Myostatin Gene Inactivation Prevents Skeletal Muscle Wasting in Cancer

Molecular and Cellular Pathobiology

Myostatin Gene Inactivation Prevents Skeletal MuscleWasting in Cancer

Yann S. Gallot1, Anne-C�ecile Durieux1, Josiane Castells1, Marine M. Desgeorges1, Barbara Vernus2,L�ea Plantureux1, Didier R�emond3, Vanessa E. Jahnke1, Etienne Lefai4, Dominique Dardevet3,Georges Nemoz4, Laurent Schaeffer5, Anne Bonnieu2, and Damien G. Freyssenet1

AbstractCachexia is a muscle-wasting syndrome that contributes significantly to morbidity and mortality of many

patients with advanced cancers. However, little is understood about how the severe loss of skeletal musclecharacterizing this condition occurs. In the current study, we tested the hypothesis that the muscle proteinmyostatin is involved in mediating the pathogenesis of cachexia-induced muscle wasting in tumor-bearingmice.Myostatin gene inactivation prevented the severe loss of skeletal muscle mass induced in mice engraftedwith Lewis lung carcinoma (LLC) cells or in ApcMin/þ mice, an established model of colorectal cancer andcachexia. Mechanistically, myostatin loss attenuated the activation of muscle fiber proteolytic pathways byinhibiting the expression of atrophy-related genes, MuRF1 and MAFbx/Atrogin-1, along with autophagy-related genes. Notably, myostatin loss also impeded the growth of LLC tumors, the number and the size ofintestinal polyps in ApcMin/þ mice, thus strongly increasing survival in both models. Gene expression analysisin the LLC model showed this phenotype to be associated with reduced expression of genes involved in tumormetabolism, activin signaling, and apoptosis. Taken together, our results reveal an essential role formyostatin in the pathogenesis of cancer cachexia and link this condition to tumor growth, with implicationsfor furthering understanding of cancer as a systemic disease. Cancer Res; 74(24); 7344–56. �2014 AACR.

IntroductionCancer cachexia is a wasting syndrome characterized by the

uncontrolled loss of body weight that results from depletion ofadipose tissue and skeletal muscle, while the nonmuscleprotein compartment is relatively preserved (1). Tumor- andhost-derived factors induce cachexia in up to 80% of patientswith advanced cancers, particularly in pancreatic and gastriccancers (2). Cancer cachexia accounts for 20% of cancer-related deaths (3), and dramatically contributes to reducingthe quality of life, increasing toxicity from chemotherapy, anddecreasing survival of patients with cancer (4).

Different strategies have been developed to limit cancercachexia, but much interest has been devoted to the thera-

peutic potential of inhibiting type-II activin receptor (ActRII)-mediated signaling triggered by activins and myostatin (Mstn;reviewed in ref. 5). Mstn is a highly conserved member of theTGF-b superfamily,mainly secreted fromskeletalmusclefibers(6). Mstn acts in an autocrine/paracrine manner by binding toActRIIA and ActRIIB, which then recruit and activate type-Iactivin receptors, also known as activin receptor-like kinase(Alk) 4 and 5. This, in turn, causes phosphorylation of Smad2and Smad3, the association with Smad4 into a Smad2/3/4complex, which then enters into the nucleus to trigger genetranscription.

Mstn is a master regulator of skeletal muscle mass. Mstngene inactivation induces skeletal muscle hypertrophy (6, 7),whereas forced overexpression of Mstn induces skeletal mus-cle atrophy (8–10). An increase in Mstn expression is themolecular signature of multiple conditions leading to skeletalmuscle wasting (5), including cancer cachexia (11–16). Inmouse models of cancer cachexia, administration of a solubleform of ActRIIB preserves skeletal muscle mass (14, 15),restores muscle strength (15, 16), and increases lifespan(15). However, a shortcoming of targeting ActRIIB ligands hasbeen the difficulty in discriminating the respective effects ofMstn and activins (15), whose expression is also upregulated inmany catabolic disease states, and likely contributes to thewasting syndrome (5).

In this study, we set out (i) to determine the molecularmechanisms by which Mstn gene inactivation may alter thecourse of cancer cachexia and (ii) to examine the possible

1Laboratoire de Physiologie de l'Exercice, Universit�e de Lyon, Saint Eti-enne, France. 2INRA UMR 866 Dynamique Musculaire et M�etabolisme,Montpellier, France. 3INRA UMR 1019, Unit�e de Nutrition Humaine, Cler-mont-Ferrand, France. 4INSERM U1060, INRA USC1235, CarMeN Labo-ratory, Universit�e de Lyon, Oullins, France. 5CNRS UMR 5239, Laboratoirede Biologie Mol�eculaire de la Cellule, Ecole Normale Sup�erieure de Lyon,Lyon, France.

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

Corresponding Author: Damien G. Freyssenet, Laboratoire de Physiolo-gie de l'Exercice, Facult�e deM�edecine, 15 rueAmbroise Par�e, 42023Saint-Etienne Cedex 2, France. Phone: 033-4-77-42-14-77; Fax: 033-4-77-42-14-78; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-14-0057

�2014 American Association for Cancer Research.

CancerResearch

Cancer Res; 74(24) December 15, 20147344

on December 15, 2014. © 2014 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst October 21, 2014; DOI: 10.1158/0008-5472.CAN-14-0057

http://cancerres.aacrjournals.org/

therapeutic impact of Mstn gene inactivation. Mice with adeletion of the third exon of the Mstn gene were used in twodifferent animal models of cancer cachexia, the Lewis lungcarcinoma (LLC) tumor-bearing mouse and the ApcMin/þ

mouse, a model of colorectal cancer and cachexia (17, 18).

Materials and MethodsAnimalsAll experiments were conducted in accordance with the

European Community guidelines for the care and use oflaboratory animals for scientific purposes. ConstitutiveC57BL/6 Mstn knockout (Mstn�/�) mice were previouslydescribed as MstnD/D mice (7). The floxed Mstn allele (wherethe third exon of the Mstn gene is flanked with a pair of loxPsites) has been deleted by transiently expressed Cre recombi-nase at the zygote stage. C57BL/6 ApcMin/þ male micewere purchased from Jackson Laboratory. To generate Apc-Min/þ/Mstn�/� mice, ApcMin/þ males were first mated toMstn�/� females. ApcMin/þMstnþ/� male mice from the prog-eny were then mated to Mstn�/� females. ApcMin/þMstnþ/þ,ApcMin/þMstnþ/�, and ApcMin/þMstn�/� mice were identifiedby genotype analysis.

