1119Research Article

IntroductionCachexia consists of marked muscle wasting and atrophy associatedwith tumor load (Morley et al., 2006; Tisdale, 2002). This conditionlowers responsiveness to treatment, contributing to a unfavorableprognosis and poor quality of life.

Several mediators of muscle wasting have been identified. Theseinclude immune and tumor-derived cytokines such as tumor necrosisfactor (TNF!), interleukin (IL)-1, IFN" and IL6 (Argiles et al.,2006); in addition, angiotensin-mediated activation of caspases andp53 transcription factors have all been shown to have a role (Tisdale,2005). Most of these pathways mediate their effects by reducingthe rate of protein synthesis at the level of protein translation orRNA content and by stimulating protein catabolism through theactivation of the ubiquitin-proteasome pathway accompanied byinduction of the ubiquitin E3 atrophy markers, muscle RING finger-1 (MURF1 or TRI63) and muscle atrophy F-Box (known as MAFbx,atrogin-1; official symbol FBX32) (Tisdale, 2005). Theheterogeneity of these factors and their potentially synergistic modeof action has made targeting them a real challenge and has yieldedlittle clinical benefit: therapeutic options against the cachecticsyndrome are in fact still lacking.

Defective skeletal muscle regeneration also substantiallycontributes to muscle wasting (Coletti et al., 2005; Moresi et al.,2008). The key cells in muscle regeneration are satellite cells, whichare activated and undergo myogenic differentiation and fusion withdamaged muscle fibers or with themselves to produce new fibers(Charge and Rudnicki, 2004). A balance between cell proliferation,differentiation and fusion is required for correct muscle regenerationto occur. The mechanisms leading to impaired muscle regenerationin cachexia have not been fully investigated.

We recently found that necdin (NECD), a member of themelanoma antigen gene (MAGE) family of proteins, has key rolesin regeneration (Brunelli et al., 2004; Deponti et al., 2007). Here,

we show that necdin is central to the strategy operated by the muscleto physiologically counteract tumor-induced muscle wasting andidentify the mechanisms of necdin action that suggests it as a suitabletarget for therapeutic intervention in cachexia.

ResultsNecdin is a physiological inhibitor of colon-carcinoma-inducedmuscle wastingTo investigate whether necdin has a role in tumor-induced muscleatrophy, we initially measured its expression in muscles of cachecticmice. Two-month-old wild-type (WT) mice (F1C57!BalbC) wereinoculated with 106 colon-26 carcinoma cells (C26), a well-described model of cachexia-inducing tumor cells (Tanaka et al.,1990). Muscles were collected 6, 8, 10, 12 days after the injection.Necdin expression, measured by real-time PCR, was significantlyupregulated during the first phases of cancer-induced cachexia,followed by a decrease from day 10 onwards (Fig. 1A), suggestingthat upregulation of necdin is a physiological response of muscleto wasting.

To verify this hypothesis, MlcNec mice overexpressing theprotein in the muscle (Deponti et al., 2007), Ndn–/– (Muscatelli etal., 2000) and WT mice were inoculated with C26 cells as above.Treated mice underwent hypoglycemia and a substantial weight lossthat was at least partially due to loss of skeletal muscle mass (seebelow) (Fig. 1B,C). There were no significant differences in themean weight of the tumors in the three genotypes (mean weight,484.86±34 mg). The lack of necdin in the muscle of Ndn–/– miceundergoing cachexia resulted in a substantial and significantincrease of these events. By contrast, overexpression of necdin inMlcNec mice clearly protected against the systemic effects relatedto the growing tumor (Fig. 1B,C).

The mean muscle weight in tumor-bearing Ndn–/– mice wassignificantly lower than in WT animals, although it was significantly

Skeletal muscles of subjects with advanced cancer undergoprogressive wasting, referred to as cachexia. Cachexia is animportant area for medical research because strategies proposeduntil now have yielded little benefit. We have recently identifiednecdin as a key player in fetal and postnatal physiologicalmyogenesis and in muscle regeneration. Here we show thatnecdin is selectively expressed in muscles of cachetic mice andprove that its expression is causally linked to a protectiveresponse of the tissue against tumor-induced wasting, inhibition

of myogenic differentiation and fiber regeneration. Necdincarries out this role mainly via interference with TNF! signalingat various levels, including regulation of expression of TNFR1and p53, and regulation of the activity of caspase 3 and caspase9. These data suggest that inhibition of muscle wasting usingnecdin is a feasible approach to treat cachexia in neoplasticpatients.

Key words: Necdin, Cachexia, Muscle regeneration, TNF!

