Necdin is expressed in cachectic skeletal muscle to protect fibers from tumor-induced wasting

Cachexia consists of marked muscle wasting and atrophy associated with tumor load (Morley et al., 2006; Tisdale, 2002). This condition lowers responsiveness to treatment, contributing to a unfavorable prognosis and poor quality of life.

Several mediators of muscle wasting have been identified. These include immune and tumor-derived cytokines such as tumor necrosis factor (TNFα), interleukin (IL)-1, IFNγ and IL6 (Argiles et al., 2006); in addition, angiotensin-mediated activation of caspases and p53 transcription factors have all been shown to have a role (Tisdale, 2005). Most of these pathways mediate their effects by reducing the rate of protein synthesis at the level of protein translation or RNA content and by stimulating protein catabolism through the activation of the ubiquitin-proteasome pathway accompanied by induction 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). The heterogeneity of these factors and their potentially synergistic mode of action has made targeting them a real challenge and has yielded little clinical benefit: therapeutic options against the cachectic syndrome are in fact still lacking.

Defective skeletal muscle regeneration also substantially contributes to muscle wasting (Coletti et al., 2005; Moresi et al., 2008). The key cells in muscle regeneration are satellite cells, which are activated and undergo myogenic differentiation and fusion with damaged 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 regeneration to occur. The mechanisms leading to impaired muscle regeneration in cachexia have not been fully investigated.

We recently found that necdin (NECD), a member of the melanoma antigen gene (MAGE) family of proteins, has key roles in regeneration (Brunelli et al., 2004; Deponti et al., 2007). Here, we show that necdin is central to the strategy operated by the muscle to physiologically counteract tumor-induced muscle wasting and identify the mechanisms of necdin action that suggests it as a suitable target for therapeutic intervention in cachexia.

To investigate whether necdin has a role in tumor-induced muscle atrophy, we initially measured its expression in muscles of cachectic mice. Two-month-old wild-type (WT) mice (F1C57×BalbC) were inoculated 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 significantly upregulated during the first phases of cancer-induced cachexia, followed by a decrease from day 10 onwards (Fig. 1A), suggesting that upregulation of necdin is a physiological response of muscle to wasting.

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

The mean muscle weight in tumor-bearing Ndn^–/– mice was significantly lower than in WT animals, although it was significantly higher in MlcNec mice. (Fig. 1D). This was also true, although to a lesser extent, for the fat and liver weight (Fig. 1D).

The muscles of Ndn^–/– mice appeared to be less well preserved and showed a lower density of fibers with respect to WT and MlcNec mice, with increased cell infiltrates, collagen accumulation and necrotic fibers (Fig. 2A). Reduction in muscle weight was accompanied by a reduction of the fiber cross-section area, which was more marked in Ndn^–/– mice and was less severe in MlcNec mice compared with the WT (Fig. 2B,C). This effect is not only a consequence of the slight hypotrophy of the untreated Ndn^–/– mice or the hypertrophy of the untreated MlcNec mice with respect to the untreated WT. The decrease in fiber cross-section area of Ndn^–/– C26-treated mice compared with Ndn^–/– PBS-treated mice is more evident than the decrease observed in WT and MlcNec mice, and the 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 accompanied by decreased levels of the muscle-specific proteins myosin heavy chain, myogenin and MyoD in C26-inoculated WT mice (Fig. 2D,E), consistent with the increased protein catabolism that occurs during tumor load (Acharyya et al., 2004; Langen et al., 2004; Tisdale, 2005). Changes were also associated with increased expression of atrogin-1 and MuRF1, which are specific markers of muscle wasting (Fig. 2F). In C26-inoculated Ndn^–/– mice, loss of muscle-specific proteins increased, and levels of atrogin-1 and MuRF1 mRNA transcripts were also reduced. Protein catabolism and 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 compared with the WT and were reduced in Ndn^–/– mice (Fig. 2F), indicating that regeneration occurs at some extent in all mice but that it is significantly enhanced when necdin is overexpressed. Increased regeneration is also supported by the presence of centronucleated fibers in MlcNec muscle (Fig. 2A, black arrows).

Thus, necdin overexpression in muscle is sufficient to counteract tumor-induced muscle wasting and its absence leads to an exacerbated phenotype.