Culture of Lewis lung carcinoma cells and inoculationLLC cells (ATCC) were cultured in DMEM supplemented

with 10% FBS and 1% penicillin-streptomycin at 37�C and 5%CO2 in air. Mstn�/� (14.54 � 0.80 months; n ¼ 11) and wild-type (14.60 � 0.95 months; n ¼ 12) male mice were subcu-taneously inoculated with 5 � 106 LLC cells in 150 mL ofsterile DPBS. Control Mstn�/� (14.67 � 1.13 months; n ¼ 8)and wild-type (14.66 � 0.95 months; n ¼ 9) male micereceived sterile DPBS. Mice were allowed food and waterad libitum. Body weight, food intake, and tumor size weremeasured daily.

Survival rateSurvival rate was determined in wild-type (15.06 � 0.62

months; n ¼ 20) and Mstn�/� (15.15 � 0.16 months; n ¼ 17)mice inoculated with LLC cells. Body weight, food intake, andtumor size were measured daily until death.Survival rate was also determined in ApcMin/þMstnþ/þ (n ¼

30), ApcMin/þMstnþ/� (n ¼ 15), and ApcMin/þMstn�/� (n ¼ 12)male mice. ApcMin/þMstnþ/þ was bred until death. Survivalrates of ApcMin/þMstnþ/� and ApcMin/þMstn�/� mice weredetermined until the age of 45 weeks.

Removal of tissuesMice were anesthetized (i.p. injection of 90 mg/kg ketamine

and 10 mg/kg xylazine) 35 days after the inoculation of LLCcells. Extensor digitorum longus, gastrocnemius, soleus, andtibialis anteriormuscles, as well as tumor and visceral adiposetissueswere rapidly excised andweighed.Micewere then killedby cervical dislocation.

Plasma amino acid levelPlasma (250 mL) was homogenized in 50 mL of sulfosalicylic

acid solution (1 mol/L in ethanol with thioglycolate 0.5 mol/L)

that had previously completely evaporated. Norleucine wasadded as an internal standard. Samples were incubated on icefor 15 minutes and centrifuged at 10,000 g at 4�C for 15minutes. The supernatant (200 mL) was then combined with100 mL of 0.1 mol/L lithium acetate buffer (pH ¼ 2.2). Aminoacid concentrations were determined by ion-exchange chro-matography (Bio-Tek Instruments A.R.L.) using post-columnderivation with ninhydrin.

Real-time PCRTotal RNA was collected from gastrocnemius muscle and

tumor tissue by using the RNeasy Fibrous Tissue Mini Kit(Qiagen). RNA (400 ng) was reverse transcribed using theiScript cDNA synthesis kit (Bio-Rad). The selected forwardand reverse primer sequences are listed in SupplementaryTable S1. Real-time PCRwas performed in a 20-mL final volumeusing the SsoFast EvaGreen Supermix (Bio-Rad). Fluorescenceintensity was recorded using a CFX96 Real-Time PCR Detec-tion System (Bio-Rad). Data were analyzed using the DDCtmethod of analysis. Reference genes (hypoxanthine guaninephosphoribosyl transferase, ribosomal protein large P0, anda-tubulin) were used to normalize the expression levels ofgenes of interest (8).

Protein extractionGastrocnemius muscles and tumor tissues were homoge-

nized (1:20 dilution wt:vol) in an ice-cold 50 mmol/L TrisHCl buffer (pH 7.4) containing 100 mmol/L NaCl, 2 mmol/LEDTA, 2 mmol/L EGTA, 50 mmol/L b-glycerophosphate,50 mmol/L sodium fluoride, 1 mmol/L sodium orthovana-date, 120 nmol/L okadaic acid, and 1% Triton X-100, andthen centrifuged at 12,000 g for 10 minutes at 4�C. Proteinconcentration of the supernatant was determined at 750 nm(Bio-Rad).

Protein analysesFor Luminex analysis, fluorescent capturing beads cou-

pled to antibodies directed against IkB-a, IkB-aSer32/Ser36,and p70S6KThr421/Ser424 (Bio-Rad) were incubated for 2 hourswith 50 mL of protein fractions (1:10 dilution vol:vol) in 96-well plates. Samples were then washed, incubated withbiotinylated antibodies for 30 minutes, followed by theincubation with a streptavidin–phycoerythrin solution for10 minutes. The analysis consisted of a double-laser fluo-rescence detection, which allowed simultaneous identifica-tion of the target protein through the red fluorescenceemission signal of the bead and quantification of the targetprotein through the fluorescence intensity of phycoerythrin.Fluorescence intensities were recorded on a Bio-Plex 200System instrument (Bio-Rad). Data were analyzed using Bio-Plex Manager software.

ForWestern blot analysis, 50 mg of proteins were resolved on12.5% SDS-polyacrylamide gels, then blotted onto 0.45 mmnitrocellulose membranes (GE Healthcare Life Sciences),and incubated overnight with the appropriate antibody.Antibodies against 4E-BP1 (1:500 vol/vol), NF-kB p65(1:600), NF-kB p65Ser536 (1:200), ribosomal protein (rp)S6 (1:600), rpS6Ser235/236 (1:800), Smad2/3 (1:500), and

Myostatin Gene Invalidation Prevents Cancer Cachexia

www.aacrjournals.org Cancer Res; 74(24) December 15, 2014 7345




Smad2Ser465/467/3Ser423/425 (1:200) were from Cell SignalingTechnology. Antibodies against Bax (1:1,000) and Bcl-2(1:800) were from Santa Cruz Biotechnology. Antibodiesagainst Atg5–Atg12 complex (1:800), FoxO3aSer253 (1:500),LC3b (1:800), anda-tubulin (1:2,000) were from Sigma-Aldrich.Incubationwith horseradish peroxidase-conjugated secondaryantibody (Dako) allowed the chemiluminescent detection ofimmunocomplexes (GE Healthcare Life Sciences). LabeledWestern blot analyses were quantified using ImageJ softwareanalysis (NIH). a-tubulin immunoblot analyses were used tocheck for equal protein loading.

Caspase-3 enzyme activity was fluorometrically (lexc ¼ 380nm and lexc ¼ 460 nm) determined on tumor protein extractsby using Acetyl-Asp-Glu-Val-Asp-7-amido-4-methylcoumarin(Bachem) as a fluorescent substrate (19).