Summary

Necdin is expressed in cachectic skeletal muscle toprotect fibers from tumor-induced wastingClara Sciorati1, Thierry Touvier1, Roberta Buono1, Patrizia Pessina2, Stephanie François2, Cristiana Perrotta1,Raffaella Meneveri2, Emilio Clementi3,4,* and Silvia Brunelli1,2,*1Division of Regenerative Medicine, San Raffaele Scientific Institute, 20132 Milan, Italy2Department of Experimental Medicine, University of Milano-Bicocca, 20052 Monza, Italy3Department of Preclinical Sciences, LITA-Vialba, University of Milano, 20157 Milan, Italy4E. Medea Scientific Institute, 23842 Bosisio Parini, Italy*Authors for correspondence (e-mails: brunelli.silvia@hsr.it; emilio.clementi@unimi.it)

Accepted 27 December 2008Journal of Cell Science 122, 1119-1125 Published by The Company of Biologists 2009doi:10.1242/jcs.041665

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higher in MlcNec mice. (Fig. 1D). This was also true, although toa lesser extent, for the fat and liver weight (Fig. 1D).

The muscles of Ndn–/– mice appeared to be less well preservedand showed a lower density of fibers with respect to WT and MlcNecmice, with increased cell infiltrates, collagen accumulation andnecrotic fibers (Fig. 2A). Reduction in muscle weight wasaccompanied by a reduction of the fiber cross-section area, whichwas more marked in Ndn–/– mice and was less severe in MlcNecmice compared with the WT (Fig. 2B,C). This effect is not only aconsequence of the slight hypotrophy of the untreated Ndn–/– miceor the hypertrophy of the untreated MlcNec mice with respect to theuntreated WT. The decrease in fiber cross-section area of Ndn–/–

C26-treated mice compared with Ndn–/– PBS-treated mice is moreevident than the decrease observed in WT and MlcNec mice, andthe difference between the two groups is statistically significant (WT:+C26 vs +PBS, –22.63%; Ndn–/–: +C26 vs +PBS, –35.23%; MlcNec:+C26 vs +PBS, –15.58%; P<0.001 vs +PBS and WT) (Fig. 2C).

Changes in muscle weight and morphology were accompaniedby decreased levels of the muscle-specific proteins myosin heavychain, myogenin and MyoD in C26-inoculated WT mice (Fig.2D,E), consistent with the increased protein catabolism that occursduring tumor load (Acharyya et al., 2004; Langen et al., 2004;Tisdale, 2005). Changes were also associated with increasedexpression of atrogin-1 and MuRF1, which are specific markers ofmuscle wasting (Fig. 2F). In C26-inoculated Ndn–/– mice, loss ofmuscle-specific proteins increased, and levels of atrogin-1 andMuRF1 mRNA transcripts were also reduced. Protein catabolismand expression of atrogin-1 and MuRF1 were reduced in C26-inoculated MlcNec mice (Fig. 2D-F).

Real-time PCR analysis showed expression of embryonic myosin,a molecular hallmark of fiber regeneration, in C26-inoculated mice.Embryonic myosin levels were increased in MlcNec mice comparedwith the WT and were reduced in Ndn–/– mice (Fig. 2F), indicating

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that regeneration occurs at some extent in all mice but that it issignificantly enhanced when necdin is overexpressed. Increasedregeneration is also supported by the presence of centronucleatedfibers in MlcNec muscle (Fig. 2A, black arrows).

Thus, necdin overexpression in muscle is sufficient to counteracttumor-induced muscle wasting and its absence leads to anexacerbated phenotype.

The protective action of necdin is mediated through inhibitionof TNF!-dependent cachectogenic signalingWe next investigated the molecular mechanism underlying the effectof necdin in tumors. To this end, we designed an in vitro approachmimicking the situation observed in vivo. We exposeddifferentiating primary myoblasts (WT, MlcNec and Ndn–/–) andC2C12 cells (transfected with either pIRESGFPNecdin orpIRESGFP plasmid) (Deponti et al., 2007) to the supernatant ofconfluent C26 cells, or 3T3 fibroblasts as a control. The C26-conditioned medium inhibited myogenic differentiation, decreasedthe expression of the differentiation markers MyoD, myogenin andMyHC (Fig. 3A,C) and reduced the number of MyHC-positivemyotubes (Fig. 3B). This effect was counteracted by theoverexpression of necdin in C2C12 and MlcNec myoblasts andenhanced by necdin ablation (Ndn–/– myoblasts).

