Sunitinib-mediated inhibition of STAT3 in skeletal muscle and spinal cord does not affect the disease in a mouse model of ALS.

Variability in disease onset and progression is a hallmark of amyotrophic lateral sclerosis (ALS), both in sporadic and genetic forms. Recently, we found that SOD1-G93A transgenic mice expressing the same amount of mutant SOD1 but with different genetic backgrounds, C57BL/6JOlaHsd and 129S2/SvHsd, show slow and rapid muscle wasting and disease progression, respectively. Here, we investigated the different molecular mechanisms underlying muscle atrophy. Although both strains showed similar denervation-induced degradation of muscle proteins, only the rapidly progressing mice exhibited early and sustained STAT3 activation that preceded atrophy in gastrocnemius muscle. We therefore investigated the therapeutic potential of sunitinib, a tyrosine kinase inhibitor known to inhibit STAT3 and prevent cancer-induced muscle wasting. Although sunitinib treatment reduced STAT3 activation in the gastrocnemius muscle and lumbar spinal cord, it did not preserve spinal motor neurons, improve neuromuscular impairment, muscle atrophy and disease progression in the rapidly progressing SOD1-G93A mice. Thus, the effect of sunitinib is not equally positive in different diseases associated with muscle wasting. Moreover, given the complex role of STAT3 in the peripheral and central compartments of the neuromuscular system, the present study suggests that its broad inhibition may lead to opposing effects, ultimately preventing a potential positive therapeutic action in ALS.

Integrated procedures for accelerating, deepening, and leading genetic inquiry: A first application on human muscle secretome.

PURPOSE: Beyond classical procedures, bioinformatic-assisted approaches and computational biology offer unprecedented opportunities for scholars. However, these amazing possibilities still need epistemological criticism, as well as standardized procedures. Especially those topics with a huge body of data may benefit from data science (DS)-assisted methods. Therefore, the current study dealt with the combined expert-assisted and DS-assisted approaches to address the broad field of muscle secretome. We aimed to apply DS tools to fix the literature research, suggest investigation targets with a data-driven approach, predict possible scenarios, and define a workflow. METHODS: Recognized scholars with expertise on myokines were invited to provide a list of the most important myokines. GeneRecommender, GeneMANIA, HumanNet, and STRING were selected as DS tools. Networks were built on STRING and GeneMANIA. The outcomes of DS tools included the top 5 recommendations. Each expert-led discussion has been then integrated with an DS-led approach to provide further perspectives. RESULTS: Among the results, 11 molecules had already been described as bona-fide myokines in literature, and 11 molecules were putative myokines. Most of the myokines and the putative myokines recommended by the DS tools were described as present in the cargo of extracellular vesicles. CONCLUSIONS: Including both supervised and unsupervised learning methods, as well as encompassing algorithms focused on both protein interaction and gene represent a comprehensive approach to tackle complex biomedical topics. DS-assisted methods for reviewing existent evidence, recommending targets of interest, and predicting original scenarios are worth exploring as in silico recommendations to be integrated with experts' ideas for optimizing molecular studies.

The p97-Nploc4 ATPase complex plays a role in muscle atrophy during cancer and amyotrophic lateral sclerosis.

BACKGROUND: The p97 complex participates in the degradation of muscle proteins during atrophy upon fasting or denervation interacting with different protein adaptors. We investigated whether and how it might also be involved in muscle wasting in cancer, where loss of appetite occurs, or amyotrophic lateral sclerosis (ALS), where motoneuron death causes muscle denervation and fatal paralysis. METHODS: As cancer cachexia models, we used mice bearing colon adenocarcinoma C26, human renal carcinoma RXF393, or Lewis lung carcinoma, with breast cancer 4T1-injected mice as controls. As ALS models, we employed 129/SvHsd mice carrying the mutation G93A in human SOD1. The expression of p97 and its adaptors was analysed in their muscles by quantitative real-time polymerase chain reaction (qPCR) and western blot. We electroporated plasmids into muscles or treated mice with disulfiram (DSF) to test the effects of inhibiting p97 and nuclear protein localization protein 4 (Nploc4), one of its adaptors, on atrophy. RESULTS: The mRNA levels of p97 were induced by 1.5-fold to 2-fold in tibialis anterior (TA) of all the cachectic models but not in the non-cachectic 4T1 tumour-bearing mice (P ≤ 0.05). Similarly, p97 was high both in mRNA and protein in TA from 17-week-old SOD1G93A mice (P ≤ 0.01). Electroporation of a shRNA for murine p97 into mouse muscle reduced the fibre atrophy caused by C26 (P = 0.0003) or ALS (P ≤ 0.01). When we interrogated a microarray, we had previously generated for the expression of p97 adaptors, we found Derl1, Herpud1, Nploc4, Rnf31, and Hsp90ab1 induced in cachectic TA from C26-mice (Fold change > 1.2, adjusted P ≤ 0.05). By qPCR, we validated their inductions in TA of cachectic and ALS models and selected Nploc4 as the one also induced at the protein level by 1.5-fold (P ≤ 0.01). Electroporation of a CRISPR/Cas9 vector against Nploc4 into muscle reduced the fibre atrophy caused by C26 (P = 0.01) or ALS (P ≤ 0.0001). Because DSF uncouples p97 from Nploc4, we treated atrophying myotubes with DSF, and found accumulated mono and polyubiquitinated proteins and reduced degradation of long-lived proteins by 35% (P ≤ 0.0001), including actin (P ≤ 0.05). DSF halves Nploc4 in the soluble muscle fraction (P ≤ 0.001) and given to C26-bearing mice limited the body and muscle weight loss (P ≤ 0.05), with no effect on tumour growth. CONCLUSIONS: Overall, cancer cachexia and ALS seem to display similar mechanisms of muscle wasting at least at the catabolic level. The p97-Nploc4 complex appears to have a crucial role in muscle atrophy during these disorders and disrupting this complex might serve as a novel drug strategy.