Count of intestinal polyps in ApcMin/þMstnþ/þ andApcMin/þMstn�/� mice

The small intestine was carefully dissected distally to thestomach and proximally to the cecum, and was cut intothree equal sections (upper, middle, and lower intestine).The colon was also removed. All sections were flushed withPBS, opened longitudinally, and fixed in 10% paraformalde-hyde for 24 hours. Sections were then rinsed with PBS (3 �12 hours) and stained in 0.1% methylene blue for 3 hours.Polyps were counted under a dissecting microscope andwere categorized as <1 mm, 1 to 2 mm, 2 to 3 mm, and >3mm diameter.

Statistical analysisStatistical comparisons were assessed across multiple con-

ditions using a two-way ANOVA, with the Student–Newman–Keuls posthoc test used to identify specific differences betweenmeans (GraphPad Prism 5.03). Student's t test was used forcomparisons between two conditions. Difference in survivalrates was determined by the c2 test. To account for differencesin tumor weight at death between wild-type and Mstn�/�

tumor-bearing mice, an analysis of covariance was performed(SuperANOVA; Abacus Concepts). The a-level of significancewas set at 0.05 for all comparisons. Data are presented asmean � SE.

ResultsMstn gene inactivation prevents loss of skeletal musclemass in LLC tumor-bearing mice

Body mass, fat mass, gastrocnemius muscle mass, and pro-tein content were all decreased in wild-type tumor-bearingmice (Fig. 1A–D). In contrast, Mstn loss attenuated thedecrease in body mass (Fig. 1A), fat mass (Fig. 1B), andcompletely prevented loss of skeletal muscle mass (Fig. 1C)and protein content (Fig. 1D). Protection against musclewasting in Mstn�/� tumor-bearing mice was also illustratedby the preservation of plasma amino acid concentration (Fig.1E and Supplementary Fig. S1). Prevention of skeletal musclemass loss was also observed in extensor digitorum longus,tibialis anterior, and soleusmuscles ofMstn�/� tumor-bearingmice (Supplementary Fig. S2). Importantly, caloric intake was

not different between groups (Supplementary Fig. S3). Mstnloss thus protects hind limb skeletal muscle against atrophyduring cancer cachexia.

Mstn gene inactivation decreases LLC tumor growthTumor growth was observed in 100% of mice, but tumor

weight was significantly lower in Mstn�/� mice comparedwith wild-type mice (Fig. 2A). Importantly, Mstn per se didnot alter the in vitro proliferative characteristics of LLC cells(Supplementary Fig. S4A–S4C). Expression of angiogenesisand tumorigenesis markers was therefore determined intumor tissue. mRNA levels of vascular endothelial growthfactor (VEGF) A, a major driver of tumor angiogenesis andgrowth (20), glucose transporter 1 (GLUT1), the predomi-nant glucose transporter in many types of cancer cells (21),and hypoxia-inducible factor (HIF)-1a, a transcriptionalactivator of VEGF and GLUT1 gene expression (22), weresignificantly lowered in the tumors of Mstn�/� mice (Fig.2B). Targeting activin signaling has recently been shown toinhibit angiogenesis and tumorigenesis (23). Activins arehomo- or heterodimeric proteins of various b-subunit iso-forms referred to as activin A (bA-bA), activin AB (bA/bB),and activin B (bB/bB; ref. 24). In the present study, mRNAlevels of both bA and bB subunits were significantly lower inthe tumors of Mstn�/� mice (Fig. 2C). Expression of bothtype II (ActRIIB) and type I (Alk4) receptors, which trans-duce activin signal (24), was also lowered in the tumors ofMstn�/� mice (Fig. 2C).

The possibility that Mstn loss reduces tumor growth byincreasing apoptosis of tumor cells was also investigated.Consistent with this hypothesis, mRNA level of TWEAK, aninducer of apoptosis (25), Bax protein content, Bax-to-Bcl-2protein ratio, and caspase-3 enzyme activity were higher in thetumors of Mstn�/� mice (Fig. 2D–G and Supplementary Fig.S4D). Altogether, these results indicate that Mstn lossrepresses the expression of genes involved in angiogenesis andtumorigenesis, and increases apoptosis.

Mstn gene inactivation attenuates the activation ofproteolytic pathways in skeletal muscle of LLC tumor-bearing mice

Mstn loss can prevent muscle wasting by regulating thebalance between protein synthesis and/or degradation. Wefirst explored the regulation of the Akt/mTOR pathway, acrucial regulator of skeletal muscle protein synthesis whoseactivation prevents muscle atrophy in vivo (26, 27). However,phosphorylation levels of downstream effectors of the path-way, including p70S6K, rpS6, and the translation repressor 4E-binding protein (4E-BP) 1, were all significantly decreased inresponse to tumor growth in the gastrocnemiusmuscle of bothwild-type and Mstn�/� mice (Fig. 3).

We next investigated whether Mstn loss in tumor-bearingmice was associated with an inhibition of the ubiquitin-proteasome pathway. Increase in the expression of muscle-specific E3 ubiquitin ligases (MAFbx/Atrogin-1 and MuRF1)induced by tumor growth in the gastrocnemius muscle ofwild-type mice was completely blunted in Mstn�/� tumor-bearing mice (Fig. 4A and B). Transcript level of forkhead

Gallot et al.

Cancer Res; 74(24) December 15, 2014 Cancer Research7346




box O (FoxO) 3, a transcription factor involved in theexpression of MAFbx/Atrogin-1 and MuRF1 (28, 29), wasalso markedly reduced in Mstn�/� tumor-bearing mice (Fig.4C). However, FoxO3 phosphorylation level remainedunchanged (Fig. 4D). Active phosphorylated form of p65, aconstituent of NF-kB transcription factor involved in the

expression of MuRF1 (30), was also 3-fold lower in Mstn�/�

tumor-bearing mice compared with wild-type tumor-bear-ing mice (Fig. 4E). Phosphorylation of IkBa on Ser32 andSer36 allows the release of NF-kB by IkBa. IkBa phosphor-ylation, which was markedly increased in the gastrocnemiusmuscle of wild-type mice in response to tumor growth,

Figure 1. Mstn gene inactivation prevents loss of skeletal muscle mass in tumor-bearing mice. A, changes in body weight during the course of cancercachexia. B, visceral fat mass. C, gastrocnemius muscle mass. D, gastrocnemius muscle protein content. E, plasma concentration of total amino acids.Wild-type and Mstn�/� mice were inoculated with LLC cells or received DPBS. Tissues were removed 35 days after the inoculation of LLC cells. Data aremeans � SE (n ¼ 8–12/group). �, P < 0.05; ��, P < 0.01; ��, P < 0.001.






remained unchanged in Mstn�/� tumor-bearing mice (Fig.4F). Accordingly, transcript levels of TWEAK, a skeletalmuscle-wasting cytokine (31), and TRAF6, an E3 ubiquitinligase and adaptator protein required for the activation ofNF-kB in response to TWEAK (32), were downregulated inMstn�/� tumor-bearing mice (Fig. 4G and H).

