Humoral and tumoral factors collectively promote cancer-induced skeletal muscle wasting by increasing protein degradation. Although several humoral proteins, namely TNFα (tumour necrosis factor α) and IL (interleukin)-6, have been shown to induce skeletal muscle wasting, there is a lack of information regarding the tumoral factors that contribute to the atrophy of muscle during cancer cachexia. Therefore, in the present study, we have characterized the secretome of C26 colon cancer cells to identify the tumoral factors involved in cancer-induced skeletal muscle wasting. In the present study, we show that myostatin, a procachectic TGFβ (transforming growth factor β) superfamily member, is abundantly secreted by C26 cells. Consistent with myostatin signalling during cachexia, treating differentiated C2C12 myotubes with C26 CM (conditioned medium) resulted in myotubular atrophy due to the up-regulation of muscle-specific E3 ligases, atrogin-1 and MuRF1 (muscle RING-finger protein 1), and enhanced activity of the ubiquitin–proteasome pathway. Furthermore, the C26 CM also activated ActRIIB (activin receptor type II B)/Smad and NF-κB (nuclear factor κB) signalling, and reduced the activity of the IGF-I (insulin-like growth factor 1)/PI3K (phosphoinositide 3-kinase)/Akt pathway, three salient molecular features of myostatin action in skeletal muscles. Antagonists to myostatin prevented C26 CM-induced wasting in muscle cell cultures, further confirming that tumoral myostatin may be a key contributor in the pathogenesis of cancer cachexia. Finally, we show that treatment with C26 CM induced the autophagy–lysosome pathway and reduced the number of mitochondria in myotubes. These two previously unreported observations were recapitulated in skeletal muscles collected from C26 tumour-bearing mice.

INTRODUCTION

Cachexia is associated with numerous chronic diseases, including cancer, renal failure, AIDS, diabetes and COPD (chronic obstructive pulmonary disease) [13]. Weight loss owing to cancer, termed cancer cachexia, is exhibited by approximately 80% of all patients who possess advanced tumours of pancreatic, lung and gastric origin, and it is responsible for over 30% of cancer-related fatalities [1,4]. Studies have established that skeletal muscle wasting may be dependent on the elevated activity of two muscle-specific E3 ligases, atrogin-1 and MuRF1 (muscle RING-finger protein 1), both of which target myofibrillar and intracellular proteins for degradation through the ubiquitin–proteasome system [58]. Currently, there are three well-documented upstream signalling cascades that regulate the expression of atrogin-1 and MuRF1 during cancer cachexia [1,9,10]. First, reduced signalling through the IGF-I (insulin-like growth factor 1)/PI3K (phosphoinositide 3-kinase)/Akt pathway; secondly, elevated activity of FoxO (forkhead box O) transcription factors, such as FoxO1 and FoxO3, the master regulators of muscle-specific E3 ligases [1113]; and thirdly, increased NF-κB (nuclear factor κB)-mediated signalling, which augments the expression of several components of the ubiquitin–proteasome system, including MuRF1 [1417].

Pro-inflammatory cytokines, including IL (interleukin)-1β, IL-6 and TNFα (tumour necrosis factor α), as well as tumoral factors such as PIF (proteolysis-inducing factor), promote the development of a cachectic phenotype in skeletal muscle [1]. In addition, TGFβ (transforming growth factor β) ligands, such as Mstn (myostatin), which function through ActRIIB (activin receptor type II B)-mediated signalling, are implicated in the development of skeletal muscle wasting in conditions such as denervation and fasting [12,18]. In fact, inactivation of Mstn and other TGFβ family of proteins by treatment with sActRIIB (a soluble form of ActRIIB) ablates the symptoms of cancer cachexia in tumour-bearing mice [12,19].

Despite the overwhelming evidence suggesting the importance of Mstn in manifesting cachectic-like muscle wasting, it is currently unclear whether cachexia-inducing neoplasms themselves secrete Mstn into the circulation. Therefore our first objective was to characterize the secretome of the C26 colon cancer cell line, an unequivocal inducer of cachexia in mice. The results of the present study confirm that Mstn is synthesized and secreted by not only C26 cells, but also other human and murine neoplasms. To determine whether tumoral Mstn from C26 cells was sufficient to induce skeletal muscle wasting, we treated differentiated C2C12 myotubes, used as a surrogate for adult skeletal muscle, with CM (conditioned medium) collected from C26 cells. Using this in vitro screen, we have demonstrated that many of the molecular hallmarks associated with Mstn-induced skeletal muscle wasting are simulated in muscle cell cultures exposed to C26 CM.

Although cancer cachexia induces cardiomyocytes to undergo atrophy through the autophagy–lysosome pathway [20,21], it is unclear whether skeletal muscle wasting during cancer also demonstrates enhanced autophagy–lysosome pathway activity. Our in vitro screen revealed that the C26 CM was able to induce the activity of the autophagy–lysosome pathway. Additionally, we also observed a reduction in mitochondrial number in muscle cell cultures exposed to C26 CM. Moreover, skeletal muscles isolated from C26 tumour-bearing mice confirmed the elevated activity of the autophagy–lysosome pathway and reduced mitochondrial number, further suggesting that tumoral factors secreted by C26 were sufficient to induce the activity of the proteolytic pathway and increase mitophagy. Although current literature has demonstrated that the removal of mitochondria and increased activity of the autophagy–lysosome pathway amplifies skeletal muscle wasting, such observations have not been recorded in skeletal muscles undergoing cancer-induced muscle wasting.

EXPERIMENTAL

Cell culture

Murine C2C12 (A.T.C.C.) myoblasts and C26 colon carcinoma cells were maintained according to previously published protocols [22]. Primary human myoblasts (hMb15) were gifts of Dr Vincent Mouly and Dr Gillian Butler-Browne (both PMC Université Paris 6, Institut de Myologie, Paris, France). MCF-7, MDA-MB-231 and T47D cells were a gift of Dr Valerie Lin, and U205, HeLa, HT1080 and A549 cells were gifts of Dr Eugene Makeyev (both School of Biological Sciences, Nanyang Technological University, Singapore).

C26 CM preparation and drug treatment

C26 cells were grown in DMEM (Dulbecco's modified Eagle's medium) with 5% FBS (fetal bovine serum) and 1% P/S (penicillin/streptomycin). For CM collection, cells were plated at a density of 50000 cells/cm2 and, after overnight attachment, the cells were washed three times with PBS, followed by two washes with serum-free DMEM, and grown in serum-free DMEM containing 1% P/S for 24 h. The resulting CM was centrifuged at 500 g for 10 min, followed by an additional centrifugation at 5000 g for 10 min. The CM was filtered using a 0.2 μm syringe filter and either stored at −80°C or used immediately. C26 CM was diluted at a 1:5 ratio with either DMEM containing 10% FBS and 1% P/S for myoblast treatment, or DMEM containing 2% HS (horse serum) and 1% P/S for myotube treatment. An appropriate quantity of FBS, HS and P/S was added to CM prior to dilution. The concentration of sActRIIB, SB 431542 and BAY 11-7085 used for the drug treatment experiments was 5 μg/ml, 1 μM and 2 μM respectively. For the neutralization of Mstn in C26 CM, anti-Mstn antibodies were pre-incubated at 5 and 10 μg/ml in C26 CM for 30 min. Similarly, activin A was neutralized by the pre-incubation of anti-activin A antibodies at 5 and 10 μg/ml in C26 CM for 30 min. The pre-incubated C26 CM was then diluted at a 1:5 ratio with either DMEM containing 10% FBS and 1% P/S for myoblast treatment, or DMEM containing 2% HS and 1% P/S for myotube treatment.

Animal handling, C26 cell implantation, tumour dissection and preparation of C26 TCM (tumour CM)

All experiments performed on mice adhered to the approved protocols stated by the Institute Animal Ethics Committee (IACUC), Singapore. Anaesthetized CD2F1 mice were injected subcutaneously with 0.5×106 C26 cells in 100 μl of sterile PBS. After solid tumour formation (approximately 10 days), mice were killed by CO2 asphyxiation. The tumour was dissected and placed in sterile PBS. The tumour was diced into approximately 2 mm3 cubes, washed twice with PBS and incubated in serum-free DMEM containing 1% P/S. TCM was collected after a 48 h incubation, and preparation of the TCM was performed as described above for C26 CM. For the in vivo autophagy–lysosome activity validation experiments, CD2F1 mice were injected subcutaneously with 0.5×106 C26 cells in 100 μl of sterile PBS, and after 15 days the mice were killed by CO2 asphyxiation. Protein extract was prepared by homogenizing the TA (tibialis anterior) muscle isolated from the tumour-bearing and control mice.