We next investigated the molecular mechanism underlying the effect of necdin in tumors. To this end, we designed an in vitro approach mimicking the situation observed in vivo. We exposed differentiating primary myoblasts (WT, MlcNec and Ndn^–/–) and C2C12 cells (transfected with either pIRESGFPNecdin or pIRESGFP plasmid) (Deponti et al., 2007) to the supernatant of confluent C26 cells, or 3T3 fibroblasts as a control. The C26-conditioned medium inhibited myogenic differentiation, decreased the expression of the differentiation markers MyoD, myogenin and MyHC (Fig. 3A,C) and reduced the number of MyHC-positive myotubes (Fig. 3B). This effect was counteracted by the overexpression of necdin in C2C12 and MlcNec myoblasts and enhanced by necdin ablation (Ndn^–/– myoblasts).

TNFα significantly contributes to cachexia (Zhou et al., 2003; Coletti et al., 2005); in addition, low concentrations of TNFα inhibit myogenic differentiation without causing apoptosis, which ensues at high concentrations of the cytokine (Alter et al., 2008; Coletti et al., 2002; Moresi et al., 2008). Oxidative stress (reactive oxygen species, ROS) accompanies and sustains the action of TNFα (Barreiro et al., 2005; Li et al., 2003).

Myoblasts from MlcNec, Ndn^–/– and WT mice were differentiated in the presence or absence of TNFα (5-20 ng/ml) or the ROS-generating As₂O₃ (2-5 μM). TNFα inhibited myogenic differentiation and decreased expression of MyoD, myogenin and MyHC. The effect of the cytokine was significantly enhanced in Ndn^–/– cells (Fig. 3B,C,E) and inhibited in MlcNec cells. These results were confirmed in C2C12 cells, where necdin overexpression overcame the inhibition of differentiation induced by TNFα and As₂O₃, and maintained the expression of MyoD, myogenin and MyHC (Fig. 3B,C,E).

We decided next to investigate how necdin interacts with the TNFα pathway. We examined whether the reduced effect of TNFα in necdin-overexpressing cells was related to a differential expression of its p55kD receptor (TNFRI). The amount of TNFRI mRNA transcript measured by real-time PCR in myoblasts from the three mouse genotypes was comparable (Fig. 4A). By contrast, the amount of TNFRI exposed at the plasma membrane of myoblasts cells measured by FACS analysis, was decreased in MlcNec cells, and increased in Ndn^–/– mice with respect to the WT (Fig. 4B). The role of necdin was confirmed by overexpression of the protein in C2C12 cells (Fig. 4B).

A key protein mediating TNFα-induced cachexia and its inhibition of myogenesis is p53 (Schwarzkopf et al., 2006). Exposure of myoblasts to TNFα during differentiation increased the expression of p53 (Fig. 4C,D). Overexpression of necdin in C2C12 and MlcNec myoblasts counterbalanced p53 overexpression, whereas significantly higher levels of p53 induced by TNFα were observed in Ndn^–/– myoblasts (Fig. 4C,D). A higher level of expression of p53 transcripts was also observed in muscles of Ndn^–/– mice compared with the WT, with a lower level of expression observed in MlcNec mice (Fig. 4E). Of importance, we also observed higher levels of p53 mRNA in Ndn^–/– untreated myoblasts (Fig. 4E) indicating that necdin acts on p53 independently of its action on TNFRI.

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

Thus, necdin protects skeletal muscle from tumor-induced cachexia by inhibiting the action of TNFα; such inhibition occurs on the TNFα-activated cachectogenic signaling pathways at various levels.

The aim of this study was to identify new candidate targets for cachexia, for which still no valid therapy has been proposed (Morley et al., 2006; Tisdale, 2006). Our results clearly indicate that such a candidate molecule is the MAGE protein necdin.

We previously demonstrated that in vivo necdin increases the ability of myoblasts to survive in presence of toxic stimuli and improves their myogenic differentiation, acting at the transcriptional level, regulating the expression of myogenin, and by inhibiting apoptosis (Deponti et al., 2007). Now, we show in vivo that necdin counteracts the muscle wasting and inhibition of differentiation specifically induced by the cachectogenic C26 cells. Necdin overexpression inhibited the drastic weight reduction, the loss of muscle mass and the decrease in cross-sectional fiber area, and the reduction of myosin, myogenin and MyoD protein levels. These effects of necdin were not artifacts due to its overexpression: necdin expression was physiologically upregulated in the muscle by the tumor in vivo and when such an increase was prevented, such as in Ndn^–/– mice, the cachectic phenotype was exacerbated. Thus, necdin expression is a response by muscles to tumor load of biological significance.