We also monitored the expression of autophagy-relatedgenes (33, 34). Cytoplasmic and membrane-bound forms ofmicrotubule-associated protein light chain 3 (LC3) b, a pro-totypical marker of autophagy (35), were significantlyincreased in gastrocnemiusmuscle of wild-type tumor-bearingmice. No variation was observed in Mstn�/� tumor-bearingmice (Fig. 5A). Ulk1 mRNA level and Atg5–Atg12 proteincomplex were also lowered in Mstn�/� mice in response to

tumor growth compared with wild-type mice (Fig. 5B and C).Collectively, all these findings indicate that Mstn loss attenu-ates the activation of proteolytic pathways in skeletal muscleduring cancer cachexia.

Mstn/activin signaling in skeletal muscle of LLC tumor-bearing mice

Elevated expression of activins promotes muscle wastingand cachexia by upregulating the expression of MAFbx/Atrogin-1 (36), and activation of activin signaling are alsocritical in triggering cachexia (5, 37). We therefore deter-mined whether Mstn loss possibly altered activin signaling.Surprisingly, Mstn transcript level remained unchanged inwild-type tumor-bearing mice (Fig. 6A). Transcript levels of

Figure 2. Mstn gene inactivationreduces tumor growth andexpression of genes involved intumor metabolism andangiogenesis, and activin signalingwhile increasing apoptosis. A,tumor weight. B, VEGF-A, GLUT1,and HIF-1a transcript levels in thetumors of wild-type and Mstn�/�

mice. C, activin bA, activin bB,ActRIIB, and Alk4 transcript levelsin the tumors of wild-type andMstn�/�mice.D, TWEAK transcriptlevel in the tumors of wild-type andMstn�/�mice. E andF, immunoblotanalysis of Bax protein content (E)and Bax-to-Bcl-2 protein ratio(F) in the tumors of wild-type andMstn�/� mice. G, fluorometricmeasurement of caspase-3enzyme activity in the tumors ofwild-type and Mstn�/� mice.Wild-type and Mstn�/� mice wereinoculated with LLC cells. Tissueswere removed 35 days after theinoculation of LLC cells. Dataare means � SE (n ¼ 8–12/group).�, P < 0.05; ��, P < 0.01;��, P < 0.001.

Gallot et al.





activin bA and ActRIIB were decreased in response to tumorgrowth both in wild-type and Mstn�/� mice (Fig. 6B and C),but transcript levels of Alk4 and Alk5 were reduced inMstn�/� mice by tumor growth (Fig. 6D and E). Finally,Smad2/3 phosphorylation and Smad2/3 protein contentwere similar in wild-type and Mstn�/� mice, and remainedunchanged in response to tumor growth (Fig. 6F). There-fore, Mstn loss does not seem to markedly alter Mstn/activin signaling in skeletal muscle 35 days after inoculationof LLC cells.

Mstn gene inactivation dramatically prolongs survival ofLLC tumor-bearing mice and ApcMin/þ miceLLC tumor-bearing mice manifested a lethal wasting

syndrome characterized by progressive weight loss (Fig.1A) and death (Fig. 7A). Notably, Mstn loss was associatedwith a significant prolongation of animal survival (Fig. 7A).We therefore examined the efficacy of Mstn gene inactiva-tion in ApcMin/þ mouse, an established model of colorectalcancer (17, 38) and cachexia (18) that carries a mutation inthe adenomatous polyposis coli (Apc) gene, leading to mul-tiple intestinal neoplasia (Min; ref. 17). In human, mutationin Apc gene is responsible for the initiation and development

of familial adenomatous polyposis (38), an autosomal dom-inant inherited disorder characterized by cancer of the largeintestine. The advantage of ApcMin/þ mice is the regularprogression of tumor growth and muscle wasting (18) that ismore closely related to human cancer cachexia, comparedwith the inoculation of tumor cells, which triggers extremelyfast tumor growth and muscle wasting. On heterozygousMstnþ/� and homozygous Mstn�/� genetic backgrounds,survival of ApcMin/þ mouse was remarkably prolonged(Fig. 7B). When 100% of ApcMin/þMstnþ/þ mice had died29 weeks after birth, about 90% of ApcMin/þMstnþ/� andApcMin/þMstn�/� mice were still alive. Of note, wild-type andMstn�/� mice had similar lifespan under normal conditions(Supplementary Fig. S5A). Increase in the survival rate ofApcMin/þMstn�/� mice was associated with a reduction inthe number and size of intestinal polyps (Fig. 7C and D andSupplementary Fig. S5B). Development of polyposis is usu-ally associated with anemia and bloody feces (17). Measure-ment of hematocrit is therefore a good criterion to assess thehealth status of the animals. Although hematocrit wascollapsed in ApcMin/þMstnþ/þ mice, it was completely pre-served in ApcMin/þMstn�/� mice (Fig. 7E). Finally, Mstn losson the ApcMin/þ background attenuated the decrease in body

Figure 3. Akt/mTOR pathway isinhibited in gastrocnemius muscleof wild-type and Mstn�/� miceduring cancer cachexia. A,immunoblot analysis of 4E-BP1phosphorylation on Thr37 andThr46. B, Luminex analysis ofp70S6K phosphorylation onThr421 and Ser424. C, immunoblotanalysis of rpS6 phosphorylationon Ser235 and Ser236 and rpS6protein content. Wild-type andMstn�/� mice were inoculated withLLC cells or received DPBS.Tissues were removed 35 daysafter the inoculation of LLCcells. Data are means � SE(n ¼ 8–12/group). �, P < 0.05;��, P < 0.01; ��, P < 0.001.