TNF! significantly contributes to cachexia (Zhou et al., 2003;Coletti et al., 2005); in addition, low concentrations of TNF! inhibitmyogenic differentiation without causing apoptosis, which ensuesat high concentrations of the cytokine (Alter et al., 2008; Coletti etal., 2002; Moresi et al., 2008). Oxidative stress (reactive oxygenspecies, ROS) accompanies and sustains the action of TNF!(Barreiro et al., 2005; Li et al., 2003).

Myoblasts from MlcNec, Ndn–/– and WT mice were differentiatedin the presence or absence of TNF! (5-20 ng/ml) or the ROS-generating As2O3 (2-5 µM). TNF! inhibited myogenic differentiation

Fig. 1. Necdin inhibits colon carcinoma-induced muscle wasting. (A) Necdinexpression measured by real-time PCR on TAmuscle from PBS-treated (‘C’) or C26-treatedanimals sacrificed 6, 8, 10 and 12 days aftertumor injection. Five animals were analyzedin parallel for each time. Results werenormalized to levels of the GAPDH mRNA.Error bars represent s.e.m.; **P<0.005 vsPBS-treated mice. (B) Body weight of bothPBS- and C26-treated animals was measuredthe day of tumor injection and every 2 days.Ten animals were analyzed in parallel foreach group: wild-type mice (wt), MlcNecmice and Ndn–/– mice treated with C26.Results are expressed as % ± s.e.m. of weightobtained for PBS-treated animals of eachgroup (PBS) (n=10 per group). The meanweight ± s.d. of all the PBS-treated animalswas 21.60±0.52 g on the day of injection and24.95±0.88 g 10 days later. *P<0.01 vs WT.(C) Glycemia measurement. Serum glucoselevel of PBS- and C26-treated animals wasmeasured 8 days after tumor injection. Tenanimals were analyzed in parallel for eachC26-treated group: wild-type mice (wt),MlcNec mice and Ndn–/– mice. Error barsrepresent s.e.m.; *P<0.01 vs WT; +P<0.01 vsPBS treated mice. (D) TA, gastrocnemius (Gst) and quadriceps (Quad) muscles and liver and epydidimal fat (Ep.fat) were isolated from both PBS- and C26-treatedanimals 12 days after tumor injection and weighed. Ten animals were analyzed in parallel for each group: wild-type mice (wt), MlcNec mice and Ndn–/– mice.Error bars represent s.e.m.; *P<0.01 and **P<0.005 vs WT; +P<0.01 vs PBS-treated mice. PBS results in B-D are mean values from PBS-treated animals of allthree genotypes.

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and decreased expression of MyoD, myogenin and MyHC. The effectof the cytokine was significantly enhanced in Ndn–/– cells (Fig.3B,C,E) and inhibited in MlcNec cells. These results were confirmedin C2C12 cells, where necdin overexpression overcame the inhibitionof differentiation induced by TNF! and As2O3, and maintained theexpression of MyoD, myogenin and MyHC (Fig. 3B,C,E).

We decided next to investigate how necdin interacts with theTNF! pathway. We examined whether the reduced effect of TNF!in necdin-overexpressing cells was related to a differentialexpression of its p55kD receptor (TNFRI). The amount of TNFRI

mRNA transcript measured by real-time PCR in myoblasts fromthe three mouse genotypes was comparable (Fig. 4A). By contrast,the amount of TNFRI exposed at the plasma membrane of myoblastscells measured by FACS analysis, was decreased in MlcNec cells,and increased in Ndn–/– mice with respect to the WT (Fig. 4B). Therole of necdin was confirmed by overexpression of the protein inC2C12 cells (Fig. 4B).

A key protein mediating TNF!-induced cachexia and itsinhibition of myogenesis is p53 (Schwarzkopf et al., 2006).Exposure of myoblasts to TNF! during differentiation increased

Fig. 2. Necdin inhibits muscle proteincatabolism induced by colon carcinoma.(A) Histology of TA muscle.Representative histological images ofH&E (top two rows) or Azan-Mallory(bottom row) stained sections of TAmuscles of mice sacrificed 12 days afterPBS (top row) or tumor injection (middleand bottom rows). Ndn–/– C26-treatedmuscles show increased cell infiltrates,collagen accumulation and necrotic fibers(brown arrow). Centronucleated fiberscan be seen in MlcNec C26-treatedmuscle (black arrows). Scale bar:100 µm. (B) Distribution of cross-sectional area of TA fibers was analyzedon sections obtained from PBS- (red, blueand green) and C26-treated (yellow, lightblue and light green) animals, 12 daysafter tumor injection (n=5). Ten H&Estained sections and a total of 300 fiberswere measured for the different groups(WT mice, red and yellow lines; MlcNec,blue and light blue lines; Ndn–/– mice,green and light green lines). (C) Meancross-section area (XSA) of TA musclefibers from PBS- and C26-treatedanimals, 12 days after tumor injection(n=5). *P<0.05 and **P<0.01 vs WTmice; ++P<0.01 vs PBS-treated mice.(D) Expression of myosin heavy chain(MHC), myogenin, MyoD orglyceraldehyde phosphate dehydrogenase(GAPDH) in TA of animals sacrificed 12days after PBS or C26 tumor cellinjection. Images are representative ofthree different experiments with musclesisolated from five animals for each groupof wild type (WT), MlcNec or Ndn–/–