An important characteristic of necdin that could be useful for therapy, is that it exerts positive effects systemically, even if overexpressed by only skeletal muscle. When we overexpressed necdin in the muscle, we found that it also reduced the wasting effects of the tumor on epididymal fat and the liver. It remains to be investigated whether necdin-expressing muscles maintain a higher metabolic state, resulting in a reduced need for fat and liver energetic reserve, thus limiting wasting of these tissues, or act by releasing some anti-wasting factors for other tissues.

The beneficial effect of necdin appears to reside in the enhancement of both satellite cell resistance to tumor-generated toxic cues and their ability to differentiate. Indeed, muscles of cachectic Ndn^–/– mice were not only smaller, but appeared less well preserved, and showed a diminished expression of embryonic myosin, which is a molecular marker of regeneration. Other effects might contribute to the less-severe loss in muscle mass observed in the gain-of-function mice, including a certain degree of fiber hypertrophy in vivo, and the increased fusion index in myoblasts in vitro, which we have also previously described (Deponti et al., 2007). This is of importance in view of a possible therapeutic application: it has been shown that hypertrophy, such as IGF1-dependent muscle hypertrophy, protects from a different kind of muscle wasting (Musaro et al., 2001; Song et al., 2005; Stitt et al., 2004), and stimulation of protein anabolism, in addition to inhibition of protein catabolism, has been proposed as a clinical approach to muscle cachexia (Muscaritoli et al., 2006).

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

A second level of interaction between necdin and TNFα signaling occurs on p53, which is the key downstream mediator of the effect of TNFα. Several studies suggest a role for p53 in regulating muscle homeostasis. Increased p53 levels are found in skeletal muscle during unloading-induced muscle atrophy, as well as in aging skeletal muscle (Chung and Ng, 2006; Siu et al., 2006). Importantly, tumor-bearing p53-null mice are resistant to cachexia (Schwarzkopf et al., 2006). We have previously shown that necdin binds p53 (Deponti et al., 2007). We found that p53 mRNA and protein levels were increased by TNFα and that such increases were lower in necdin-overexpressing myoblasts, and higher in Ndn^–/– myoblasts; thus necdin might influence not only p53 abundance or activity by direct binding, but also might reduce TNFα-dependent transcription and translation of p53. Of importance, we also found that p53 transcript levels were regulated by necdin independently of TNFα, since untreated Ndn^–/– myoblasts expressed higher basal levels of p53; thus, regulation of this protein is not entirely a consequence of the effect of necdin on TNFRI expression. These results indicate that p53 is a major target of necdin and that necdin has a dual action upon it, decreasing not only its activity, but also its expression.

Important effectors downstream of p53 in skeletal muscle are caspases (Moresi et al., 2008). We detected increased activation of caspases in Ndn^–/– myoblasts treated with TNFα and the opposite in MlcNec mice. Consistent with such protective role of necdin, is the observation that this protein also counteracts the deleterious role of ROS, which leads to reduced antioxidant gene expression associated with muscle catabolism (Li et al., 2003).

In conclusion, our data show that necdin is part of the biological response of muscle to tumor-induced cachexia in vivo, and that it acts through a multitargeted inhibition of a relevant cachectogenic pathway: that of TNFα. Muscle-specific overexpression of necdin showed that this protein not only has a high degree of efficacy in preventing tumor-induced muscle wasting, but also exerts beneficial effects on other tissues affected by the disease. We conclude that necdin can be considered as a candidate molecule to design new approaches to cachexia.

Necdin gain-of-function mice (MlcNec) and necdin loss-of-function mice Ndn^–/– have been described elsewhere (Deponti et al., 2007; Muscatelli et al., 2000). Male BALB/c × C57Bl/6 F and BALB/c × MlcNec F₁ or BALB/c × Ndn^–/– F1 mice were used at the age of 6 weeks, maintained in a temperature-controlled room under a 12 hour light, 12 hour dark cycle and fed on water and food ad libitum. In the BALB/c × Ndn^–/– F1, male homozygous mice were used to obtain Ndn^–p/+m offspring, that do not express necdin as a result of maternal imprinting of the gene (Deponti et al., 2007).

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

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

Serum glucose level was measured in both PBS and C26-treated animals 8 days after the injection using peripheral tail blood and the Ascensia Breeze 2 glucose-monitoring system from Bayer (Mishawaka, IN).