Figure 4. Mstn gene inactivation attenuates the activation of ubiquitin-proteasome pathway and NF-kB pathway in gastrocnemius muscle duringcancer cachexia. A–C, transcript levels of MAFbx/Atrogin-1 (A), MuRF1 (B), and FoxO3 (C). D, immunoblot analysis of FoxO3 phosphorylationon Ser253. E, immunoblot analysis of NF-kB p65 phosphorylation on Ser536 and NF-kB p65 protein content. F, Luminex analysis of IkBaphosphorylation on Ser32 and Ser36 and IkBa protein content. G and H, transcript levels of TWEAK (G) and TRAF6 (H). Wild-type and Mstn�/�

mice were inoculated with LLC cells or received DPBS. Tissues were removed 35 days after the inoculation of LLC cells. Data are means � SE(n ¼ 8–12/group). �, P < 0.05; ��, P < 0.01; ��, P < 0.001.

Gallot et al.





mass (Fig. 7F), and completely prevented the loss of skeletalmuscle mass (Fig. 7G).

DiscussionCancer-associated cachexia is a multifactorial devastating

syndrome that increases toxicity from chemotherapy,increases surgery risk, reduces physical performance andpatient's quality of life, and ultimately decreases survival inpatients with cancer (4). Although muscle wasting, the keyfeature of cancer-associated cachexia, is recognized as a factorresponsible for the death of many patients with cancer (3), itremains a poorly understood process whose molecularmechanisms are only beginning to emerge. The present studyclearly indicates that targeting Mstn is an effective strategy toprevent skeletal muscle wasting, to slow tumor growth, and toincrease survival rate in LLC tumor-bearingmice and ApcMin/þ

mice.Mechanistically, Mstn can promote muscle catabolism by

repressing protein synthesis (9, 39) and increasing proteindegradation (39). Our findings showing that Mstn loss did

not attenuate the inhibition of the Akt/mTOR pathwayinduced by tumor growth, but drastically attenuated theactivation of proteolytic pathways, clearly indicate thatMstn loss provides protection against cancer-induced mus-cle wasting by repressing the activation of proteolytic path-ways. However, we cannot exclude the possibility thatprotein synthesis could be regulated differently earlierduring the time course of cancer cachexia. The mechanismssignaling the cachectic state have been controversial, butcancer cachexia is considered to result, at least in part, frominteractions between the host and the tumor through theproduction of tumor- and host-derived cachectic factors (2).In the present study, transcript levels of TWEAK, a powerfulskeletal muscle-wasting cytokine (31), and that of TRAF6, itsdownstream effector (32), were markedly reduced in skel-etal muscle of Mstn�/� tumor-bearing mice. Furthermore,we also observed that circulating levels of catabolic cyto-kines are lowered in Mstn�/� mice compared with wild-typemice under normal conditions (Supplementary Fig. S6).Therefore, Mstn loss could contribute favorably to regulat-ing the level of cachectic factors. However, additional

Figure 5. Mstn gene inactivationattenuates the activation ofautophagy-lysosome pathway ingastrocnemius muscle duringcancer cachexia. A, immunoblotanalysis of LC3b-I and LC3b-IIprotein content. B, Ulk1 transcriptlevel. C, immunoblot analysis ofAtg5–Atg12protein complex.Wild-type and Mstn�/� mice wereinoculated with LLC cells orreceived DPBS. Tissues wereremoved 35 days after theinoculation of LLC cells. Dataare means � SE (n ¼ 8–12/group).�, P < 0.05; ��, P < 0.01;��, P < 0.001.






investigations will be necessary to clearly determine therole of Mstn in the regulation of the production of cachecticfactors.

Myostatin and activins signal by binding to ActRIIA orActRIIB, which then recruits and activates type-I activinreceptors, Alk 4 and 5. Neither transcript levels of activin bA,

Figure 6. Mstn/activin signaling ingastrocnemius muscle duringcancer cachexia. A, Mstntranscript level. B–E, transcriptlevels of activin bA (B), ActRIIB(C), Alk4 (D), and Alk5 (E). F,immunoblot analysis of Smad2/3phosphorylation on Ser465/467and Ser423/425 and Smad2/3protein content. Wild-type andMstn�/� mice were inoculatedwith LLC cells or received DPBS.Tissues were removed 35 daysafter the inoculation of LLCcells. Data are means � SE(n ¼ 8–12/group). �, P < 0.05;��, P < 0.01; ��, P < 0.001.

Gallot et al.





ActRIIB, Alk4 and Alk5, nor Smad2/3 phosphorylation, wereconsistently regulated in wild-type and Mstn�/� mice inresponse to tumor growth, suggesting that myostatin/activinsignaling was not impaired 35 days after inoculation of LLCcells. However, it is possible that myostatin/activin signalingwas differently regulated between wild-type and Mstn�/�

mice earlier during the time course of tumor growth. Insupport of this assumption, myostatin expression also

remained unchanged in skeletal muscle of wild-type micein response to tumor growth at day 35, an observation thatcontrasts with all the studies that measured myostatin expres-sion in response to tumor growth, but at earlier time points(11–16). Therefore, a stimulation of myostatin/activin signal-ing inwild-typemice could occur earlier during the time courseof tumor growth. Finally, a similar level of Smad 2/3 phos-phorylation was observed between wild-type and Mstn�/�

Figure 7. Mstn gene inactivationprolongs lifespan of LLC tumor-bearing mice and ApcMin/þ mice.A, survival rate of wild-type andMstn�/� tumor-bearing mice.B, Kaplan–Meier plot showingthat Mstn gene inactivationdramatically prolongs lifespanof ApcMin/þMstnþ/� andApcMin/þMstn�/� mice. C,representative longitudinalsections of intestine stained withmethylene blue showingmultiple intestinal polyps inApcMin/þMstnþ/þ. D, the numberand size of intestinal polyps arereduced in ApcMin/þMstn�/� mice.E, hematocrit in ApcMin/þMstnþ/þ

andApcMin/þMstn�/�mice. F,Mstngene inactivation prevents bodyweight loss in ApcMin/þ mice. G,prevention of skeletal musclewasting in ApcMin/þMstn�/� mice.Mice were 23.3 � 0.9 weeks old atthe time of analysis. Data aremeans� SE (n ¼ 4–7/group). ��, P < 0.01;��, P < 0.001.






mice at day 35, suggesting that other signaling influences mayexert a stimulatory effect on this pathway in Mstn�/� mice.