mice. (E) Graphs showing the meanvalues ± s.e.m. obtained for the ratio ofprotein bands:GAPDH band values of theexperiments represented in Fig. 2D (n=3).Error bars represent s.e.m. *P<0.05 and**P<0.01 respectively vs WT; +P<0.01vs PBS. (F) Embryonic myosin(eMyHC), atrogin-1 (Atg1) and MuRF1expression was measured by real-timePCR on TA muscle from C26-treatedanimals sacrificed 10 days after tumorinjection. Five animals were analyzed inparallel for each group: wild-type mice(WT), MlcNec mice and Ndn–/– mice.Results are normalized to levels ofGAPDH RNA. Error bars represent s.e.m.**P<0.01 vs WT.

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the expression of p53 (Fig. 4C,D). Overexpression of necdin inC2C12 and MlcNec myoblasts counterbalanced p53 overexpression,whereas significantly higher levels of p53 induced by TNF! wereobserved in Ndn–/– myoblasts (Fig. 4C,D). A higher level ofexpression of p53 transcripts was also observed in muscles of Ndn–/–

mice compared with the WT, with a lower level of expressionobserved in MlcNec mice (Fig. 4E). Of importance, we alsoobserved higher levels of p53 mRNA in Ndn–/– untreated myoblasts(Fig. 4E) indicating that necdin acts on p53 independently of itsaction on TNFRI.

TNF! activates caspases that can mediate proteolysis of muscleproteins (Moresi et al., 2008). In both myoblasts and C2C12 cells,exposure to TNF! led to the activation of caspase 3 and caspase9. Overexpression of necdin significantly reduced this activation,whereas in its absence, we observed an increased activation ofcaspase 3 and caspase 9 (Fig. 4C,D).

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Thus, necdin protects skeletal muscle from tumor-inducedcachexia by inhibiting the action of TNF!; such inhibition occurson the TNF!-activated cachectogenic signaling pathways at variouslevels.

DiscussionThe aim of this study was to identify new candidate targets forcachexia, for which still no valid therapy has been proposed (Morleyet al., 2006; Tisdale, 2006). Our results clearly indicate that sucha candidate molecule is the MAGE protein necdin.

We previously demonstrated that in vivo necdin increases theability of myoblasts to survive in presence of toxic stimuli andimproves their myogenic differentiation, acting at the transcriptionallevel, regulating the expression of myogenin, and by inhibitingapoptosis (Deponti et al., 2007). Now, we show in vivo that necdincounteracts the muscle wasting and inhibition of differentiation

Fig. 3. Necdin counteracts inhibition of myogenicdifferentiation induced by TNF! and ROS.(A) Expression of MHC, myogenin, MyoD orglyceraldehyde phosphate dehydrogenase(GAPDH) in primary myoblasts isolated fromwild-type (WT), transgenic (MlcNec) or null(Ndn–/–) newborn mice (myoblasts) or C2C12 cellstransfected with a plasmid containing pIRES2-EGFP (EGFP) or pIRESEGF-necdin (EGFPNdn)(C2C12), and differentiated in the presence ofculture medium from the fibroblast 3T3 cell line(3T3) or C26 colon carcinoma cells (C26). Imagesare representative of three reproducibleexperiments. Quantifications are shown in D.(B) Expression of sarcomeric myosin heavy chainby immunofluorescence on C2C12 cellstransfected with pIRES2-EGFP (EGFP) orpIRESEGF-necdin (EGFPNdn) and differentiatedin presence of culture medium of the fibroblast3T3 cell line (3T3), C26 colon carcinoma cells, or5 ng/ml TNF! or 2 µM As2O3; nuclei are stainedwith Hoechst 33258. Scale bar: 200 µm.(C) Expression of MHC, Myogenin, MyoD orGAPDH in primary myoblasts isolated from WT,MlcNec or Ndn–/– newborn mice (myoblasts) orC2C12 cells transfected with a plasmid containingpIRES2-EGFP (EGFP) or pIRESEGF-necdin(EGFPNdn) (C2C12), and differentiated inpresence of TNF! (20 ng/ml myoblasts; 5 ng/ml,C2C12) or As2O3 (5 µM myoblasts; 2 µM C2C12).Images are representative of three reproducibleexperiments. Quantifications are shown in E.(D) Graphs of mean values ± s.e.m. obtained fromthe densitometric analysis of the gel band of theprotein normalized on the GAPDH expression ofthe experiments shown in A (n=3). (E) Graphs ofmean values ± s.e.m. obtained from thedensitometric analysis of the gel band of theprotein normalized on the GAPDH expression ofthe experiments shown in panel B (n=3).

