Myostatin is a novel tumoral factor that induces cancer cachexia

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.     

Lung cancer-derived extracellular vesicles induced myotube atrophy and adipocyte lipolysis via the extracellular IL-6-mediated STAT3 pathway

Cancer-associated cachexia (CAC) constitutes a metabolic dysfunction characterized by systemic inflammation and body weight loss. Muscle atrophy and adipose tissue lipolysis might explain weight loss in CAC. Specific functions of numerous hormones and cytokines derived from tumours can provoke cachexia. Extracellular vesicles (EVs) can be involved in intercellular communication. However, whether EVs participate in this process has not been investigated thoroughly. Using Lewis lung carcinoma (LLC) cell cultures, we tested whether LLC-derived EVs induced C2C12 myotube atrophy and 3T3-L1 adipocyte lipolysis. EVs derived from LLC cells and serum from patients with lung cancer, non-lung cancer controls, tumour-bearing mice, and non-tumour-bearing control mice were isolated and characterized biochemically and biophysically. LLC cell-derived EVs induced dose-dependent effects of atrophy in C2C12 myotubes and lipolysis in 3T3-L1 adipocytes. Mechanistically, EVs directly fused with target C2C12 myotubes and 3T3-L1 adipocytes, and transferred interleukin-6 (IL-6) activates the STAT3 signalling pathway in C2C12 myotubes and 3T3-L1 adipocytes. Neutralization of extracellular IL-6 prevented the atrophy and lipolysis effects of EVs. Inhibiting the STAT3 signalling pathway also prevented the atrophy and lipolysis effects of EVs. PKH67-labelled (PKH 67 is a lipid dye that can be used to label extracellular vesicles) LLC-EVs were readily internalized into myotubes and adipocytes. Our data showed that LLC cell-derived EVs induced myotube atrophy and adipocyte lipolysis via the extracellular IL-6-mediated STAT3 pathway in target cells. These findings represent a potentially novel basis for further research in this field towards identifying targets and developing strategies for maintaining weight in CAC.     

A novel role for CD4+ T cells in the control of cachexia

Cachexia is the dramatic weight loss and muscle atrophy seen in chronic disease states, including autoimmunity, cancer, and infection, and is often associated with lymphopenia. We have previously shown that CD4(+) T cells that express the lowest density of CD44 (CD4(+)CD44(v.low)) are significantly reduced in diabetic NOD mice that are cachexic compared with diabetic mice that are not cachexic. Using this model, and a model of cancer cachexia, we test the hypothesis that CD4(+)CD44(v.low) cells play an active role in protecting the host from cachexia. CD4(+)CD44(v.low) cells, but not CD4(+) cells depleted of CD44(v.low) cells, delay the onset of wasting when infused into either diabetic or prediabetic NOD recipients. However, no significant effect on the severity of diabetes was detected. In a model of cancer cachexia, they significantly reduce muscle atrophy, and inhibit muscle protein loss and DNA loss, even when given after the onset of cachexia. Protection from wasting and muscle atrophy by CD4(+)CD44(v.low) cells is associated with protection from lymphopenia. These data suggest, for the first time, a role for an immune cell subset in protection from cachexia, and further suggest that the mechanism of protection is independent of protection from autoimmunity.     

JAK/STAT3 pathway inhibition blocks skeletal muscle wasting downstream of IL-6 and in experimental cancer cachexia

Cachexia, the metabolic dysregulation leading to sustained loss of muscle and adipose tissue, is a devastating complication of cancer and other chronic diseases. Interleukin-6 and related cytokines are associated with muscle wasting in clinical and experimental cachexia, although the mechanisms by which they might induce muscle wasting are unknown. One pathway activated strongly by IL-6 family ligands is the JAK/STAT3 pathway, the function of which has not been evaluated in regulation of skeletal muscle mass. Recently, we showed that skeletal muscle STAT3 phosphorylation, nuclear localization, and target gene expression are activated in C26 cancer cachexia, a model with high IL-6 family ligands. Here, we report that STAT3 activation is a common feature of muscle wasting, activated in muscle by IL-6 in vivo and in vitro and by different types of cancer and sterile sepsis. Moreover, STAT3 activation proved both necessary and sufficient for muscle wasting. In C(2)C(12) myotubes and in mouse muscle, mutant constitutively activated STAT3-induced muscle fiber atrophy and exacerbated wasting in cachexia. Conversely, inhibiting STAT3 pharmacologically with JAK or STAT3 inhibitors or genetically with dominant negative STAT3 and short hairpin STAT3 reduced muscle atrophy downstream of IL-6 or cancer. These results indicate that STAT3 is a primary mediator of muscle wasting in cancer cachexia and other conditions of high IL-6 family signaling. Thus STAT3 could represent a novel therapeutic target for the preservation of skeletal muscle in cachexia.     