Mice were sacrificed 6, 8, 10, and 12 days after cancer cell injection by cervical dislocation and gastrocnemius, tibialis anterior (TA), quadriceps muscles were collected and weighed. Liver, epididymal fat pads and tumors were also isolated and weighed while wet at 12 days. Tumor weights did not show any statistical significance between the mouse lines.

Total RNA from TA and gastrocnemius muscles dissected from PBS- or C26-injected mice (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 and dNTPs were from Invitrogen). Each cDNA sample was amplified in duplicate using the 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 the GAPDH or 28S ribosomal RNA. The following primers were used: GAPDH forward, 5′-TGAAGGTCGGTGTGAACGGATTTG-3′; GAPDH reverse, 5′-CATGTGGGCCATGAGGTCCACCAC-3′; NecdinF1, 5′-GTCCTGCTCTGATCCGAAGG-3′; NecdinR1A, 5′-GGTCAACATCTTCTATCCGTTC-3′; TNFR1aF, 5′-TCAAAGAGGAGAAGGCTGGA-3′; TNFR1R, 5′-GCAGAGTGATTCGTAGAGCAGA-3′; Atrogin1 forward, 5′-CAGAGAGGCAGATTCGCAAG-3′; Atrogin-1 reverse, 5′-GGGAAAGTGAGACGGAGCA-3′; MuRF1 forward, 5′-GTTAAACCAGAGGTTCGG-3′; MuRF1 reverse 5′-ATGGTTCGCAACATTTCGG-3′; p53 forward, 5′-ACCTCACTGCATGGACGATCT-3′; p53 reverse, 5′-GACACTCGGAGGGCTTCACTT-3′; embMHC forward 5′-TGAAGAAGGAGCAGGACACCAG-3′; embMHC reverse, 5′-CACTTGGAGTTTATCCACCAGATCC-3′.

TA 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 centrifuged at 13,000 g for 5 minutes at 4°C to discard cellular debris.

Sample preparation and western blot analyses were performed as described (Deponti et al., 2007). After electrophoresis, polypeptides were transferred to nitrocellulose filters and antigens were revealed by incubation with primary antibodies and followed by 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-myogenin from Developmental Studies Hybridoma Bank, anti-p53 antibody from Calbiochem, anti-activated caspase-3 pAb and anti-caspase-9 mAb (recognizing both activated and non-activated forms) from Cell Signaling.

TA and gastrocnemius muscles were dissected from PBS- or C26-injected mice sacrificed 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 sections of tibialis anterior and gastrocnemius using and the Image 1.63 program (Scion Corporation) to determine the cross-sectional area. 10 sections and 300 fibers were analyzed for each group.

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

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

Primary myoblasts from newborn mice of the different strains were isolated as described (Deponti et al., 2007) and plated at clonal density using collagen-coated dishes. 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/ml gentamycin). To induce myotube formation, cells were cultured in differentiation medium for 24 hours in the presence of medium obtained from confluent culture of 3T3 or C26 cells (1:2 dilution), 20 ng/ml TNFα or 5 μM As₂O₃. The doses of TNFα or As₂O₃ were previously determined as those resulting in no cell death (by TUNEL assay, not shown). In the case of caspases or p53 experiments, the incubation in differentiation medium was reduced to 16 hours.

Immunohistochemistry was performed as described (Deponti et al., 2007) using the antibody specific for sarcomeric myosin MF20 (Developmental Studies Hybridoma Bank).

The 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 TNF receptor 1 antibody (Cat. no. 19139, Abcam). After two washes with PBS, the cells were stained at 4°C for 30 minutes with an Alexa Fluor 488 dye-conjugated donkey anti-rabbit antibody (A-21206, Molecular Probes). After three washes, analysis of the fluorescence was performed using a FACScan flow cytometer (Becton Dickinson). Cells stained with the secondary antibody alone were used as a control.

Fluorescent and phase contrast images were taken on the Nikon microscope Eclipse E600 with Pan Fluor: ×10 0.33 NA and ×20 0.50 NA lenses. Images were acquired using the Nikon digital camera DXM1200, and the acquisition software Nikon ACT-1. The imaging medium was PBS buffer and images were recorded at room temperature. Images and scanned films were assembled in panels using Adobe Photoshop 7.0. Images showing double fluorescence were separately acquired using the appropriate filters, and the different layers were then merged with Adobe Photoshop 7.0.

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