In-gel digestion, nano-LC-MS/MS (nano-liquid chromatography tandem MS) and data analysis

CM and TCM were concentrated using the Amicon Ultra 3K spin column (Millipore). The concentrated CM was precipitated with filtered sterile 80% acetone solution, and incubated overnight at −80°C. The precipitated proteins were collected by centrifugation at 12500 g for 15 min at 4°C. Acetone was aspirated and the pellet was air-dried. The pellet was homogenized and dissolved in protein lysis buffer [50 mM Tris/HCl (pH 7.5), 250 mM NaCl, 5 mM EDTA, 0.1% Nonidet P40, Complete™ protease inhibitor cocktail (Roche), 2 mM sodium fluoride, 1 mM sodium orthovanadate and 1 mM PMSF] containing Complete™ protease inhibitor tablet (Roche) [18], and was stored at −80°C. Approximately 30 μg of protein lysate was run on a 4–12% NuPAGE gradient gel (Invitrogen) and stained with Coomassie Brilliant Blue. The gel was destained and in-gel digestion was performed using porcine trypsin (Promega) [23]. The nano-LC-MS/MS was set-up to perform data acquisition in the positive-ion mode with a selected mass range of 300–2000 m/z. Protein identification and mouse database searching was performed according to previously described protocols [23]. IPA (Ingenuity Systems) and DAVID (Database for Annotation, Visualization, and Integrated Discovery) Bioinformatics Resources 6.7 [NIAID (National Institute of Allergy and Infectious Diseases), NIH (National Institutes of Health), Bethesda, MD, U.S.A.] were used to analyse the cellular localization and function of identified proteins present in secretome.

Mstn ELISA

An ELISA (Immuno Diagnostik) was performed according to the manufacturer's recommended protocols. Briefly, 100 μl of C26 in vitro CM and ex vivo TCM were used for the ELISA.

Cell cycle analysis

Cell cycle analysis was performed using PI (propidium iodide) according to a previously published protocol [24]. Actively growing myoblasts were treated with or without CM at a dilution of 1:5 for 24 h. After treatment, myoblasts were trypsinized and 100000 cells were labelled with PI. FACS, set on single-colour mode, was used to detect the cell cycle stages. The experiment was performed three times, and values are represented as means±S.D.

Myoblast proliferation assay

The myoblast proliferation assay has been described previously [25]. Briefly, C2C12 myoblasts were plated at a density of 1000 cells/well in a 96-well plate and, after overnight attachment, the cells were treated with or without CM and incubated at 37°C, 5% CO2 for 0, 24, 48 and 72 h. At each time point, cells were fixed with 100 μl of fixative (10% formaldehyde and 0.9% NaCl). The fixative was removed and the cells were washed twice with PBS. Cells were then incubated with 100 μl of Methylene Blue stain [1% Methylene Blue and 0.01 M sodium tetraborate (pH 8.5)] for 30 min at room temperature (25°C). After removing the Methylene Blue stain, cells were washed four times with 0.01 M sodium tetraborate (pH 8.5) and 200 μl of 0.1 M HCl/70% ethanol (1:1) was added into each well. The absorbance was read on a microplate reader at 655 nm.

Transfection and luciferase assay

Transfection was performed using the Gene Pulser MXcell Electroporation System (Bio-Rad Laboratories). Actively growing C2C12 myoblasts (~1×106 cells) were transfected with 25 μg of plasmid construct (for the luciferase assay, we used a 20:1 ratio of reporter and Renilla respectively) at 110 V and 500 Ω resistance on exponential mode using 0.2 mm cuvettes. After electroporation, myoblasts were transferred to a plate containing pre-warmed medium and left for overnight attachment. Cells were then plated in a 96-well plate at a density of 7500 cells/well. After overnight myoblast attachment, cells were then treated with CM, and incubated at 37°C, 5% CO2 for 24 h. Cells were lysed using 1× passive lysis buffer (Promega) for 30 min on an orbital shaker at room temperature. The plate was centrifuged at 4000 g for 30 min at 4°C, and 25 μl of the supernatant was transferred into opaque luminescence plates (Nunc). Plates were stored at −80°C until ready for luciferase assay measurement using the GlowMax Luminometer (Promega).

Total RNA extraction and RT (reverse transcription)-qPCR (quantitative PCR)

RNA was isolated from myotubes using TRIzol® reagent (Invitrogen) and 1 μg of total RNA was used to synthesize cDNA with the iScript kit (Bio-Rad Laboratories), as per the manufacturer's instructions. qPCR was performed using the CFX96 Real-Time System (Bio-Rad). Each RT-qPCR reaction (10 μl) contained 3 μl of 5× diluted cDNA, 5 μl of 2× SsoFast Evagreen (Bio-Rad Laboratories) and primers at a final concentration of 200 nM. All reactions were performed using the following thermal cycle conditions: 98°C for 3 min followed by 45 cycles of a three-step reaction, denaturation at 98°C for 3 s, annealing at 60°C for 20 s, followed by a melting curve from 60 to 95°C in 5 s increments of 0.5°C to ensure amplification specificity. Transcript levels of the target genes were normalized to Gapdh (glyceraldehyde-3-phosphate dehydrogenase), Actb (β-actin) and Hprt (hypoxanthine–guanine phosphoribosyltransferase). The relative fold change in expression was calculated using the ΔΔCT method) All primers pertaining to the present study were purchased from Sigma–Aldrich. The sequences of the primers used are available upon request.

Protein extraction and immunoblotting

Myoblasts and myotubes were re-suspended in protein lysis buffer, incubated for 15 min, homogenized and centrifuged at 12500 g for 10 min at 4°C. Proteins were quantified using Bradford reagent (Bio-Rad Laboratories). A total of 20 μg of each protein lysate was subjected to SDS/PAGE using a 4–12% pre-cast NuPAGE gel (Invitrogen), and the proteins were transferred on to 0.2 μm nitrocellulose membranes (Bio-Rad Laboratories). The membrane was blocked overnight at 4°C in blocking buffer {5% (w/v) non-fat dried skimmed milk in 1× TBST [Tris-buffered saline (137 mM NaCl and 20 mM Tris, pH 7.5) containing 0.1% Tween 20]}, and incubated with primary antibodies in blocking buffer for 3 h. The membranes were washed in 1× TBST for 5 min (five times), and incubated with the appropriate secondary antibody [IgG–HRP (horseradish peroxidase) conjugate] in blocking buffer for 1 h at room temperature. The membranes were finally washed in 1× TBST for 5 min (five times) before detection of HRP activity using Western Lightning™ Chemiluminescence Reagent Plus (PerkinElmer). The developed films were scanned and analysed using Quantity One imaging software (Bio-Rad Laboratories). Information pertaining to the antibodies and working dilutions are given in Supplementary Table S4 (at http://www.BiochemJ.org/bj/446/bj4460023add.htm).

Haematoxylin and eosin staining, and measurement of myotube number and area

Differentiating and differentiated myotubes were stained with haematoxylin and eosin according to a previous described protocol [6]. The images were acquired using the Leica DM60000 B microscope and analysed using Image-Pro software. Differentiated myotubes containing equal or more than three myonuclei were considered for measuring the myotube number and area. The myotube area was represented as μm2.

ICC (immunocytochemistry)

ICC was performed on C26 tumour (day 10) cryosections (8 μm). Sections were incubated at room temperature for 1 h with specific anti-Mstn and anti-activin A antibodies at dilutions of 1:50 and 5 μg/ml respectively, in 10% normal serum/PBS. C26 cells were fixed with 1% paraformaldehyde in PBS for 5 min followed by incubation with the primary antibody as mentioned above for 1 h at room temperature with gentle shaking. Fluorescently labelled secondary antibodies [AF-488 (Alexa Fluor® 488)-conjugated anti-rat and AF-488-conjugated anti-goat] were added at a dilution of 1:400. The slides were washed three times with PBS for 5-min intervals each. After washing, slides were incubated with DAPI (4′,6-diamidino-2-phenylindole) for 2 min and washed once with PBS. All slides were mounted with ProLong Gold Antifade Reagent (Invitrogen). Images were acquired using the LSM710 Carl Zeiss confocal microscope.

Measuring protein synthesis in C2C12 myotubes

Total protein synthesis was assessed through quantifying the amount of [3H]tyrosine incorporation into C2C12 myotube cultures [26]. C2C12 myotubes were treated with or without CM for 24 h. The myotubes were incubated with medium containing 5 μCi/ml [3H]tyrosine for 2 h. After incubation, the medium was discarded, cells were washed twice with PBS, and 1 ml of 10% TCA (trichloroacetate) was added to each well. Total cell lysates were collected into 1.5 ml centrifuge tubes and centrifuged at 12500 g for 10 min at 4°C. The resulting pellet was washed with 95% ethanol and dissolved in 0.1 M NaOH at 25°C for 2 h. These samples were analysed for total radioactivity and protein concentration using a scintillation counter (PerkinElmer) and the Bradford assay respectively. For radioactivity measurement, 100 μl of the soluble protein was added into 1 ml of scintillation cocktail. Results are expressed as d.p.m./mg of protein, normalized to the control. The experiment was repeated four times in duplicate.