Extracellular vesicles-released parathyroid hormone-related protein from Lewis lung carcinoma induces lipolysis and adipose tissue browning in cancer cachexia

Cancer cachexia is a metabolic disorder characterized by skeletal muscle wasting and white adipose tissue browning. Specific functions of several hormones, growth factors, and cytokines derived from tumors can trigger cachexia. Moreover, adipose tissue lipolysis might explain weight loss that occurs owing to cachexia. Extracellular vesicles (EVs) are involved in intercellular communication. However, whether EVs participate in lipolysis induced by cancer cachexia has not been thoroughly investigated. Using Lewis lung carcinoma (LLC) cell culture, we tested whether LLC cell-derived EVs can induce lipolysis in 3T3-L1 adipocytes. EVs derived from LLC cells were isolated and characterized biochemically and biophysically. Western blotting and glycerol assay were used to study lipolysis. LLC cell-derived EVs induced lipolysis in vivo and vitro. EVs fused directly with target 3T3-L1 adipocytes and transferred parathyroid hormone-related protein (PTHrP), activating the PKA signaling pathway in 3T3-L1 adipocytes. Blocking PTHrP activity in LLC-EVs using a neutralizing antibody and by knocking down PTHR expression prevented lipolysis in adipocytes. Inhibiting the PKA signaling pathway also prevents the lipolytic effects of EVs. In vivo, suppression of LLC-EVs release by knocking down Rab27A alleviated white adipose tissue browning and lipolysis. Our data showed that LLC cell-derived EVs induced adipocyte lipolysis via the extracellular PTHrP-mediated PKA pathway. Our data demonstrate that LLC-EVs induce lipolysis in vitro and vivo by delivering PTHrP, which interacts with PTHR. The lipolytic effect of LLC-EVs was abrogated by PTHR knockdown and treatment with a neutralizing anti-PTHrP antibody. Together, these data show that LLC-EV-induced lipolysis is mediated by extracellular PTHrP. These findings suggest a novel mechanism of lipid droplet loss and identify a potential therapeutic strategy for cancer cachexia.Subject terms: Oncogenes, Mechanisms of disease     

Antibody-directed myostatin inhibition enhances muscle mass and function in tumor-bearing mice

Cancer cachexia describes the progressive skeletal muscle wasting and weakness in many cancer patients and accounts for >20% of cancer-related deaths. We tested the hypothesis that antibody-directed myostatin inhibition would attenuate the atrophy and loss of function in muscles of tumor-bearing mice. Twelve-week-old C57BL/6 mice received a subcutaneous injection of saline (control) or Lewis lung carcinoma (LLC) tumor cells. One week later, mice received either once weekly injections of saline (control, n = 12; LLC, n = 9) or a mouse chimera of anti-human myostatin antibody (PF-354, 10 mg·kg?1·wk?1, LLC+PF-354, n = 11) for 5 wk. Injection of LLC cells reduced muscle mass and maximum force of tibialis anterior (TA) muscles by 8-10% (P < 0.05), but the muscle atrophy and weakness were prevented with PF-354 treatment (P > 0.05). Maximum specific (normalized) force of diaphragm muscle strips was reduced with LLC injection (P < 0.05) but was not improved with PF-354 treatment (P > 0.05). PF-354 enhanced activity of oxidative enzymes in TA and diaphragm muscles of tumor-bearing mice by 118% and 89%, respectively (P < 0.05). Compared with controls, apoptosis that was not of myofibrillar or satellite cell origin was 140% higher in TA muscle cross sections from saline-treated LLC tumor-bearing mice (P < 0.05) but was not different in PF-354-treated tumor-bearing mice (P > 0.05). Antibody-directed myostatin inhibition attenuated the skeletal muscle atrophy and loss of muscle force-producing capacity in a murine model of cancer cachexia, in part by reducing apoptosis. The improvements in limb muscle mass and function highlight the therapeutic potential of antibody-directed myostatin inhibition for cancer cachexia.     