Preservation of skeletal muscle mass in Mstn�/� tumor-bearing mice and ApcMin/þMstn�/�mice is in line with studiesshowing that sequestration of extracellular activins and Mstnby administration of a soluble form of ActRIIB exerts antic-atabolic effects in mice models of cancer-cachexia (15, 16).However, our study extends these observations by showingthat Mstn per se is a critical determinant of muscle masshomeostasis during cancer cachexia. Onemay questionwheth-er the double-muscling phenotype ofMstn�/�mice is the basisfor protection against muscle wasting. Previous studies haveclearly demonstrated that the blockade of ActRIIB signaling atthe time of tumor development in mice also provides protec-tion against cancer cachexia (15, 16). Although the strategyused in these studies inhibits both Mstn and activin signaling,these data strongly suggest that Mstn inhibition confers resis-tance to skeletal muscle atrophy. This also raises the questionof whether blocking activin may provide additional protectionagainst cancer cachexia. An increase in activin level has beenreported in many catabolic states, including cancer cachexia(5). Increasing circulating level of activin A in mice alsopromotes muscle wasting, an effect that is not dependent onMstn signaling (36). Furthermore, inhibition of ActRIIB sig-naling leads to muscle hypertrophy both in wild-type andMstn�/� mice (40). Similar results have also been obtained byusing an anti-ActRII antibody (41). Together, these data sup-port an important role of activins in the regulation of skeletalmusclemass during cachexia. However, soluble ActRII can alsobe a target for other ligands, such as bone morphogeneticproteins (42), whose positive action on skeletal muscle homeo-stasis has been recently demonstrated (43, 44). Therefore, thebeneficial effect of inhibiting both Mstn and activins throughan ActRII blockade could be counterbalanced by the inhibitionof bonemorphogenetic proteins. Specific inhibition of Mstn oractivins in the context of cancer cachexia should allow dis-criminating between the respective effects of Mstn andactivins.

One striking finding of the present study was the reductionin tumor growth in Mstn�/� mice, an effect that can beattributed to a decrease in tumor metabolism and angiogen-esis, and an increase in apoptosis. Tumor growth was alsomarkedly slowed in ApcMin/þMstn�/� mice, together with apreservation of body weight and skeletal muscle mass.Although a reduction in tumor growth has not been system-atically observed (14, 15), the current findings are consistentwith a previous study demonstrating that administration of asoluble ActRIIB slowed LLC tumor growth in mice (16). Ourdata are also further supported by genetic and pharmacologicstudies showing that reduction/inhibition of ActRII signalingslows tumor development in gonads (45–48). Taken together,our results identify Mstn as a potential growth-promotingfactor in intestinal tumor progression. Therefore, the currentfindings have implications in the design of new drugs andstrategies for the treatment of tumors of the intestine andcolon cancer.

It is assumed that up to 20% of cancer-related mortalitiesmay derive from cachexia rather than direct tumor burden (2).

Here, we clearly show that prevention of muscle wasting andreduction in tumor growth in Mstn�/� mice was associatedwith an increase in survival rate. The reduction in tumorgrowth probably contributes to increasing the survival rateof Mstn�/� tumor-bearing mice. However, this antitumori-genic effect alone does not explain the prolonged survival ofLLC tumor-bearing mice: when a covariance analysis was usedto account for differences in tumor weight, survival rate wasstill significantly increased inMstn�/� tumor-bearingmice. Anassociation between preservation of skeletal muscle mass,reduction in tumor growth, and an increase in survival ratedoes not prove causality, but mechanistic links between thesefactors are biologically possible. Skeletal muscle constitutes animportant reservoir of amino acids. An increase in the circu-lating pool of amino acids, due to excessive skeletal muscleprotein degradation, may increase the availability of glucoseprecursors for liver gluconeogenesis, and ultimately provideenergy in glucose form for tumor metabolism (49). Alterna-tively, acute respiratory failure is a frequent fatal event inpatients with cancer (2). Preservation of muscle mass andfunction would therefore be beneficial by maintaining respi-ratory muscle function and could thus contribute to prolong-ing lifespan. Finally, preservation of skeletal muscle mass isalso critical to limit the decrease in physical activity that occurswith the progression of the disease, and ultimately to delaywhole body deconditioning.

The present study indicates that preserving skeletal musclemass should be one of the goals of interventions aimed atprolonging survival in patients with advanced cancers. How-ever, further studies will be necessary to precisely determinethe antitumorigenic function of Mstn inhibition. We also needmore information about the circulating level ofMstn and otherActRII ligands (activins, inhibins, bone morphogenetic pro-teins) in patients with cancer. Possible differences in theexpression of Mstn and other ActRII ligands between cancertypes should be also determined. Finally, cachexia is not onlyencountered in cancer, but also in numerous diseases, includ-ing sepsis, chronic heart failure, renal failure, pulmonarydiseases, prolonged immobilization, and neurogenic atrophy.Thus, the development of knowledge and strategies for thetreatment of cancer cachexia may also be applicable to otherpathologic situations associated with muscle wasting andexcessive Mstn signaling.

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

Authors' ContributionsConception and design: Y.S. Gallot, D.G. FreyssenetDevelopment of methodology: Y.S. Gallot, A.-C. Durieux, D.G. FreyssenetAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.):Y.S. Gallot, A.-C. Durieux, J. Castells, M.M. Desgeorges,B. Vernus, L. Plantureux, D. R�emond, V.E. Jahnke, D. Dardevet, G. Nemoz,A. Bonnieu, D.G. FreyssenetAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y.S. Gallot, A.-C. Durieux, D. R�emond, V.E. Jahnke,D. Dardevet, G. Nemoz, D.G. FreyssenetWriting, review, and/or revision of the manuscript: Y.S. Gallot, E. Lefai,G. Nemoz, L. Schaeffer, D.G. FreyssenetAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): Y.S. Gallot, J. Castells, D.G. FreyssenetStudy supervision: Y.S. Gallot, D.G. Freyssenet

Gallot et al.





AcknowledgmentsThe authors thank David Arnould for his help in animal genotyping.

Grant SupportThis work was supported by grants from the Ligue Contre le Cancer and the

Institut National du Cancer (INCa).

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 indicate thisfact.

Received January 15, 2014; revised September 12, 2014; accepted October 1,2014; published OnlineFirst October 21, 2014.