Measuring protein degradation in C2C12 myotubes

C2C12 myotubes were incubated with 5 μCi/ml [3H]tyrosine for 36 h to label cellular proteins [27]. Cells were incubated for 2 h in chase medium, which contained 2 mM unlabelled tyrosine. Myotubes were then incubated with fresh chase medium containing CM and control (dialysis buffer) for 24 h. The medium was collected at different intervals, precipitated with 10% TCA and centrifuged at 12500 g for 10 min at 4°C. The acid-soluble radioactivity was measured using a scintillation counter (PerkinElmer). The radioactivity reflects the amount of pre-labelled protein that was degraded, and is expressed as a percentage of the total radioactivity initially incorporated. The experiment was repeated four times in duplicate.

Real-time ROS (reactive oxygen species) measurements

Real-time ROS measurement was performed according to a previously described protocol [28]. Briefly, C2C12 myoblasts were plated at a density of 25000 cells/cm2 in a 24-well plate. After overnight attachment, myoblasts were treated with or without C26 CM for a further 1 or 2 h. Then, after treatment, myoblasts were washed twice with PBS, and then incubated with DCFH-DA (2′,7′-dichlorodihydrofluorescein diacetate) probe (Invitrogen) in normal growth medum (10% FBS and 1% P/S) for 5 min. The medium was replaced with PBS, and images were acquired immediately using Leica live-cell microscopy at 543 nm (excitation) and 570 nm (long-pass emission). Relative quantification of green fluorescence was measured using ImageJ (NIH) software. The significance of green fluorescence emission was calculated from three random images from each well (total four wells).

OxyBlot

Oxidized proteins were detected using the OxyBlot protein detection kit (Millipore) according to the manufacturer's protocol.

Quantification of the mtDNA (mitochondrial DNA) to nuDNA (nuclear DNA) ratio by qPCR

Relative copy number of mtDNA to nuDNA was measured using a protocol described previously [29]. The set of primers used for mtDNA were 5′-CCTATCACCCTTGCCATCAT-3′ (forward) and 5′-GAGGCTGTTGCTTGTGTGAC-3′ (reverse). The set of primers used for nuDNA were 5′-ATGGAAAGCCTGCCATCATG-3′ (forward) and 5′-TCCTTGTTGTTCAGCATCAC-3′ (reverse). Quantification of the relative copy number of mtDNA to nuDNA was analysed using the ΔΔCT method. The experiment was repeated four times in duplicate.

Statistical analysis

Statistical analysis was performed using Student's t test and one-way ANOVA available in Microsoft Excel 2007. The values are expressed as means±S.D. and significance was assessed and represented as P<0.01 (*).

RESULTS

C26 murine colon carcinoma and human secondary cell lines secrete Mstn into CM

To characterize the secreted procachectic factors produced by the cachexia-inducing C26 cells, we collected C26 CM and identified the secreted proteins (secretome) using nano-LC-MS/MS. Proteomic analysis of the C26 CM surprisingly revealed the presence of Mstn and activin A, two TGFβ superfamily members involved in skeletal muscle wasting (Supplementary Table S1 at http://www.BiochemJ.org/bj/446/bj4460023add.htm). In addition, we detected several extracellular signalling molecules in the C26 CM, namely Grn (granulin), Ogn (osteoglycin), Aimp1 (aminoacyl tRNA synthetase complex-interacting multifunctional protein 1), Csf1 (colony stimulating factor 1), Csf3 (colony stimulating factor 3), Mif (macrophage migration inhibitory factor), Nampt (nicotinamide phosphoribosyltransferase) and Spp1 (secreted phosphoprotein 1), although their role in facilitating cancer cachexia is currently unknown (Supplementary Table S1). Since there are no methodologies to assess the tumoral secretome in vivo, we established explant cultures from C26 tumours and collected TCM for the characterization of secreted procachectic factors (Supplementary Figure S1 at http://www.BiochemJ.org/bj/446/bj4460023add.htm) [30,31]. Similar to the C26 in vitro CM secretome, the TCM collected from resected C26 tumours also contained Mstn and activin A (Supplementary Table S2 at http://www.BiochemJ.org/bj/446/bj4460023add.htm). Although the number of extracellular signalling components was comparable with the in vitro C26 secretome, we detected several proteins that were unique to TCM (Supplementary Tables S1 and S2), which we postulate may arise from host cell contamination.

Since proteomic analysis of the C26 CM and TCM revealed the presence of Mstn and activin A, we performed immunoblot analysis to validate the secretion of these two proteins. As seen in Figure 1(A), we noticed active mature forms of both Mstn and activin A in the CM and TCM (Figure 1A). We quantified Mstn levels by ELISA and demonstrated that Mstn protein was present at 6±1.32 ng/ml and 8±2.25 ng/ml in the CM and TCM respectively (Figure 1B). Furthermore, ICC on the C26 tumour and C26 cells further highlighted Mstn expression (Figure 1C). Owing to the absence of normal epithelial colon secondary cell lines isolated from mice, we could not compare the expression of Mstn produced by the C26 colon cancer cell to its normal counterpart. We next tested several human cancer cell lines for the expression of Mstn and activin A by immunoblotting and discovered that both Mstn and activin A appear to be differentially expressed among the chosen human cancer cell lines (Figure 1D).

C26 CM possesses Mstn and activin A

Figure 1
C26 CM possesses Mstn and activin A

(A) Immunoblots of Mstn and activin A expression in the C26 cell lysate, C26 tumour lysate, CM and TCM. An immunoblot of tubulin performed on the same membrane shows negligible amounts of the cytoplasmic protein in the CM and TCM. The molecular mass in kDa is indicated on the left-hand side. (B) Mstn levels in the CM and TCM as quantified by sandwich ELISA. (C) Representative confocal microscopy images of isotype control (IgG), Mstn and activin A staining in C26 tumour cryosections and in C26 cells. Scale bars represent 100 μm. (D) Immunoblot analysis of Mstn and activin A expression in human primary myoblasts (hMb15), myotubes (hMt15) and human cancer cell lines [MCF-7, MDA-MB-231 (MDA), T47D, U205, HeLa, HT1080 and A549]. Ponceau S staining indicates equal loading of protein samples. IB, immunoblot.

Figure 1
C26 CM possesses Mstn and activin A

(A) Immunoblots of Mstn and activin A expression in the C26 cell lysate, C26 tumour lysate, CM and TCM. An immunoblot of tubulin performed on the same membrane shows negligible amounts of the cytoplasmic protein in the CM and TCM. The molecular mass in kDa is indicated on the left-hand side. (B) Mstn levels in the CM and TCM as quantified by sandwich ELISA. (C) Representative confocal microscopy images of isotype control (IgG), Mstn and activin A staining in C26 tumour cryosections and in C26 cells. Scale bars represent 100 μm. (D) Immunoblot analysis of Mstn and activin A expression in human primary myoblasts (hMb15), myotubes (hMt15) and human cancer cell lines [MCF-7, MDA-MB-231 (MDA), T47D, U205, HeLa, HT1080 and A549]. Ponceau S staining indicates equal loading of protein samples. IB, immunoblot.

C26 CM inhibited C2C12 myoblast proliferation

The impaired regenerative capacity of muscles as a consequence of circulating tumoral components is considered a route through which certain cancers induce muscle wasting [1]. Hence, to verify whether secreted procachectic tumoral factors influenced myogenesis, C2C12 myoblasts were exposed to C26 CM and myoblast proliferation was monitored. Exposure to C26 CM inhibited C2C12 myoblast growth in a dose- and time-dependent manner (Figure 2A). Using FACS analysis, we determined the cell-cycle distribution of proliferating myoblasts after 24 h of C26 CM exposure. The results highlighted that the addition of C26 CM profoundly reduced the percentage of myoblasts in the S-phase of the cell cycle, from 25.7±1.3 to 11.3±0.8 (Figure 2B). Although there was a concomitant increase in the percentage of myoblasts in the G0/G1-phase, from 57.5±1.4 to 72.0±0.9, the number of cells in the G2/M-phase remained unchanged following treatment with C26 CM (Figure 2B). Importantly, we observed no appreciable change in apoptotic cells between growth-medium-treated and C26-CM-treated C2C12 myoblasts (results not shown), thus confirming that C26 CM inhibits myoblast proliferation through inhibiting cell-cycle progression rather than promoting apoptosis.

C26 CM inhibited the proliferation of C2C12 myoblasts

Figure 2
C26 CM inhibited the proliferation of C2C12 myoblasts

(A) Proliferation of myoblasts when treated with 1:3 and 1:5 dilutions of CM, as measured by the Methylene Blue photometric end-point assay. Absorbance (655 nm) is directly proportional to cell number, and 48 and 72 h represent the time of myoblasts in CM. n=8; values represent means±S.D. (B) The distribution of myoblasts in each phase of the cell cycle when incubated in the absence (Ctrl) or presence of CM for 24 h as assessed by FACS analysis (top panel). A Table indicating the percentages of myoblasts found in the respective phases of the cell cycle is also shown (bottom panel). n=4; values are means±S.D. (C) Immunoblot analysis of pRb, Cdk2, cyclin D1 and p21 expression in C2C12 myoblasts treated with (+) or without (−) CM for 24 h. (D) Proliferation of myoblasts when treated with a 1:3 dilution of CM and sActRIIB (CM+sActRIIB), as measured by the Methylene Blue photometric end-point assay. Absorbance (655 nm) is directly proportional to cell number. Values represent means±S.D. Statistical significance was assessed by Student's t test when compared with the control (Ctrl). *P<0.01. Ctrl, control; IB, immunoblot.