A high-quality secretome of A549 cells aided the discovery of C4b-binding protein as a novel serum biomarker for non-small cell lung cancer

Cancer secretomes are a promising source for biomarker discovery. The analysis of cancer secretomes still faces some difficulties mainly related to the intracellular contamination, which hinders the qualification and follow-up validations. This study aimed to establish a high-quality secretome of A549 cells by using the cellular proteome as a reference and to test the merits of this refined secretome for biomarker discovery for non-small cell lung cancer (NSCLC). Using one-dimensional gel electrophoresis followed by liquid-chromatography tandem mass spectrometry, we comprehensively investigated the secretome and the concurrent cellular proteome of A549 cells. A high-quality secretome consisting of 382 proteins was refined from 889 initial secretory proteins. More than 85.3% of proteins were annotated as secreted and 76.8% as extracellular or membrane-bound. The discriminative power of the lung-cancer associated secretome was confirmed by gene expression and serum proteomic data. The elevated level of C4b-binding Protein (C4BP) in NSCLC blood was verified by enzyme-linked immunosorbent assays (ELISA, p = 6.07e-6). Moreover, the serum C4BP level in 89 patients showed a strong association with the clinical staging of NSCLC. Our reference-experiment-driven strategy is simple and widely applicable, and may facilitate the identification of novel promising biomarkers of lung cancer.     

Muscle wasting and impaired myogenesis in tumor bearing mice are prevented by ERK inhibition

Background

The onset of cachexia is a frequent feature in cancer patients. Prominent characteristic of this syndrome is the loss of body and muscle weight, this latter being mainly supported by increased protein breakdown rates. While the signaling pathways dependent on IGF-1 or myostatin were causally involved in muscle atrophy, the role of the Mitogen-Activated-Protein-Kinases is still largely debated. The present study investigated this point on mice bearing the C26 colon adenocarcinoma.

Methodology/Principal Findings

C26-bearing mice display a marked loss of body weight and muscle mass, this latter associated with increased phosphorylated (p)-ERK. Administration of the ERK inhibitor PD98059 to tumor bearers attenuates muscle depletion and weakness, while restoring normal atrogin-1 expression. In C26 hosts, muscle wasting is also associated with increased Pax7 expression and reduced myogenin levels. Such pattern, suggestive of impaired myogenesis, is reversed by PD98059. Increased p-ERK and reduced myosin heavy chain content can be observed in TNFα-treated C2C12 myotubes, while decreased myogenin and MyoD levels occur in differentiating myoblasts exposed to the cytokine. All these changes are prevented by PD98059.

Conclusions/Significance

These results demonstrate that ERK is involved in the pathogenesis of muscle wasting in cancer cachexia and could thus be proposed as a therapeutic target.     

Muscle Atrophy in Response to Cytotoxic Chemotherapy Is Dependent on Intact Glucocorticoid Signaling in Skeletal Muscle

Cancer cachexia is a syndrome of weight loss that results from the selective depletion of skeletal muscle mass and contributes significantly to cancer morbidity and mortality. The driver of skeletal muscle atrophy in cancer cachexia is systemic inflammation arising from both the cancer and cancer treatment. While the importance of tumor derived inflammation is well described, the mechanism by which cytotoxic chemotherapy contributes to cancer cachexia is relatively unexplored. We found that the administration of chemotherapy to mice produces a rapid inflammatory response. This drives activation of the hypothalamic-pituitary-adrenal axis, which increases the circulating level of corticosterone, the predominant endogenous glucocorticoid in rodents. Additionally, chemotherapy administration results in a significant loss of skeletal muscle mass 18 hours after administration with a concurrent induction of genes involved with the ubiquitin proteasome and autophagy lysosome systems. However, in mice lacking glucocorticoid receptor expression in skeletal muscle, chemotherapy-induced muscle atrophy is completely blocked. This demonstrates that cytotoxic chemotherapy elicits significant muscle atrophy driven by the production of endogenous glucocorticoids. Further, it argues that pharmacotherapy targeting the glucocorticoid receptor, given in concert with chemotherapy, is a viable therapeutic strategy in the treatment of cancer cachexia.     

