andBMI; right, migration of SKMEL28 cellsincubated or not with exosomes fromhuman adipose tissue. Ctrl, control;L, lean; OW, overweight; OB, obese.C, migration assays in the indicatedconditions. � , P < 0.05; �� , P < 0.01;��� , P < 0.001. ns, nonsignificant.

Lazar et al.

Cancer Res; 76(14) July 15, 2016 Cancer Research4056

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(11) and as adipocytes contain more lipids in obesity, theexosomes they secrete may be enriched in fatty acids, knownpositive regulators of FAO (25).

We anticipate that our findings could contribute to the devel-opment of new strategies for cancer treatment involving FAOinhibitors, especially for the treatment of obese individuals. Suchmolecules, as trimetazidine, are currently used in the context ofother diseases, such as angina pectoris (23).

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: C. Muller, L. NietoDevelopment of methodology: I. Lazar, E. ClementAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): I. Lazar, E. Clement, S. Dauvillier, D. Milhas,M. Ducoux-Petit, C. Moro, V. Soldan, S. Balor, M. Golzio, O. Burlet-SchiltzAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): I. Lazar, E. Clement, C. Moro, S. Balor, C. Muller,L. Nieto

Writing, review, and/or revision of the manuscript: I. Lazar, E. Clement,C. Moro, P. Valet, C. Muller, L. NietoAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S. LeGonidec, S. Dalle, P. ValetStudy supervision: O. Burlet-Schiltz, C. Muller, L. Nieto

AcknowledgmentsThe authors thank Lionel Larue for melanoma lines and helpful discussions

and Heinz Arnheiter for carefully proofreading and for his comments thatgreatly improved the manuscript.

Grant SupportThis work was supported by the "Ligue contre le Cancer" (5FI11679PXVJ),

the "Fondation ARC pour la recherche sur le cancer" (ARC PJA20131200457),and the "Soci�et�e Francaise deDermatologie" (140372 to L.Nieto andC.Muller)and by the European Found (FEDER to O. Burlet-Schiltz). I. Lazar andE. Clement are recipients of PhD fellowships from "Fondation ARC pour larecherche sur le cancer" and "Ligue contre le Cancer," respectively.

Received March 7, 2016; revised May 10, 2016; accepted May 18, 2016;published OnlineFirst May 23, 2016.

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2016;76:4051-4057. Published OnlineFirst May 23, 2016.Cancer Res   Ikrame Lazar, Emily Clement, Stéphanie Dauvillier, et al.   CancerFatty Acid Oxidation: A Novel Mechanism Linking Obesity and Adipocyte Exosomes Promote Melanoma Aggressiveness through

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