Figure 2
C26 CM inhibited the proliferation of C2C12 myoblasts

(A) Proliferation of myoblasts when treated with 1:3 and 1:5 dilutions of CM, as measured by the Methylene Blue photometric end-point assay. Absorbance (655 nm) is directly proportional to cell number, and 48 and 72 h represent the time of myoblasts in CM. n=8; values represent means±S.D. (B) The distribution of myoblasts in each phase of the cell cycle when incubated in the absence (Ctrl) or presence of CM for 24 h as assessed by FACS analysis (top panel). A Table indicating the percentages of myoblasts found in the respective phases of the cell cycle is also shown (bottom panel). n=4; values are means±S.D. (C) Immunoblot analysis of pRb, Cdk2, cyclin D1 and p21 expression in C2C12 myoblasts treated with (+) or without (−) CM for 24 h. (D) Proliferation of myoblasts when treated with a 1:3 dilution of CM and sActRIIB (CM+sActRIIB), as measured by the Methylene Blue photometric end-point assay. Absorbance (655 nm) is directly proportional to cell number. Values represent means±S.D. Statistical significance was assessed by Student's t test when compared with the control (Ctrl). *P<0.01. Ctrl, control; IB, immunoblot.

Accumulation of active hypophosphorylated pRb (retinoblastoma protein) prevents S-phase transition [32]. Immunoblot analysis of pRb revealed increased quantities of the hypophosphorylated (active) form of pRb in proliferating myoblasts treated with C26 CM when compared with myoblasts exposed to growth medium (Figure 2C). Consistent with this observation, the levels of cyclins and Cdks (cyclin-dependent kinases), namely cyclin D1 and Cdk2, which are known to phosphorylate and thus inhibit Rb were decreased in myoblasts treated with CM (Figure 2C). Furthermore, p21, a Cdk inhibitor that prevents S-phase transition, was strikingly up-regulated in CM-treated myoblasts (Figure 2C). These results collectively indicate that the addition of C26 CM inhibited myoblast proliferation through pRb-mediated blockade of cell-cycle progression at the G0/G1 checkpoint. Finally, the addition of sActRIIB, an inhibitor of the Mstn and other TGFβ ligands, to CM (CM+ActRIIB) facilitated myoblast proliferation (Figure 2D).

C26 CM attenuated C2C12 myoblast differentiation

Since C26 CM prevented C2C12 myoblast proliferation, we next examined whether or not the C26 CM inhibited the differentiation of C2C12 myoblasts. Thus C2C12 myoblasts were differentiated in C26 CM (diluted to a ratio of 1:5 with low-serum medium) and myotube number was quantified. C2C12 myoblasts differentiating in CM demonstrated reduced myotube number as compared with the untreated control (Figures 3A and 3B). Furthermore, after 72 h, myotube area was decreased in the C26-CM-differentiated myotubes as compared with the control (Figure 3C). Immunoblot analysis of myoblasts differentiating in C26 CM revealed a decrease in Myh (myosin heavy chain), MyoD (myogenic differentiation 1) and Myog (myogenin) expression when compared with the control-differentiating myoblasts (Figure 3D). Importantly, the inhibition of C2C12 myoblast differentiation in CM was rescued by the addition of sActRIIB (CM+sActRIIB), as a similar myotube number and myotube area was observed in C2C12 myoblasts differentiated in CM+sActRIIB when compared with the control (Figures 3E and 3F).

C26 CM prevented myogenic differentiation of C2C12 myoblasts

Figure 3
C26 CM prevented myogenic differentiation of C2C12 myoblasts

(A) Haemotoxylin-and-eosin-stained micrographs of C2C12 myoblasts differentiating in low-serum medium (Ctrl) or in the presence of CM diluted 1:5 with low-serum medium. Scale bars represent 100 μm. (B) C2C12 myotube number as quantified from the haemotoxylin-and-eosin-stained micrographs using Image Pro Plus. (C) Myotubular area (μm2) as calculated for the 72 h differentiation time point using Image Pro Plus. (D) Immunoblot analysis of Myh, MyoD and Myog expression in C2C12 myoblasts treated with (+) or without (−) CM over a period of 96 h (left-hand panel). Densitometric analysis of the relative protein levels of Myh, MyoD and Myog, represented as arbitrary units (A.U), at each time point normalized to tubulin (right-hand panel). (E) Number of myotubes formed when C2C12 myoblasts were differentiated in control, CM and CM+sActRIIB. Myotubes were stained with haemotoxylin and eosin (Supplementary Figure S2 at http://www.BiochemJ.org/bj/446/bj4460023add.htm) and counted using Image Pro Plus. (F) Area (μm2) of C2C12 myotubes. Myotube area was calculated from the 72 h differentiation time point (Supplementary Figure S2 at http://www.BiochemJ.org/bj/446/bj4460023add.htm) using Image Pro Plus. Ctrl, control; IB, immunoblot.

Figure 3
C26 CM prevented myogenic differentiation of C2C12 myoblasts

(A) Haemotoxylin-and-eosin-stained micrographs of C2C12 myoblasts differentiating in low-serum medium (Ctrl) or in the presence of CM diluted 1:5 with low-serum medium. Scale bars represent 100 μm. (B) C2C12 myotube number as quantified from the haemotoxylin-and-eosin-stained micrographs using Image Pro Plus. (C) Myotubular area (μm2) as calculated for the 72 h differentiation time point using Image Pro Plus. (D) Immunoblot analysis of Myh, MyoD and Myog expression in C2C12 myoblasts treated with (+) or without (−) CM over a period of 96 h (left-hand panel). Densitometric analysis of the relative protein levels of Myh, MyoD and Myog, represented as arbitrary units (A.U), at each time point normalized to tubulin (right-hand panel). (E) Number of myotubes formed when C2C12 myoblasts were differentiated in control, CM and CM+sActRIIB. Myotubes were stained with haemotoxylin and eosin (Supplementary Figure S2 at http://www.BiochemJ.org/bj/446/bj4460023add.htm) and counted using Image Pro Plus. (F) Area (μm2) of C2C12 myotubes. Myotube area was calculated from the 72 h differentiation time point (Supplementary Figure S2 at http://www.BiochemJ.org/bj/446/bj4460023add.htm) using Image Pro Plus. Ctrl, control; IB, immunoblot.

C26 CM simulated C2C12 myotube wasting in vitro

In addition to the reduced muscle regenerative capacity exhibited by patients who suffer from cancer cachexia, the atrophy of skeletal muscle due to the elevated activity of proteolytic pathways is another salient feature of cancer-induced muscle wasting [12]. To emulate this aspect of cancer cachexia in vitro, we exposed differentiated C2C12 myotubes to C26 CM for 24 h. The C26-CM-treated myotubes yielded a muscle wasting phenotype characterized by a reduction in myotube area when compared with the control myotubes (Figures 4A and 4B). Moreover, the amount of protein synthesis, as quantified by [3H]tyrosine incorporation, was markedly decreased after 24 h exposure to CM (Figure 4C). Furthermore, the level of protein degradation, as measured by the percentage of [3H]tyrosine lost 12, 24 and 36 h after CM addition, was elevated when compared with the control (Figure 4D). The enhanced level of proteolysis could be attributed to elevated activity of the ubiquitin–proteasome pathway, as immunoblot analysis detected increased amounts of ubiquitinated proteins in C2C12 myotubes exposed to C26 CM (Figure 4E).

C26 CM induced wasting of C2C12 myotubes

Figure 4
C26 CM induced wasting of C2C12 myotubes

(A) Representative confocal micrographs of differentiated C2C12 myotubes treated without (Ctrl) or with CM (1:5 dilution in low-serum medium) for 24 h. Scale bars represent 100 μm. DAPI (blue) highlights the nucleus and red represents Myh staining. (B) The histogram represents myotube area (μm2) in control and CM-treated C2C12 myotubes acquired by Image Pro Plus. (C) CM suppressed protein synthesis in differentiated myotubes. After 24 h of treatment with CM (1:5 and 1:10 dilutions) or control, myotubes were incubated with [3H]tyrosine for 2 h. The radioactivity incorporated was measured and normalized to total protein lysate. n=4; values are means±S.D. (D) CM increased proteolysis in differentiated myotubes. Differentiated myotubes were incubated with [3H]tyrosine for 36 h and then treated with CM (1:5 and 1:10 dilutions). Medium was collected at 12, 24 and 36 h, and the amount of degraded [3H]tyrosine-labelled protein was expressed as a percentage of the initial amount of [3H]tyrosine added. n=4; values are means±S.D. (E) Immunoblot analysis of ubiquitinated proteins in C2C12 myotubes after treatment with various concentrations of CM for 24 h (left-hand panel). Densitometric quantification of the ubiquitin immunoblot, represented as the relative ubiquitination level in arbitrary units (A.U) (right-hand panel). Values are means±S.D., *P<0.01. Ctrl, control; IB, immunoblot; Ub, ubiquitin.