The TWEAK–Fn14 system is a critical regulator of denervation-induced skeletal muscle atrophy in mice

Skeletal muscle atrophy occurs in a variety of clinical settings, including cachexia, disuse, and denervation. Inflammatory cytokines have been shown to be mediators of cancer cachexia; however, the role of cytokines in denervation- and immobilization-induced skeletal muscle loss remains unknown. In this study, we demonstrate that a single cytokine, TNF-like weak inducer of apoptosis (TWEAK), mediates skeletal muscle atrophy that occurs under denervation conditions. Transgenic expression of TWEAK induces atrophy, fibrosis, fiber-type switching, and the degradation of muscle proteins. Importantly, genetic ablation of TWEAK decreases the loss of muscle proteins and spared fiber cross-sectional area, muscle mass, and strength after denervation. Expression of the TWEAK receptor Fn14 (fibroblast growth factor–inducible receptor 14) and not the cytokine is significantly increased in muscle upon denervation, demonstrating an unexpected inside-out signaling pathway; the receptor up-regulation allows for TWEAK activation of nuclear factor κB, causing an increase in the expression of the E3 ubiquitin ligase MuRF1. This study reveals a novel mediator of skeletal muscle atrophy and indicates that the TWEAK–Fn14 system is an important target for preventing skeletal muscle wasting.     

Identification of the Acetylation and Ubiquitin-Modified Proteome during the Progression of Skeletal Muscle Atrophy

Skeletal muscle atrophy is a consequence of several physiological and pathophysiological conditions including muscle disuse, aging and diseases such as cancer and heart failure. In each of these conditions, the predominant mechanism contributing to the loss of skeletal muscle mass is increased protein turnover. Two important mechanisms which regulate protein stability and degradation are lysine acetylation and ubiquitination, respectively. However our understanding of the skeletal muscle proteins regulated through acetylation and ubiquitination during muscle atrophy is limited. Therefore, the purpose of the current study was to conduct an unbiased assessment of the acetylation and ubiquitin-modified proteome in skeletal muscle during a physiological condition of muscle atrophy. To induce progressive, physiologically relevant, muscle atrophy, rats were cast immobilized for 0, 2, 4 or 6 days and muscles harvested. Acetylated and ubiquitinated peptides were identified via a peptide IP proteomic approach using an anti-acetyl lysine antibody or a ubiquitin remnant motif antibody followed by mass spectrometry. In control skeletal muscle we identified and mapped the acetylation of 1,326 lysine residues to 425 different proteins and the ubiquitination of 4,948 lysine residues to 1,131 different proteins. Of these proteins 43, 47 and 50 proteins were differentially acetylated and 183, 227 and 172 were differentially ubiquitinated following 2, 4 and 6 days of disuse, respectively. Bioinformatics analysis identified contractile proteins as being enriched among proteins decreased in acetylation and increased in ubiquitination, whereas histone proteins were enriched among proteins increased in acetylation and decreased in ubiquitination. These findings provide the first proteome-wide identification of skeletal muscle proteins exhibiting changes in lysine acetylation and ubiquitination during any atrophy condition, and provide a basis for future mechanistic studies into how the acetylation and ubiquitination status of these identified proteins regulates the muscle atrophy phenotype.     

Inhibition of Stat3 Activation Suppresses Caspase-3 and the Ubiquitin-Proteasome System,Leading to Preservation of Muscle Mass in Cancer Cachexia