References1. Fearon K, Strasser F, Anker SD, Bosaeus I, Bruera E, Fainsinger RL,

et al. Definition and classification of cancer cachexia: an internationalconsensus. Lancet Oncol 2011;12:489–95.

2. Tisdale MJ. Mechanisms of cancer cachexia. Physiol Rev 2009;89:381–410.

3. Tisdale MJ. Cancer cachexia. Curr Opin Gastroenterol 2010;26:146–51.

4. Tisdale MJ. Cachexia in cancer patients. Nat Rev Cancer 2002;2:862–71.

5. Han HQ, Zhou X, Mitch WE, Goldberg AL. Myostatin/activin pathwayantagonism: molecular basis and therapeutic potential. Int J BiochemCell Biol 2013;45:2333–47.

6. McPherronAC, Lawler AM, LeeSJ.Regulationof skeletalmusclemassin mice by a new TGF-beta superfamily member. Nature 1997;387:83–90.

7. Grobet L, Pirottin D, Farnir F, Poncelet D, Royo LJ, Brouwers B, et al.Modulating skeletal muscle mass by postnatal, muscle-specific inac-tivation of the myostatin gene. Genesis 2003;35:227–38.

8. Durieux AC, Amirouche A, Banzet S, Koulmann N, Bonnefoy R,Pasdeloup M, et al. Ectopic expression of myostatin induces atrophyof adult skeletal muscle by decreasing muscle gene expression.Endocrinology 2007;148:3140–7.

9. Amirouche A, Durieux AC, Banzet S, KoulmannN, Bonnefoy R,MouretC, et al. Down-regulation of Akt/mammalian target of rapamycinsignaling pathway in response to myostatin overexpression in skeletalmuscle. Endocrinology 2009;150:286–94.

10. Zimmers TA, Davies MV, Koniaris LG, Haynes P, Esquela AF, Tom-kinson KN, et al. Induction of cachexia in mice by systemically admin-istered myostatin. Science 2002;296:1486–8.

11. Costelli P,MuscaritoliM, Bonetto A, Penna F, Reffo P, BossolaM, et al.Muscle myostatin signalling is enhanced in experimental cancercachexia. Eur J Clin Invest 2008;38:531–8.

12. Liu CM, Yang Z, Liu CW, Wang R, Tien P, Dale R, et al. MyostatinantisenseRNA-mediatedmuscle growth innormal andcancer cachex-ia mice. Gene Ther 2008;15:155–60.

13. Bonetto A, Penna F, Minero VG, Reffo P, Bonelli G, Baccino FM, et al.Deacetylase inhibitors modulate the myostatin/follistatin axis withoutimproving cachexia in tumor-bearing mice. Curr Cancer Drug Targets2009;9:608–16.

14. Benny KlimekME, Aydogdu T, LinkMJ, PonsM, Koniaris LG, ZimmersTA. Acute inhibition of myostatin-family proteins preserves skeletalmuscle in mouse models of cancer cachexia. Biochem Biophys ResCommun 2010;391:1548–54.

15. Zhou X, Wang JL, Lu J, Song Y, Kwak KS, Jiao Q, et al. Reversal ofcancer cachexia and muscle wasting by ActRIIB antagonism leads toprolonged survival. Cell 2010;142:531–43.

16. Busquets S, Toledo M, Orpi M, Massa D, Porta M, Capdevila E, et al.Myostatin blockage using actRIIB antagonism in mice bearing theLewis lung carcinoma results in the improvement of muscle wastingand physical performance. J Cachexia Sarcopenia Muscle 2012;3:37–43.

17. Moser AR, Pitot HC, Dove WF. A dominant mutation that predisposestomultiple intestinal neoplasia in themouse. Science 1990;247:322–4.

18. Baltgalvis KA, Berger FG, Pena MM, Davis JM, Muga SJ, Carson JA.Interleukin-6 and cachexia in ApcMin/þ mice. Am J Physiol RegulIntegr Comp Physiol 2008;294:R393–401.

19. Berthon P, Duguez S, Favier FB, Amirouche A, Feasson L, Vico L, et al.Regulation of ubiquitin-proteasome system, caspase enzyme activi-

ties, and extracellular proteinases in rat soleus muscle in response tounloading. Pflugers Arch 2007;454:625–33.

20. Rapisarda A,Melillo G. Role of the VEGF/VEGFR axis in cancer biologyand therapy. Adv Cancer Res 2012;114:237–67.

21. Macheda ML, Rogers S, Best JD. Molecular and cellular regulation ofglucose transporter (GLUT) proteins in cancer. J Cell Physiol2005;202:654–62.

22. SemenzaGL. Hydroxylation of HIF-1: oxygen sensing at themolecularlevel. Physiology 2004;19:176–82.

23. Hu-Lowe DD, Chen E, Zhang L, Watson KD, Mancuso P, Lappin P,et al. Targeting activin receptor-like kinase 1 inhibits angiogenesis andtumorigenesis through a mechanism of action complementary to anti-VEGF therapies. Cancer Res 2011;71:1362–73.

24. Antsiferova M, Werner S. The bright and the dark sides of activin inwound healing and cancer. J Cell Sci 2012;125:3929–37.

25. Chicheportiche Y, Bourdon PR, Xu H, Hsu YM, Scott H, Hession C,et al. TWEAK, a new secreted ligand in the tumor necrosis factor familythat weakly induces apoptosis. J Biol Chem 1997;272:32401–10.

26. Rommel C, Bodine SC, Clarke BA, Rossman R, Nunez L, Stitt TN, et al.Mediation of IGF-1-induced skeletal myotube hypertrophy by PI(3)K/Akt/mTOR and PI(3)K/Akt/GSK3 pathways. Nat Cell Biol 2001;3:1009–13.

27. Bodine SC, Stitt TN, Gonzalez M, Kline WO, Stover GL, Bauerlein R,et al. Akt/mTOR pathway is a crucial regulator of skeletal musclehypertrophy and can prevent muscle atrophy in vivo. Nat Cell Biol2001;3:1014–9.

28. Sandri M, Sandri C, Gilbert A, Skurk C, Calabria E, Picard A, et al. Foxotranscription factors induce the atrophy-related ubiquitin ligase atro-gin-1 and cause skeletal muscle atrophy. Cell 2004;117:399–412.

29. Stitt TN, Drujan D, Clarke BA, Panaro F, Timofeyva Y, Kline WO, et al.The IGF-1/PI3K/Akt pathway prevents expression of muscle atrophy-induced ubiquitin ligases by inhibiting FOXO transcription factors. MolCell 2004;14:395–403.