Figure 4
C26 CM induced wasting of C2C12 myotubes

(A) Representative confocal micrographs of differentiated C2C12 myotubes treated without (Ctrl) or with CM (1:5 dilution in low-serum medium) for 24 h. Scale bars represent 100 μm. DAPI (blue) highlights the nucleus and red represents Myh staining. (B) The histogram represents myotube area (μm2) in control and CM-treated C2C12 myotubes acquired by Image Pro Plus. (C) CM suppressed protein synthesis in differentiated myotubes. After 24 h of treatment with CM (1:5 and 1:10 dilutions) or control, myotubes were incubated with [3H]tyrosine for 2 h. The radioactivity incorporated was measured and normalized to total protein lysate. n=4; values are means±S.D. (D) CM increased proteolysis in differentiated myotubes. Differentiated myotubes were incubated with [3H]tyrosine for 36 h and then treated with CM (1:5 and 1:10 dilutions). Medium was collected at 12, 24 and 36 h, and the amount of degraded [3H]tyrosine-labelled protein was expressed as a percentage of the initial amount of [3H]tyrosine added. n=4; values are means±S.D. (E) Immunoblot analysis of ubiquitinated proteins in C2C12 myotubes after treatment with various concentrations of CM for 24 h (left-hand panel). Densitometric quantification of the ubiquitin immunoblot, represented as the relative ubiquitination level in arbitrary units (A.U) (right-hand panel). Values are means±S.D., *P<0.01. Ctrl, control; IB, immunoblot; Ub, ubiquitin.

C26 CM stimulated signalling pathways implicated in cancer-induced skeletal muscle wasting

We next focused on validating the expression profile of specific genes and proteins involved in myotube wasting manifested by exposure to C26 CM. We observed an increased expression of E3 ligases in myotubes after 24 h exposure to CM. In particular, Fbxo32 (atrogin-1) and MuRF1 expression was up-regulated more than 3.6-fold in myotubes exposed to C26 CM (Figure 5A and Supplementary Table S3 at http://www.BiochemJ.org/bj/446/bj4460023add.htm). A reporter assay with the promoter of Fbxo32 confirmed dose-dependent up regulation of Fbxo32 transcriptional activity 24 h after treatment with CM (Figure 5B). Consistent with the overexpression of muscle-specific E3 ligases in C2C12 myotubes, the transcription factors FoxO1 and FoxO3 were also elevated (Figure 5A and Supplementary Table S3). Subsequent FoxO1 and FoxO3 promoter–reporter assays in myotubes validated the increased activity of these genes after a 24 h exposure to C26 CM (Figure 5B). In addition, we also observed increased protein levels of atrogin-1, MuRF1, FoxO1 and FoxO3 in CM-treated myotubes (Figures 5C and 5D), which was consistent with the gene expression profile (Figure 5A).

Treatment of C2C12 myotubes with C26 CM activated signalling pathways involved in skeletal muscle wasting

Figure 5
Treatment of C2C12 myotubes with C26 CM activated signalling pathways involved in skeletal muscle wasting

(A) Heat-map analysis of RT-qPCR results, drawn with Orange Canvas, on a selected set of genes that highlight the anabolic and catabolic components affected when myotubes are treated with 1:1, 1:3, 1:5 and 1:10 dilutions of CM (n=6; numeric values are found in Supplementary Table S3 at http://www.BiochemJ.org/bj/446/bj4460023add.htm). Gene expression was normalized to three endogenous controls, Gapdh, Actb and Hprt using the ΔΔCT method. The green colour in the heat-map represents values with a change above 3.6-fold. Atg, autophagy-related; Bnip3, BCL2/adenovirus E1B interacting protein 3; Cat, catalase; Cath L, cathepsin L; Cblb, Casitas B-lineage lymphoma b; Drp1, dynamin 1-like; Fis1, fission 1; Gsr, glutathione reductase; Lc3, light chain 3; Map1lc3a, microtubule-associated protein 1 LC (light chain) 3α; March5, membrane-associated ring finger (C3HC4) 5; Mfn, mitofusin; Mul1, mitochondrial ubiquitin ligase activator of NF-κB 1; Nrf1, nuclear respiratory factor 1; Sod1, superoxide dismutase 1; Tfam, transcription factor A, mitochondrial. (B) Histogram representing the fold induction (luciferase/Renilla ratio, normalized to the control) of FoxO1, FoxO3, atrogin-1 and PGC-1α promoter–reporter constructs in the presence of 1:1, 1:3 and 1:5 dilutions of CM. n=8; values are means±S.D. (C) Immunoblot analysis of Myh, Myl, atrogin-1 and MuRF1 expression in C2C12 myotubes after treatment with (+) or without (−) CM for 24 h. (D) Immunoblot analysis of FoxO1, p-FoxO1, FoxO3, p-FoxO3, Akt, p-Akt and PGC-1α levels following treatment with (+) or without (−) CM. (E) Immunoblot analysis of Smad2/3 expression and phosphorylation status in C2C12 myotubes after treatment with CM for 24 h. (F) Fold induction (luciferase/Renilla ratio, normalized to control) of a Smad reporter construct possessing 4 SBE (4X SBE) repeats after treatment with 1:1, 1:3 and 1:5 dilutions of CM (bottom). n=8; values are means±S.D. Statistical significance was assessed by Student's t test compared with the control; *P<0.01. Ctrl, control; IB, immunoblot; luc/ren, luciferase/Renilla.

Figure 5
Treatment of C2C12 myotubes with C26 CM activated signalling pathways involved in skeletal muscle wasting

(A) Heat-map analysis of RT-qPCR results, drawn with Orange Canvas, on a selected set of genes that highlight the anabolic and catabolic components affected when myotubes are treated with 1:1, 1:3, 1:5 and 1:10 dilutions of CM (n=6; numeric values are found in Supplementary Table S3 at http://www.BiochemJ.org/bj/446/bj4460023add.htm). Gene expression was normalized to three endogenous controls, Gapdh, Actb and Hprt using the ΔΔCT method. The green colour in the heat-map represents values with a change above 3.6-fold. Atg, autophagy-related; Bnip3, BCL2/adenovirus E1B interacting protein 3; Cat, catalase; Cath L, cathepsin L; Cblb, Casitas B-lineage lymphoma b; Drp1, dynamin 1-like; Fis1, fission 1; Gsr, glutathione reductase; Lc3, light chain 3; Map1lc3a, microtubule-associated protein 1 LC (light chain) 3α; March5, membrane-associated ring finger (C3HC4) 5; Mfn, mitofusin; Mul1, mitochondrial ubiquitin ligase activator of NF-κB 1; Nrf1, nuclear respiratory factor 1; Sod1, superoxide dismutase 1; Tfam, transcription factor A, mitochondrial. (B) Histogram representing the fold induction (luciferase/Renilla ratio, normalized to the control) of FoxO1, FoxO3, atrogin-1 and PGC-1α promoter–reporter constructs in the presence of 1:1, 1:3 and 1:5 dilutions of CM. n=8; values are means±S.D. (C) Immunoblot analysis of Myh, Myl, atrogin-1 and MuRF1 expression in C2C12 myotubes after treatment with (+) or without (−) CM for 24 h. (D) Immunoblot analysis of FoxO1, p-FoxO1, FoxO3, p-FoxO3, Akt, p-Akt and PGC-1α levels following treatment with (+) or without (−) CM. (E) Immunoblot analysis of Smad2/3 expression and phosphorylation status in C2C12 myotubes after treatment with CM for 24 h. (F) Fold induction (luciferase/Renilla ratio, normalized to control) of a Smad reporter construct possessing 4 SBE (4X SBE) repeats after treatment with 1:1, 1:3 and 1:5 dilutions of CM (bottom). n=8; values are means±S.D. Statistical significance was assessed by Student's t test compared with the control; *P<0.01. Ctrl, control; IB, immunoblot; luc/ren, luciferase/Renilla.

The activation of FoxO transcription factors is regulated by upstream IGF-I/PI3K/Akt signalling [11]. Importantly, while total Akt levels remained constant, we noticed reduced levels of active p-Akt (phosphorylated Akt) in CM-treated myotube cultures (Figure 5D and Supplementary Figure S3A at http://www.BiochemJ.org/bj/446/bj4460023add.htm). As expected, we noticed reduced amounts of p-FoxO1 (phosphorylated FoxO1) and p-FoxO3 (phosphorylated FoxO3) respectively, and increased levels of active dephosphorylated FoxO1 and FoxO3 in myotubes treated for 24 h with CM (Figure 5D and Supplementary Figure S3A). Moreover, reduced protein levels of PGC-1α [PPAR (peroxisome-proliferator-activated receptor) γ co-activator 1α], a negative regulator of FoxO transcription factors, supports the accumulation of active FoxO transcription factors following treatment with C26 CM (Figure 5D). Activation of Smad2/3 through Mstn signalling also stimulates FoxO1 and FoxO3 [33]. Similarly, C2C12 myotubes treated with CM for 24 h displayed increased levels of p-Smad2/3 (phosphorylated Smad2/3), as well as increased SBE (Smad-binding element)-reporter activity (Figures 5E and 5F, and Supplementary Figure S3B).