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specifically induced by the cachectogenic C26 cells. Necdinoverexpression inhibited the drastic weight reduction, the loss ofmuscle mass and the decrease in cross-sectional fiber area, and thereduction of myosin, myogenin and MyoD protein levels. Theseeffects of necdin were not artifacts due to its overexpression: necdinexpression was physiologically upregulated in the muscle by thetumor in vivo and when such an increase was prevented, such asin Ndn–/– mice, the cachectic phenotype was exacerbated. Thus,necdin expression is a response by muscles to tumor load ofbiological significance.

An important characteristic of necdin that could be useful fortherapy, is that it exerts positive effects systemically, even ifoverexpressed by only skeletal muscle. When we overexpressednecdin in the muscle, we found that it also reduced the wastingeffects of the tumor on epididymal fat and the liver. It remains tobe investigated whether necdin-expressing muscles maintain ahigher metabolic state, resulting in a reduced need for fat and liver

energetic reserve, thus limiting wasting of these tissues, or act byreleasing some anti-wasting factors for other tissues.

The beneficial effect of necdin appears to reside in theenhancement of both satellite cell resistance to tumor-generatedtoxic cues and their ability to differentiate. Indeed, muscles ofcachectic Ndn–/– mice were not only smaller, but appeared less wellpreserved, and showed a diminished expression of embryonicmyosin, which is a molecular marker of regeneration. Other effectsmight contribute to the less-severe loss in muscle mass observedin the gain-of-function mice, including a certain degree of fiberhypertrophy in vivo, and the increased fusion index in myoblastsin vitro, which we have also previously described (Deponti et al.,2007). This is of importance in view of a possible therapeuticapplication: it has been shown that hypertrophy, such as IGF1-dependent muscle hypertrophy, protects from a different kind ofmuscle wasting (Musaro et al., 2001; Song et al., 2005; Stitt et al.,2004), and stimulation of protein anabolism, in addition to inhibition

Fig. 4. Necdin inhibits TNF!cachectogenic signaling at different levels.(A) Expression of TNFR1 in primarymyoblasts from wild-type (WT), MlcNec orNdn–/– newborn mice measured by real-time PCR. Results are normalized to levelsof the GAPDH RNA (n=3). Error barsrepresent s.e.m. (B) Flow cytometryanalysis of TNFR1 receptor in primarymyoblasts (left panel) or transfected C2C12(right panel). Satellite cells, isolated fromWT, MlcNec or Ndn–/– newborn mice, wereanalysed for TNFR1 expression (light blue,orange and light green solid lines,respectively). Controls were made usingFITC-conjugated secondary antibody alone(blue, red and green dashed lines,respectively) (RFI: MlcNec, 29.9±0.4; WT,44.23±1.1; Ndn–/–, 55.7±0.73; P<0.001 vsWT; n=3). C2C12 cells were transientlytransfected with a plasmid containingpIRES2-EGFP (EGFP) or pIRESEGF-necdin (EGFPNdn) and analysed forTNFR1 expression after gating oftransfected cells (light blue and red,respectively). Control sample was madeusing secondary antibody alone (blue andorange, respectively) (RFI: pIRESGFP,19.4966±1.15; pIRESEGFP-necdin,9.136±1.03; P<0.001 vs pIRESEGFP;n=3). (C) Primary myoblasts from wild type(WT), MlcNec or Ndn–/– newborn mice(myoblasts) or C2C12 cells transientlytransfected with a plasmid containingpIRES2-EGFP (EGFP) or pIRESEGF-necdin (EGFPNdn) were differentiated inthe absence (PBS) or in the presence of 5-20 ng/ml of TNF! (TNF). Cells wereanalyzed for p53 expression and for theactivation of caspase 9 and caspase 3 bywestern blot. Graphs show mean values ±s.e.m. obtained for ratio of protein:GAPDHband density values evaluated on the blot ofthe same experiments shown in D (n=3). Inthe case of caspase 9, only active caspasebands were analyzed. Error bars represents.e.m. *P<0.05 and **P<0.01 vs WT;+P<0.01 vs 3T3 (A) or PBS (B,C). (D) Western blot images representative of three reproducible experiments quantified above in C. (E) p53 expression wasmeasured by real-time PCR in TA muscle from C26-treated WT, MlcNec and Ndn–/– mice sacrificed 6, 8, 10 and 12 days after tumor injection (n=5) (left graph) orin primary myoblasts from wild type (WT), MlcNec or Ndn–/– newborn mice (myoblasts) (n=3) (right graph). Results are normalized to levels of the GAPDHRNA. Error bars represent s.e.m. *P<0.01 and **P<0.001 vs WT.