Cachexia occurs in patients with advanced cancers. Despite the adverse clinical impact of cancer-induced muscle wasting, pathways causing cachexia are controversial, and clinically reliable therapies are not available. A trigger of muscle protein loss is the Jak/Stat pathway, and indeed, we found that conditioned medium from C26 colon carcinoma (C26) or Lewis lung carcinoma cells activates Stat3 (p-Stat3) in C2C12 myotubes. We identified two proteolytic pathways that are activated in muscle by p-Stat3; one is activation of caspase-3, and the other is p-Stat3 to myostatin, MAFbx/Atrogin-1, and MuRF-1 via CAAT/enhancer-binding protein δ (C/EBPδ). Using sequential deletions of the caspase-3 promoter and CHIP assays, we determined that Stat3 activation increases caspase-3 expression in C2C12 cells. Caspase-3 expression and proteolytic activity were stimulated by p-Stat3 in muscles of tumor-bearing mice. In mice with cachexia caused by Lewis lung carcinoma or C26 tumors, knock-out of p-Stat3 in muscle or with a small chemical inhibitor of p-Stat3 suppressed muscle mass losses, improved protein synthesis and degradation in muscle, and increased body weight and grip strength. Activation of p-Stat3 stimulates a pathway from C/EBPδ to myostatin and expression of MAFbx/Atrogin-1 and increases the ubiquitin-proteasome system. Indeed, C/EBPδ KO decreases the expression of MAFbx/Atrogin-1 and myostatin, while increasing muscle mass and grip strength. In conclusion, cancer stimulates p-Stat3 in muscle, activating protein loss by stimulating caspase-3, myostatin, and the ubiquitin-proteasome system. These results could lead to novel strategies for preventing cancer-induced muscle wasting.     

Advances in cancer cachexia: Intersection between affected organs,mediators, and pharmacological interventions

Advanced cancer patients exhibit cachexia, a condition characterized by a significant reduction in the body weight predominantly from loss of skeletal muscle and adipose tissue. Cachexia is one of the major causes of morbidity and mortality in cancer patients. Decreased food intake and multi-organ energy imbalance in cancer patients worsen the cachexia syndrome. Cachectic cancer patients have a low tolerance for chemo- and radiation therapies and also have a reduced quality of life. The presence of tumors and the current treatment options for cancer further exacerbate the cachexia condition, which remains an unmet medical need. The onset of cachexia involves crosstalk between different organs leading to muscle wasting. Recent advancements in understanding the molecular mechanisms of skeletal muscle atrophy/hypertrophy and adipose tissue wasting/browning provide a platform for the development of new targeted therapies. Therefore, a better understanding of this multifactorial disorder will help to improve the quality of life of cachectic patients. In this review, we summarize the metabolic mediators of cachexia, their molecular functions, affected organs especially with respect to muscle atrophy and adipose browning and then discuss advanced therapeutic approaches to cancer cachexia.     

Tumor-host interactions

A number of malignant tumors interact with the host to cause a syndrome of cachexia, characterized by extensive loss of adipose tissue and skeletal muscle mass, but with preservation of proteins in visceral tissues. Although anorexia is frequently present, the body composition changes in cancer cachexia cannot be explained by nutritional deprivation alone. Loss of skeletal muscle mass is a result of depression in protein synthesis and an increase in protein degradation. The main degradative pathway that has been found to have increased expression and activity in the skeletal muscle of cachectic patients is the ubiquitin-proteasome proteolytic pathway. Cachexia-inducing tumors produce catabolic factors such as proteolysis-inducing factor (PIF), a 24 kDa sulfated glycoprotein, which inhibit protein synthesis and stimulate degradation of intracellular proteins in skeletal muscle by inducing an increased expression of regulatory components of the ubiquitin-proteasome proteolytic pathway. While the oligosaccharide chains in PIF are required to initiate protein degradation the central polypeptide core may act as a growth and survival factor. Only cachexia-inducing tumors are capable of elaborating fully glycosylated PIF, and the selectivity of production possibly rests with the acquisition of the necessary glycosylating enzymes, rather than expressing the gene for the polypeptide core. Loss of adipose tissue is probably the result of an increase in catabolism rather than a defect in anabolism. A lipid mobilizing factor (LMF), identical with the plasma protein Zn-alpha2-glycoprotein (ZAG) is found in the urine of cachectic cancer patients and is produced by tumors causing a decrease in carcass lipid. LMF causes triglyceride hydrolysis in adipose tissue through a cyclic AMP-mediated process by interaction with a beta3-adrenoreceptor. Thus, by producing circulating factors certain malignant tumors are able to interfere with host metabolism even without metastasis to that particular site.