The selective degradation of sarcomeric proteins is a salient feature of cancer-induced skeletal muscle wasting [12]. A slight increase in the expression of sarcomeric protein transcripts in CM-treated myotubes was seen (Figure 5A and Supplementary Table S3). However, in contrast, the protein expression level of Myh and Myl (myosin light chain) was reduced in myotubes exposed to C26 CM (Figure 5C), indicating that the increased transcriptional activity of sarcomeric genes is an attempt to overcome the loss of myofibrillar proteins during skeletal muscle wasting.

C26 CM promoted NF-κB activation and ROS production

NF-κB is a powerful signalling molecule implicated in muscle-wasting disorders [14,34]. We used immunoblot and ICC analysis to characterize the protein expression, phosphorylation status and translocation of the p65 subunit of NF-κB in the presence of CM. As seen in Figure 6(A), we observed increased levels of active p-p65 (phosphorylated p65) in CM-treated myotubes when compared with the control. Furthermore, ICC analysis revealed increased nuclear translocation of both p65 and p-p65 in myoblasts exposed to C26 CM for 24 h (Figure 6B).

C26 CM induced NF-κB activation

Figure 6
C26 CM induced NF-κB activation

(A) Immunoblot analysis of p65 and p-p65 levels in C2C12 myotubes in the absence (−) or presence (+) of CM (left-hand panel). Densitometric quantification of the relative protein levels of p-p65, expressed as a proportion of total p65 (right-hand panel). (B) Confocal micrographs highlighting p65 (green) and p-p65 (green) nuclear localization in control and CM-treated C2C12 myoblasts. Nuclei are stained with DAPI (blue). Scale bars represent 100 μm. Surface plots drawn by ImageJ as calculated from the corresponding confocal images. The images highlight the exclusive nuclear localization of p65 and p-p65 subunits of NF-κB upon treatment with CM. Ctrl, control; IB, immunoblot.

Figure 6
C26 CM induced NF-κB activation

(A) Immunoblot analysis of p65 and p-p65 levels in C2C12 myotubes in the absence (−) or presence (+) of CM (left-hand panel). Densitometric quantification of the relative protein levels of p-p65, expressed as a proportion of total p65 (right-hand panel). (B) Confocal micrographs highlighting p65 (green) and p-p65 (green) nuclear localization in control and CM-treated C2C12 myoblasts. Nuclei are stained with DAPI (blue). Scale bars represent 100 μm. Surface plots drawn by ImageJ as calculated from the corresponding confocal images. The images highlight the exclusive nuclear localization of p65 and p-p65 subunits of NF-κB upon treatment with CM. Ctrl, control; IB, immunoblot.

ROS are potent activators of the NF-κB signalling cascade [35,36]. To test for the generation of ROS, we used real-time ROS measurement using the DCFH-DA probe and found that CM-treated myoblasts demonstrated significantly increased ROS production when compared with the control (Figures 7A and 7B). An OxyBlot assay also revealed increased levels of oxidized proteins in myotubes treated with C26 CM as compared with the control (Figure 7C).

C26 CM promoted ROS induction, mitophagy and increased autophagy–lysosome pathway activity.

Figure 7
C26 CM promoted ROS induction, mitophagy and increased autophagy–lysosome pathway activity.

(A) Representative micrographs demonstrating ROS induction in myoblasts treated with CM for 1 or 2 h. Scale bars represent 100 μm. (B) Histogram represents the relative green fluorescence intensity in arbitrary units (A.U) calculated using ImageJ software. Values are means±S.D. (C) An OxyBlot assay demonstrating the amounts of oxidized proteins in myotubes after treatment with (+) or without (−) CM. (D) Histogram represents the qPCR quantification of the mtDNA/nuDNA ratio in control or CM-treated myoblasts. n=4; values are means±S.D. (E) Histogram represents the qPCR quantification of the mtDNA/nuDNA ratio in TA muscle isolated from day 17 C26 tumour-bearing mice (C26 tumour) and control mice. n=5; values are means±S.D. (F) Immunoblot analysis of LC3-I into LC3-II conversion in control (−) and CM-treated (+) C2C12 myotubes incubated for 24 and 36 h. (G) Immunoblot analysis of LC3-I into LC3-II conversion in C2C12 myotubes treated with CM and CM+sActRIIB for 24 h. (H) Immunoblot analysis of LC3-I into LC3-II conversion in TA muscle isolated from control CD2F1 mice (−) and 17 day C26 tumour-bearing CD2F1 mice (+). Statistical significance was assessed by Student's t test compared with the control; *P<0.01. Ctrl, control; IB, immunoblot.

Figure 7
C26 CM promoted ROS induction, mitophagy and increased autophagy–lysosome pathway activity.

(A) Representative micrographs demonstrating ROS induction in myoblasts treated with CM for 1 or 2 h. Scale bars represent 100 μm. (B) Histogram represents the relative green fluorescence intensity in arbitrary units (A.U) calculated using ImageJ software. Values are means±S.D. (C) An OxyBlot assay demonstrating the amounts of oxidized proteins in myotubes after treatment with (+) or without (−) CM. (D) Histogram represents the qPCR quantification of the mtDNA/nuDNA ratio in control or CM-treated myoblasts. n=4; values are means±S.D. (E) Histogram represents the qPCR quantification of the mtDNA/nuDNA ratio in TA muscle isolated from day 17 C26 tumour-bearing mice (C26 tumour) and control mice. n=5; values are means±S.D. (F) Immunoblot analysis of LC3-I into LC3-II conversion in control (−) and CM-treated (+) C2C12 myotubes incubated for 24 and 36 h. (G) Immunoblot analysis of LC3-I into LC3-II conversion in C2C12 myotubes treated with CM and CM+sActRIIB for 24 h. (H) Immunoblot analysis of LC3-I into LC3-II conversion in TA muscle isolated from control CD2F1 mice (−) and 17 day C26 tumour-bearing CD2F1 mice (+). Statistical significance was assessed by Student's t test compared with the control; *P<0.01. Ctrl, control; IB, immunoblot.

C26 CM induced the activity of the autophagy–lysosome pathway and promoted mitophagy

Studies have demonstrated that deregulated ROS generation is an indicator of mitochondrial dysfunction [37,38]. Interestingly, the transcription factors Ppargc1a (PGC-1α) and Tfam (transcription factor A, mitochondrial), which are involved in mitochondrial biogenesis and maintenance, were down-regulated upon C26 CM treatment (Figure 5A and Supplementary Table S3). Furthermore, we also observed increased expression of Mul1 (mitochondrial ubiquitin ligase activator of NF-κB 1) and March5 [membrane-associated ring finger (C3HC4) 5], two E3 ligases involved in mitochondrial fragmentation [39,40], following the addition of CM (Figure 5A and Supplementary Table S3). Genes involved in mitochondrial fission, such as Drp1 (dynamin 1-like) and Fis1 (fission 1), increased marginally when compared with the control, whereas the mitochondrial fusion genes Mfn1 and Mfn2 (Mfn is mitofusin) remained unchanged (Figure 5A and Supplementary Table S3). By quantifying mtDNA with respect to nuDNA, we observed a marked decrease in mitochondrial number in myotubes exposed to CM for 24 and 48 h (Figure 7D). In vitro observations were replicated in the skeletal muscle of C26 tumour-bearing mice (Figure 7E), further confirming that mitochondrial depletion and dysfunction may be due to circulating tumoral factors, such as Mstn.

Large depressions in mitochondrial number are indicative of mitophagy, the selective loss of mitochondria through the autophagy–lysosome pathway. However, to date, there are no reports of increased autophagy–lysosome pathway activity in wasting skeletal muscle, induced through addition of neoplasms. RT-qPCR analysis of C2C12 myotubes exposed to C26 CM for 24 h revealed the up-regulation of several autophagy genes, namely Map1lc3a [microtubule-associated protein 1 LC (light chain) 3α], Atg5 (autophagy-related 5), Atg7 (autophagy-related 7) and Bnip3 (BCL2/adenovirus E1B interacting protein 3) (Figure 5A and Supplementary Table S3). Furthermore, differentiated C2C12 myotubes incubated with C26 CM for 24 and 36 h displayed a greater conversion of the unlipidated LC3 species (LC3-I) into the PE (phosphatidylethanolamine)-conjugated form (LC3-II), which typically binds to autophagosomes (Figure 7F). Moreover, sActRIIB prevented the conversion of LC3 in differentiated C2C12 myotubes incubated in CM (Figure 7G). The in vitro observations of increased autophagy–lysosome pathway activity during cancer cachexia correlated with skeletal muscles isolated from C26 tumour-bearing mice, as we noted elevated conversion of LC3-I into LC3-II in cachectic mice when compared with the controls (Figure 7H).