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of protein catabolism, has been proposed as a clinical approach tomuscle cachexia (Muscaritoli et al., 2006).

The molecular mechanism of the beneficial effect of necdinincludes its specific action on TNF!-activated signaling, becausenecdin interferes with this pathway at various levels. We observeda decrease of TNFRI expression on the surface of necdin-overexpressing myoblasts, and an increase in Ndn–/– cells. Sincethe relative abundance of TNFRI mediates the extent of TNF!signaling (Neznanov et al., 2001; Paland et al., 2008), this mightin part explain the protective effect of necdin. TNFRI belongs tothe TNF-NGF death domain family, and necdin has been describedto bind to the death domain of p75NTR in endosomes and tomodulate its signaling (Bronfman et al., 2003; Kuwako et al., 2005).Other proteins have been shown to interact with several deathdomains and mediate or inhibit signaling of different receptors(Yazidi-Belkoura et al., 2003). It is conceivable that necdin bindsthe death domain of TNFR1 to modulate the TNF!-signalingpathway. Further investigations will be required to clarify this issue.

A second level of interaction between necdin and TNF! signalingoccurs on p53, which is the key downstream mediator of the effectof TNF!. Several studies suggest a role for p53 in regulating musclehomeostasis. Increased p53 levels are found in skeletal muscleduring unloading-induced muscle atrophy, as well as in agingskeletal muscle (Chung and Ng, 2006; Siu et al., 2006). Importantly,tumor-bearing p53-null mice are resistant to cachexia (Schwarzkopfet al., 2006). We have previously shown that necdin binds p53(Deponti et al., 2007). We found that p53 mRNA and protein levelswere increased by TNF! and that such increases were lower innecdin-overexpressing myoblasts, and higher in Ndn–/– myoblasts;thus necdin might influence not only p53 abundance or activity bydirect binding, but also might reduce TNF!-dependent transcriptionand translation of p53. Of importance, we also found that p53transcript levels were regulated by necdin independently of TNF!,since untreated Ndn–/– myoblasts expressed higher basal levels ofp53; thus, regulation of this protein is not entirely a consequenceof the effect of necdin on TNFRI expression. These results indicatethat p53 is a major target of necdin and that necdin has a dual actionupon it, decreasing not only its activity, but also its expression.

Important effectors downstream of p53 in skeletal muscle arecaspases (Moresi et al., 2008). We detected increased activation ofcaspases in Ndn–/– myoblasts treated with TNF! and the oppositein MlcNec mice. Consistent with such protective role of necdin, isthe observation that this protein also counteracts the deleterious roleof ROS, which leads to reduced antioxidant gene expressionassociated with muscle catabolism (Li et al., 2003).

In conclusion, our data show that necdin is part of the biologicalresponse of muscle to tumor-induced cachexia in vivo, and that itacts through a multitargeted inhibition of a relevant cachectogenicpathway: that of TNF!. Muscle-specific overexpression of necdinshowed that this protein not only has a high degree of efficacy inpreventing tumor-induced muscle wasting, but also exerts beneficialeffects on other tissues affected by the disease. We conclude thatnecdin can be considered as a candidate molecule to design newapproaches to cachexia.

Materials and MethodsMiceNecdin gain-of-function mice (MlcNec) and necdin loss-of-function mice Ndn–/– havebeen described elsewhere (Deponti et al., 2007; Muscatelli et al., 2000). Male BALB/c! C57Bl/6 F1 and BALB/c ! MlcNec F1 or BALB/c ! Ndn–/– F1 mice were usedat the age of 6 weeks, maintained in a temperature-controlled room under a 12 hourlight, 12 hour dark cycle and fed on water and food ad libitum. In the BALB/c !

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Ndn–/– F1, male homozygous mice were used to obtain Ndn–p/+m offspring, that donot express necdin as a result of maternal imprinting of the gene (Deponti et al., 2007).