30. Cai D, Frantz JD, Tawa NE Jr, Melendez PA, Oh BC, Lidov HG, et al.IKKbeta/NF-kappaB activation causes severemuscle wasting inmice.Cell 2004;119:285–98.

31. Mittal A, Bhatnagar S, Kumar A, Lach-Trifilieff E, Wauters S, Li H, et al.The TWEAK-Fn14 system is a critical regulator of denervation-inducedskeletal muscle atrophy in mice. J Cell Biol 2010;188:833–49.

32. Paul PK, Gupta SK, Bhatnagar S, Panguluri SK, Darnay BG, Choi Y,et al. Targeted ablation of TRAF6 inhibits skeletal muscle wasting inmice. J Cell Biol 2010;191:1395–411.

33. Mammucari C, Milan G, Romanello V, Masiero E, Rudolf R, Del PiccoloP, et al. FoxO3 controls autophagy in skeletal muscle in vivo. CellMetab 2007;6:458–71.

34. Zhao J, Brault JJ, Schild A, Cao P, Sandri M, Schiaffino S, et al. FoxO3coordinately activates protein degradation by the autophagic/lyso-somal and proteasomal pathways in atrophying muscle cells. CellMetab 2007;6:472–83.

35. KlionskyDJ,Abdalla FC,AbeliovichH,AbrahamRT,Acevedo-ArozenaA, Adeli K, et al. Guidelines for the use and interpretation of assays formonitoring autophagy. Autophagy 2012;8:445–544.

36. Chen JL, Walton KL, Winbanks CE, Murphy KT, Thomson RE, MakanjiY, et al. Elevated expression of activins promotes muscle wasting andcachexia. FASEB J 2014;28:1711–23.

37. Matzuk MM, Finegold MJ, Mather JP, Krummen L, Lu H, Bradley A.Development of cancer cachexia-like syndrome and adrenal tumors ininhibin-deficient mice. Proc Natl Acad Sci U S A 1994;91:8817–21.






38. Groden J, Thliveris A, Samowitz W, Carlson M, Gelbert L, Albertsen H,et al. Identification and characterization of the familial adenomatouspolyposis coli gene. Cell 1991;66:589–600.

39. Lokireddy S, Mouly V, Butler-Browne G, Gluckman PD, Sharma M,Kambadur R, et al. Myostatin promotes the wasting of human myo-blast cultures through promoting ubiquitin-proteasome pathway-mediated loss of sarcomeric proteins. Am J Physiol Cell Physiol2011;301:C1316–24.

40. Lee SJ, Reed LA, Davies MV, Girgenrath S, Goad ME, Tomkinson KN,et al. Regulation of muscle growth by multiple ligands signalingthrough activin type II receptors. Proc Natl Acad Sci U S A2005;102:18117–22.

41. Lach-Trifilieff E, Minetti GC, Sheppard K, Ibebunjo C, Feige JN,Hartmann S, et al. An antibody blocking activin type II receptorsinduces strong skeletal muscle hypertrophy and protects from atro-phy. Mol Cell Biol 2014;34:606–18.

42. Souza TA, Chen X, Guo Y, Sava P, Zhang J, Hill JJ, et al. Proteomicidentification and functional validation of activins and bone morpho-genetic protein 11 as candidate novel muscle mass regulators. MolEndocrinol 2008;22:2689–702.

43. Sartori R, Schirwis E, Blaauw B, Bortolanza S, Zhao J, Enzo E, et al.BMP signaling controls muscle mass. Nat Genet 2013;45:1309–18.

44. Winbanks CE, Chen JL, Qian H, Liu Y, Bernardo BC, Beyer C, et al. Thebone morphogenetic protein axis is a positive regulator of skeletalmuscle mass. J Cell Biol 2013;203:345–57.

45. Cipriano SC, Chen L, Kumar TR, Matzuk MM. Follistatin is amodulator of gonadal tumor progression and the activin-inducedwasting syndrome in inhibin-deficient mice. Endocrinology 2000;141:2319–27.

46. Coerver KA, Woodruff TK, Finegold MJ, Mather J, Bradley A, MatzukMM. Activin signaling through activin receptor type II causes thecachexia-like symptoms in inhibin-deficient mice. Mol Endocrinol1996;10:534–43.

47. Li Q, Kumar R, Underwood K, O'Connor AE, Loveland KL, Seehra JS,et al. Prevention of cachexia-like syndrome development and reduc-tion of tumor progression in inhibin-deficient mice following adminis-tration of a chimeric activin receptor type II-murine Fcprotein.MolHumReprod 2007;13:675–83.

48. Kumar TR, Palapattu G, Wang P, Woodruff TK, Boime I, Byrne MC,et al. Transgenic models to study gonadotropin function: the role offollicle-stimulating hormone in gonadal growth and tumorigenesis.MolEndocrinol 1999;13:851–65.

49. Argiles JM, Lopez-Soriano FJ. The energy state of tumor-bearing rats.J Biol Chem 1991;266:2978–82.


Gallot et al.




2014;74:7344-7356. Published OnlineFirst October 21, 2014.Cancer Res Yann S. Gallot, Anne-Cécile Durieux, Josiane Castells, et al. CancerMyostatin Gene Inactivation Prevents Skeletal Muscle Wasting in

Updated version

10.1158/0008-5472.CAN-14-0057doi:

Access the most recent version of this article at:

Material

Supplementary

http://cancerres.aacrjournals.org/content/suppl/2014/10/21/0008-5472.CAN-14-0057.DC1.html

Access the most recent supplemental material at:

Cited Articles

http://cancerres.aacrjournals.org/content/74/24/7344.full.html#ref-list-1

This article cites by 49 articles, 18 of which you can access for free at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

[email protected]

To request permission to re-use all or part of this article, contact the AACR Publications Department at



http://cancerres.aacrjournals.org/lookup/doi/10.1158/0008-5472.CAN-14-0057

http://cancerres.aacrjournals.org/content/suppl/2014/10/21/0008-5472.CAN-14-0057.DC1.html

http://cancerres.aacrjournals.org/content/74/24/7344.full.html#ref-list-1

http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

mailto:[email protected]


Myostatin Gene Inactivation Prevents Skeletal Muscle Wasting in Cancer

Documents

Transcript of Myostatin Gene Inactivation Prevents Skeletal Muscle Wasting in Cancer