Mstn, activin A and NF-κB inhibitors ameliorated C26 CM-induced muscle wasting

Inhibitors of Mstn, activin A and NF-κB signalling have previously been shown to alleviate cancer-induced cachexia in vivo [12,14]. In order to investigate whether these inhibitors are effective in alleviating the cachectic-like muscle wasting manifested by C26 CM, C2C12 myotubes were challenged with C26 CM either in the presence or absence of sActRIIB, SB 431542 (a Smad2/3 inhibitor) and BAY 11-7085 (an NF-κB inhibitor). Histological analysis qualitatively demonstrated the partial rescue of myotube atrophy conferred by inhibitor treatment (Figure 8A). Additionally, the myotube area of C2C12 myotubes exposed to CM and sActRIIB (CM+sActRIIB) was similar to that of the control-treated myotubes, suggesting that sActRIIB prevented CM-induced myotube atrophy (Figure 8B). Subsequent immunoblot analysis indicated that sActRIIB, SB 431542 and BAY 11-7085 blocked Myh and Myl degradation in C2C12 myotubes treated with CM (Figure 8C).

Mstn, activin A and NF-κB inhibitors ameliorated C26 CM-induced muscle wasting

Figure 8
Mstn, activin A and NF-κB inhibitors ameliorated C26 CM-induced muscle wasting

(A) Treatment with sActRIIB, SB 431542 and BAY 11-7085 attenuates C2C12 myotube atrophy induced by C26 CM. Haemotoxylin-and-eosin-stained micrographs of C2C12 myotubes with or without CM in the absence or presence of sActRIIB, SB 431542 and BAY 11-7085. Scale bars represent 100 μm. Note: the wasting phenotype observed in myotubes exposed to CM was reversed by the addition of the Mstn (sActRIIB), Smad2/3 (SB 431542) and NF-κB (BAY 11-7085) inhibitors. (B) Histogram represents the myotube area (μm2) in control, CM-, sActRIIB- and CM+ActRIIB-treated C2C12 myotubes as quantified by Image Pro Plus. (C) Immunoblot analysis of Myh and Myl expression showing rescue in response to treatment with (+) or without (−) sActRIIB, SB 431542 and BAY 11-7085 in the presence (+) and absence (−) of CM. Ctrl, control; IB, immunoblot.

Figure 8
Mstn, activin A and NF-κB inhibitors ameliorated C26 CM-induced muscle wasting

(A) Treatment with sActRIIB, SB 431542 and BAY 11-7085 attenuates C2C12 myotube atrophy induced by C26 CM. Haemotoxylin-and-eosin-stained micrographs of C2C12 myotubes with or without CM in the absence or presence of sActRIIB, SB 431542 and BAY 11-7085. Scale bars represent 100 μm. Note: the wasting phenotype observed in myotubes exposed to CM was reversed by the addition of the Mstn (sActRIIB), Smad2/3 (SB 431542) and NF-κB (BAY 11-7085) inhibitors. (B) Histogram represents the myotube area (μm2) in control, CM-, sActRIIB- and CM+ActRIIB-treated C2C12 myotubes as quantified by Image Pro Plus. (C) Immunoblot analysis of Myh and Myl expression showing rescue in response to treatment with (+) or without (−) sActRIIB, SB 431542 and BAY 11-7085 in the presence (+) and absence (−) of CM. Ctrl, control; IB, immunoblot.

Since sActRIIB inhibits both Mstn and activin A function, we next investigated the individual contribution of tumoral Mstn and activin A in manifesting cancer cachexia. Treating differentiated C2C12 myotubes with C26 CM pre-incubated with anti-Mstn antibody (CM+anti-Mstn) partially rescued myotube atrophy when compared with myotubes treated with C26 CM (Figure 9A). A similar rescue of myotube atrophy was recorded when C2C12 myotubes were treated with C26 CM pre-incubated with anti-activin A antibody (CM+anti-activin A) (Figure 9A). However, the myotube area in C2C12 myotubes treated with CM+anti-Mstn was approximately similar to the control myotubes (Figures 9B and 9C). Although C2C12 myotubes treated with CM+anti-activin A demonstrated an improvement in myotube area when compared with CM-treated myotubes, a significant decrease in myotube area was observed when compared with the control myotubes (Figures 9B and 9C). Furthermore, immunoblot analysis demonstrated that anti-Mstn and anti-activin A prevented Myh and Myl degradation in C2C12 myotubes treated with CM (Figure 9D).

Specific inhibitors of Mstn and activin A reduced C26 CM-induced cancer cachexia

Figure 9
Specific inhibitors of Mstn and activin A reduced C26 CM-induced cancer cachexia

(A) Haemotoxylin-and-eosin-stained micrographs of C2C12 differentiated myotubes treated for 24 h with low-serum medium (Ctrl), CM, CM pre-incubated with anti-Mstn antibody (CM+anti-Mstn) and CM pre-incubated with anti-activin A antibody (CM+anti-activin A). Scale bars represent 100 μm. (B) The line graph represents the myotube area (μm2) of control, CM-, CM+anti-Mstn- and CM+anti-activin A-treated C2C12 myotubes as calculated by Image Pro Plus. (C) Average myotube area (μm2) of C2C12 myotubes treated with control, CM, CM+anti-Mstn and CM+anti-activin A. (D) Immunoblot analysis of Myh and Myl expression in C2C12 myotubes exposed to control, CM, CM+anti-Mstn and CM+anti-activin A. Statistical significance was assessed by Student's t test compared with the control; *P<0.01. Ctrl, control; IB, immunoblot.

Figure 9
Specific inhibitors of Mstn and activin A reduced C26 CM-induced cancer cachexia

(A) Haemotoxylin-and-eosin-stained micrographs of C2C12 differentiated myotubes treated for 24 h with low-serum medium (Ctrl), CM, CM pre-incubated with anti-Mstn antibody (CM+anti-Mstn) and CM pre-incubated with anti-activin A antibody (CM+anti-activin A). Scale bars represent 100 μm. (B) The line graph represents the myotube area (μm2) of control, CM-, CM+anti-Mstn- and CM+anti-activin A-treated C2C12 myotubes as calculated by Image Pro Plus. (C) Average myotube area (μm2) of C2C12 myotubes treated with control, CM, CM+anti-Mstn and CM+anti-activin A. (D) Immunoblot analysis of Myh and Myl expression in C2C12 myotubes exposed to control, CM, CM+anti-Mstn and CM+anti-activin A. Statistical significance was assessed by Student's t test compared with the control; *P<0.01. Ctrl, control; IB, immunoblot.

DISCUSSION

Although murine models of cancer cachexia have established that cancer-induced muscle wasting is a direct consequence of circulating protein components produced by both the host and tumour, a comprehensive understanding of the impact of tumoral factors in manifesting cancer cachexia is currently lacking. In the present study, we have identified that proteins secreted by murine C26 colon cancer cells facilitated skeletal muscle wasting through the increased activity of ubiquitin–proteasome and autophagy–lysosome proteolytic pathways (Supplementary Figure S4 at http://www.BiochemJ.org/bj/446/bj4460023add.htm). Furthermore, we have also validated that Mstn, a TGFβ superfamily member involved in the negative regulation of skeletal muscle growth and differentiation, is secreted by murine and human neoplasms. To our knowledge, the expression of Mstn in murine and human cancer cells has not been reported previously, and as such, we propose that Mstn is a novel tumoral factor that stimulates dramatic skeletal muscle wasting during cancer cachexia.

On the basis of the results of the present study, it appeared that cachexia-inducing C26 cells produced vast amounts of Mstn (over 6 ng/ml) when compared with C2C12 myoblasts (2 ng/ml) (S. Lokireddy, I.W. Wijesoma, S. Bonala, C. McFarlane, R. Kambadur and M. Sharma, unpublished work) (Figure 1B). However, we were unable to compare the amount of Mstn secretion by C26 cells with normal non-cancerous colon cells owing to the unavailability of primary or secondary colon cell lines. Nonetheless, the inactivation of Mstn in the C26 CM by a Mstn-specific antibody and sActRIIB prevented the loss of Myh and Myl in muscle cell cultures exposed to C26 CM, further implicating the involvement of Mstn in manifesting C26-induced skeletal muscle wasting (Figures 8 and 9). The specific inhibition of activin A in the C26 CM also prevented the reduction of Myh and Myl in C2C12 myotubes (Figure 9D). However, the average myotube area of CM+activin A-treated myotubes when compared with control myotubes was still significantly lower, suggesting that activin A may play a secondary role in the onset of C26 CM-induced cancer cachexia (Figures 9B and 9C).

Although tumoral Mstn in the C26 CM was sufficient to induce muscle atrophy, the production of Mstn by C2C12 myotubes upon the exposure to C26 CM would imply a feed-forward mechanism of cancer-induced skeletal muscle wasting (Figure 5A and Supplementary Table S3). Indeed, the elevated expression of Mstn has been recorded in the skeletal muscles of tumour-bearing mice, further suggesting that muscle-derived Mstn might be inextricably linked to the pathogenesis of cancer cachexia [12,19].