C26 colon carcinoma cells and injectionMurine colon 26 adenocarcinoma cells (C26) (Tanaka et al., 1990) were cultured inDMEM containing 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/mlstreptomycin. For in vivo inoculation, a single suspension of 106 cells in 100 µl ofPBS was injected subcutaneously into the inguinal flank of mice. The same volumeof PBS was injected into the control groups.

Body weight measurementsMice body weight was measured the day of C26 cell injection and every 48 hoursthereafter. The amount of food consumed by one mouse per cage was calculated fromthe weight of the food pellet that remained every 48 hours.

Glucose level measurementsSerum glucose level was measured in both PBS and C26-treated animals 8 days afterthe injection using peripheral tail blood and the Ascensia Breeze 2 glucose-monitoringsystem from Bayer (Mishawaka, IN).

Tumor and organ isolationMice were sacrificed 6, 8, 10, and 12 days after cancer cell injection by cervicaldislocation and gastrocnemius, tibialis anterior (TA), quadriceps muscles werecollected and weighed. Liver, epididymal fat pads and tumors were also isolated andweighed while wet at 12 days. Tumor weights did not show any statistical significancebetween the mouse lines.

Real-time PCR analysisTotal RNA from TA and gastrocnemius muscles dissected from PBS- or C26-injectedmice (or myoblasts) were extracted using TRIzol protocol (Invitrogen, Carlsbad, CA).RNA (2 µg) was reverse transcribed using SuperScript III First-strand synthesis system(Invitrogen) according to the manufacturer’s instructions (random hexamers anddNTPs were from Invitrogen). Each cDNA sample was amplified in duplicate usingthe iQ SYBR Green SuperMix (Bio-Rad, Hercules, CA) on a real-time PCR system(Mx3000P, Stratagene, La Jolla, CA). All results were normalized to levels of theGAPDH or 28S ribosomal RNA. The following primers were used: GAPDH forward,5"-TGAAGGTCGGTGTGAACGGATTTG-3"; GAPDH reverse, 5"-CATGTGGGC-CATGAGGTCCACCAC-3"; NecdinF1, 5"-GTCCTGCTCTGATCCGAAGG-3";NecdinR1A, 5"-GGTCAACATCTTCTATCCGTTC-3"; TNFR1aF, 5"-TCAAA-GAGGAGAAGGCTGGA-3"; TNFR1R, 5"-GCAGAGTGATTCGTAGAGCAGA-3"; Atrogin1 forward, 5"-CAGAGAGGCAGATTCGCAAG-3"; Atrogin-1 reverse, 5"-GGGAAAGTGAGACGGAGCA-3"; MuRF1 forward, 5"-GTTAAACCAGAGGT -TCGG-3"; MuRF1 reverse 5"-ATGGTTCGCAACATTTCGG-3"; p53 forward, 5"-ACCTCACTGCATGGACGATCT-3"; p53 reverse, 5"-GACACTCGGAGG GC -TTCACTT-3"; embMHC forward 5"-TGAAGAAGGAGCAGGACACCAG-3";embMHC reverse, 5"-CACTTGGAGTTTATCCACCAGATCC-3".

Western blot analysisTA and gastrocnemius muscles dissected from PBS- or C26-injected mice or cells(primary myoblasWTs and C2C12) were homogenized in 50 mM Tris-HCl, pH 7.4,1 mM EGTA, 1 mM EDTA, 1% Triton X-100 or in 100 mM NaHCO3, 1 mM EDTA,2% sodium dodecyl sulfate and protease inhibitor cocktail (Sigma) and centrifugedat 13,000 g for 5 minutes at 4°C to discard cellular debris.

Sample preparation and western blot analyses were performed as described (Depontiet al., 2007). After electrophoresis, polypeptides were transferred to nitrocellulosefilters and antigens were revealed by incubation with primary antibodies and followedby the appropriate secondary antibodies. The antibodies used were: anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mAb from Biogenesis, anti-MyoD mAb from DakoCytomation, anti-sarcomeric myosin MF20 and anti-myogeninfrom Developmental Studies Hybridoma Bank, anti-p53 antibody from Calbiochem,anti-activated caspase-3 pAb and anti-caspase-9 mAb (recognizing both activatedand non-activated forms) from Cell Signaling.