Mstn elicits its catabolic effects on adult skeletal muscles by elevating the levels of muscle-specific E3 ligases, MuRF1 and atrogin-1, and increasing intracellular protein degradation through the ubiquitin–proteasome pathway [5,6,18]. Likewise, when C2C12 myotubes were exposed to C26 CM for 24 h, the expression of muscle-specific E3 ligases, levels of polyubiquitinated proteins, and the rate of protein degradation were significantly increased when compared with control myotubes (Figures 4 and 5). IGF-I/PI3K/Akt and TGFβ superfamily members regulate the expression of atrogin-1 and MuRF1 during muscle wasting. The results of the present study revealed that C26 CM reduced the levels of active Akt in muscle cell cultures (Figure 5D). Low amounts of active Akt correspond with reduced protein synthesis, an observation that was recapitulated in C2C12 myotubes treated with C26 CM (Figure 4C). Previous studies have shown that low amounts of active Akt also induce the activity of FoxO transcription factors [11]. Unsurprisingly, we observed enhanced levels of active FoxO1 and FoxO3 in C2C12 myotubes exposed to C26 CM (Figure 5D). The FoxO transcription factors, once activated, induce the expression of muscle-specific E3 ligases to enhance skeletal muscle catabolism during cancer cachexia [11,41].

Consistent with enhanced Mstn activity we found elevated levels of active p-Smad2/3 in myotubes treated with C26 CM (Figures 5E and 5F). Increased p-Smad2/3 levels, as shown in the present study, would also enhance the activity of both FoxO1 and FoxO3, and further promote the expression of muscle-specific E3 ligases [5,6].

An overwhelming body of evidence suggests that Mstn signalling inhibits skeletal muscle myogenesis [42]. Given that Mstn was present in large quantities in the C26 CM, it was not surprising to observe inhibition of myoblast proliferation and differentiation upon C26 CM treatment (Figures 2 and 3). In particular, C26 CM halted myoblast proliferation at the G0/G1-phase of the cell cycle through an pRb-dependent manner, a remarkably similar phenomenon to that observed during Mstn-mediated inhibition of myoblast proliferation [43] (Figure 2). Moreover, we also speculate that the reduced levels of MyoD and Myog in differentiating myotubes treated with C26 CM (Figure 3D) were due to the action of Mstn signalling, as a previous study has suggested that Mstn suppresses the expression of MyoD and Myog during skeletal muscle differentiation [43]. Furthermore, sActRIIB addition into the C26 CM facilitated both myoblast proliferation and differentiation, hence confirming the involvement of Mstn and other TGFβ family ligands that bind to ActRIIB in promoting C26-induced cancer cachexia (Figures 2D, 3E and 3F).

Along with the elevated activity of the ubiquitin–proteasome pathway, we detected that the C26 CM activated the autophagy–lysosome pathway (Figure 7F). Since sActRIIB halted the activity of the autophagy–lysosome pathway in C2C12 myotubes exposed to C26 CM, we postulate that Mstn or other ActRIIB-binding ligands in C26 CM may promote the activity of this degradative pathway (Figure 7G). Skeletal muscle from C26 tumour-bearing mice also displayed elevated activity of the autophagy–lysosome pathway, further confirming the involvement of secreted tumoral factors in stimulating autophagy/lysosomal-mediated degradation (Figure 7H). The elevated activity of the autophagy–lysosome pathway is indicative of dysfunctional mitochondria [44]. Increased levels of ROS detected in myoblasts and myotubes exposed to C26 CM may further indicate mitochondrial instability (Figures 7A–7C). The autophagy–lysosome pathway selectively removes damaged mitochondria through a process termed mitophagy [45,46]. Greatly reduced mtDNA copy number per nuDNA, a marker of mitophagy, was observed in muscle cultures treated with C26 CM and in skeletal muscles isolated from C26 tumour-bearing mice (Figures 7D and 7E). Although mitophagy and mitochondrial dysfunction have been previously reported as potent inducers of fasting- and denervation-induced skeletal muscle wasting [44], it remains to be elucidated whether or not similar events further promote or amplify cancer-induced skeletal muscle atrophy.

In conclusion, we have shown in the present study that Mstn is a novel tumoral factor. Given the potent functions conferred by Mstn, we surmise that Mstn secretion by cachexia-inducing neoplasms would initiate the pathogenesis of cancer cachexia. Even though enhanced autophagy has been observed in the heart of tumour-bearing mice [21], in the present study we demonstrated that tumoral factors secreted by C26 cells stimulate the autophagy–lysosomal pathway and increased mitophagy in skeletal muscle.

Abbreviations

     
  • Actb

    β-actin

  •  
  • ActRIIB

    activin receptor type II B

  •  
  • AF-488

    Alexa Fluor® 488

  •  
  • Cdk

    cyclin-dependent kinase

  •  
  • CM

    conditioned medium

  •  
  • DAPI

    4′,6-diamidino-2-phenylindole

  •  
  • DCFH-DA

    2′,7′-dichlorodihydrofluorescein diacetate

  •  
  • DMEM

    Dulbecco's modified Eagle's medium

  •  
  • FBS

    fetal bovine serum

  •  
  • FoxO

    forkhead box O

  •  
  • Gapdh

    glyceraldehyde-3-phosphate dehydrogenase

  •  
  • Hprt

    hypoxanthine–guanine phosphoribosyltransferase

  •  
  • HRP

    horseradish peroxidase

  •  
  • HS

    horse serum

  •  
  • ICC

    immunocytochemistry

  •  
  • IGF-I

    insulin-like growth factor 1

  •  
  • IL

    interleukin

  •  
  • LC

    light chain

  •  
  • Mfn

    mitofusin

  •  
  • Mstn

    myostatin

  •  
  • mtDNA

    mitochondrial DNA

  •  
  • MuRF1

    muscle RING-finger protein 1

  •  
  • Myh

    myosin heavy chain

  •  
  • Myl

    myosin light chain

  •  
  • MyoD

    myogenic differentiation 1

  •  
  • Myog

    myogenin

  •  
  • nano-LC-MS/MS

    nano-liquid chromatography tandem MS

  •  
  • NF-κB

    nuclear factor κB

  •  
  • nuDNA

    nuclear DNA

  •  
  • p-

    phosphorylated

  •  
  • PGC-1α

    PPAR (peroxisome-proliferator-activated receptor) γ co-activator 1α

  •  
  • PI

    propidium iodide

  •  
  • PI3K

    phosphoinositide 3-kinase

  •  
  • pRb

    retinoblastoma protein

  •  
  • P/S

    penicillin/streptomycin

  •  
  • qPCR

    quantitative PCR

  •  
  • ROS

    reactive oxygen species

  •  
  • RT

    reverse transcription

  •  
  • sActRIIB

    soluble form of ActRIIB

  •  
  • SBE

    Smad-binding element

  •  
  • TA

    tibialis anterior

  •  
  • TBST

    Tris buffered saline containing 0.1% Tween 20

  •  
  • TCA

    trichloroacetate

  •  
  • TCM

    tumour CM

  •  
  • TGFβ

    transforming growth factor β

AUTHOR CONTRIBUTION

Sudarsanareddy Lokireddy, Isuru Wijesoma, Sabeera Bonala, Craig McFarlane, Ravi Kambadur and Mridula Sharma conceived and designed the study; Sudarsanareddy Lokireddy, Isuru Wijesoma, Sabeera Bonala and Meng Wei performed the experiments; Sudarsanareddy Lokireddy, Isuru Wijesoma, Sabeera Bonala, Meng Wei, Siu Kwan Sze, Craig McFarlane, Ravi Kambadur and Mridula Sharma analysed the data and interpreted the results of the experiments; Sudarsanareddy Lokireddy, Isuru Wijesoma, Craig McFarlane, Ravi Kambadur and Mridula Sharma prepared the Figures, drafted the paper, and edited and revised the paper prior to submission; and Sudarsanareddy Lokireddy, Isuru Wijesoma, Sabeera Bonala, Meng Wei, Siu Kwan Sze, Craig McFarlane, Ravi Kambadur and Mridula Sharma approved the final version of the paper.

We thank Esther Latres (Regeneron Pharmaceuticals, Tarrytown, NJ, U.S.A.) for providing the anti-atrogin-1 and anti-MuRF1 antibodies used in the present study. The MF20 and T14 monoclonal antibodies developed by Donald A. Fischman and Frank E. Stockdale respectively were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD (National Institute of Child Health and Human Development) and maintained by Department of Biology, The University of Iowa, Iowa City, IA, U.S.A. Finally, we thank Addgene (Cambridge, MA, U.S.A.) for providing the reporter vectors [FoxO3 (Dr Michael Greenberg, #1789), PGC-1α (Dr Bruce Spiegelman, #8887) and 4X SBE (Dr Bert Vogelstein, #16495)].

FUNDING

This work was supported by the MOE (Ministry of Education), Singapore [grant number T208B3211] and BMRC (Biomedical Research Council), Singapore [grant number 07/1/21/19/521].

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Author notes

1

These authors contributed equally to the work.

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