Histology and morphometric analysisTA and gastrocnemius muscles were dissected from PBS- or C26-injected micesacrificed 12 days after inoculation and frozen in liquid-nitrogen-cooled isopentane.8-µm-thick serial muscle sections were stained with hematoxylin and eosin (H&E)as described (Deponti et al., 2007). Morphometric analyses were performed on sectionsof tibialis anterior and gastrocnemius using and the Image 1.63 program (ScionCorporation) to determine the cross-sectional area. 10 sections and 300 fibers wereanalyzed for each group.

Cell culture and transfectionC2C12 and fibroblast 3T3 cell line (3T3) were obtained from American Type CultureCollection and cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin,and 100 µg/ml streptomycin (proliferation medium). C2C12 cells were differentiatedin DMEM supplemented with 2% horse serum, 100 U/ml penicillin and 100 µg/mlstreptomycin (differentiation medium) as described (Deponti et al., 2007).

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1125Necdin protects against muscle wasting

C2C12 cells were transiently transfected with Lipofectamine Plus reagent(Invitrogen) as described (Deponti et al., 2007), using pIRESEGFPNecdin plasmidor pIRESEGPF plasmid. Cells were grown for 24 hours in proliferation medium andthan incubated for an additional 72 hours in differentiation medium in the presenceof medium obtained from confluent culture of 3T3 or C26 cells (1:2 dilution), 5ng/ml of TNF! or 2 µM of As2O3. The doses of TNF! or As2O3 were previouslydetermined as those resulting in no cell death (by TUNEL assay, not shown). In thecase of caspases or p53 experiments, the incubation in differentiation medium wasreduced to 24 hours.

Primary myoblast culturePrimary myoblasts from newborn mice of the different strains were isolated asdescribed (Deponti et al., 2007) and plated at clonal density using collagen-coateddishes. Cells were grown in proliferation medium (DMEM supplemented with 20%FBS, 3% chick embryo, 100 U/ml penicillin, 100 µg/ml streptomycin and 50 µg/mlgentamycin). To induce myotube formation, cells were cultured in differentiationmedium for 24 hours in the presence of medium obtained from confluent culture of3T3 or C26 cells (1:2 dilution), 20 ng/ml TNF! or 5 µM As2O3. The doses of TNF!or As2O3 were previously determined as those resulting in no cell death (by TUNELassay, not shown). In the case of caspases or p53 experiments, the incubation indifferentiation medium was reduced to 16 hours.

ImmunohistochemistryImmunohistochemistry was performed as described (Deponti et al., 2007) using theantibody specific for sarcomeric myosin MF20 (Developmental Studies HybridomaBank).

Flow cytometryThe cells were scraped from the Petri dish and resuspended in PBS containing 1%BSA and 0.05% NaN3. Then, cells were stained at 4°C for 45 minutes with the TNFreceptor 1 antibody (Cat. no. 19139, Abcam). After two washes with PBS, the cellswere stained at 4°C for 30 minutes with an Alexa Fluor 488 dye-conjugated donkeyanti-rabbit antibody (A-21206, Molecular Probes). After three washes, analysis ofthe fluorescence was performed using a FACScan flow cytometer (Becton Dickinson).Cells stained with the secondary antibody alone were used as a control.

Image acquisition and manipulationFluorescent and phase contrast images were taken on the Nikon microscope EclipseE600 with Pan Fluor: !10 0.33 NA and !20 0.50 NA lenses. Images were acquiredusing the Nikon digital camera DXM1200, and the acquisition software Nikon ACT-1. The imaging medium was PBS buffer and images were recorded at roomtemperature. Images and scanned films were assembled in panels using AdobePhotoshop 7.0. Images showing double fluorescence were separately acquired usingthe appropriate filters, and the different layers were then merged with Adobe Photoshop7.0.

Statistical analysisThe results are expressed as means ± s.e.m.; n represents the number of individualexperiments. Statistical analysis was carried out using the ANOVA test. Asterisks inthe figure panels refer to statistical probabilities versus WT controls. Statisticalprobability values (P) of less than 0.05 were considered significant.

We are grateful to Emanuele Azzoni and Greta Milani (San RaffaeleScientific Institute, Milan, Italy) for help with histology and to GiulioCossu and Angelo Manfredi (San Raffaele Scientific Institute, Milan,Italy) for discussion and critical reading of the manuscript. We thankMario Colombo (Instituto Nazionale Tumori, Malian, Italy) forsupplying the C26 cells and Françoise Muscatelli (IBDM, Marseille,France) for the Ndn–/– mice. This work was supported by grants fromTelethon (GGP07013, S.B. and GGP07006, E.C.), Fondazione Cariplo(to E.C. and S.B.), the Italian Association for Cancer Research (AIRC;to E.C.) and the Italian Ministry of Health (to E.C. and S.B.,PSX56/05/76).

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