Apelin Resistance Contributes to Muscle Loss during Cancer Cachexia in Mice.

Cancer cachexia consists of dramatic body weight loss with rapid muscle depletion due to imbalanced protein homeostasis. We found that the mRNA levels of apelin decrease in muscles from cachectic hepatoma-bearing rats and three mouse models of cachexia. Furthermore, apelin expression inversely correlates with MuRF1 in muscle biopsies from cancer patients. To shed light on the possible role of apelin in cachexia in vivo, we generated apelin 13 carrying all the last 13 amino acids of apelin in D isomers, ultimately extending plasma stability. Notably, apelin D-peptides alter cAMP-based signaling in vitro as the L-peptides, supporting receptor binding. In vitro apelin 13 protects myotube diameter from dexamethasone-induced atrophy, restrains rates of degradation of long-lived proteins and MuRF1 expression, but fails to protect mice from atrophy. D-apelin 13 given intraperitoneally for 13 days in colon adenocarcinoma C26-bearing mice does not reduce catabolic pathways in muscles, as it does in vitro. Puzzlingly, the levels of circulating apelin seemingly deriving from cachexia-inducing tumors, increase in murine plasma during cachexia. Muscle electroporation of a plasmid expressing its receptor APJ, unlike apelin, preserves myofiber area from C26-induced atrophy, supporting apelin resistance in vivo. Altogether, we believe that during cachexia apelin resistance occurs, contributing to muscle wasting and nullifying any possible peptide-based treatment.

Transforming Growth Factor-Beta Signaling in Cancer-Induced Cachexia: From Molecular Pathways to the Clinics.

Cachexia is a metabolic syndrome consisting of massive loss of muscle mass and function that has a severe impact on the quality of life and survival of cancer patients. Up to 20% of lung cancer patients and up to 80% of pancreatic cancer patients are diagnosed with cachexia, leading to death in 20% of them. The main drivers of cachexia are cytokines such as interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), macrophage inhibitory cytokine 1 (MIC-1/GDF15) and transforming growth factor-beta (TGF-ß). Besides its double-edged role as a tumor suppressor and activator, TGF-ß causes muscle loss through myostatin-based signaling, involved in the reduction in protein synthesis and enhanced protein degradation. Additionally, TGF-ß induces inhibin and activin, causing weight loss and muscle depletion, while MIC-1/GDF15, a member of the TGF-ß superfamily, leads to anorexia and so, indirectly, to muscle wasting, acting on the hypothalamus center. Against this background, the blockade of TGF-ß is tested as a potential mechanism to revert cachexia, and antibodies against TGF-ß reduced weight and muscle loss in murine models of pancreatic cancer. This article reviews the role of the TGF-ß pathway and to a minor extent of other molecules including microRNA in cancer onset and progression with a special focus on their involvement in cachexia, to enlighten whether TGF-ß and such other players could be potential targets for therapy.

Costameric integrin and sarcoglycan protein levels are altered in a Drosophila model for Limb-girdle muscular dystrophy type 2H.

Mutations in two different domains of the ubiquitously expressed TRIM32 protein give rise to two clinically separate diseases, one of which is Limb-girdle muscular dystrophy type 2H (LGMD2H). Uncovering the muscle-specific role of TRIM32 in LGMD2H pathogenesis has proven difficult, as neurogenic phenotypes, independent of LGMD2H pathology, are present in TRIM32 KO mice. We previously established a platform to study LGMD2H pathogenesis using Drosophila melanogaster as a model. Here we show that LGMD2H disease-causing mutations in the NHL domain are molecularly and structurally conserved between fly and human TRIM32. Furthermore, transgenic expression of a subset of myopathic alleles (R394H, D487N, and 520fs) induce myofibril abnormalities, altered nuclear morphology, and reduced TRIM32 protein levels, mimicking phenotypes in patients afflicted with LGMD2H. Intriguingly, we also report for the first time that the protein levels of ßPS integrin and sarcoglycan δ, both core components of costameres, are elevated in TRIM32 disease-causing alleles. Similarly, murine myoblasts overexpressing a catalytically inactive TRIM32 mutant aberrantly accumulate α- and ß-dystroglycan and α-sarcoglycan. We speculate that the stoichiometric loss of costamere components disrupts costamere complexes to promote muscle degeneration.

Nutraceuticals and Exercise against Muscle Wasting during Cancer Cachexia.

Cancer cachexia (CC) is a debilitating multifactorial syndrome, involving progressive deterioration and functional impairment of skeletal muscles. It affects about 80% of patients with advanced cancer and causes premature death. No causal therapy is available against CC. In the last few decades, our understanding of the mechanisms contributing to muscle wasting during cancer has markedly increased. Both inflammation and oxidative stress (OS) alter anabolic and catabolic signaling pathways mostly culminating with muscle depletion. Several preclinical studies have emphasized the beneficial roles of several classes of nutraceuticals and modes of physical exercise, but their efficacy in CC patients remains scant. The route of nutraceutical administration is critical to increase its bioavailability and achieve the desired anti-cachexia effects. Accumulating evidence suggests that a single therapy may not be enough, and a bimodal intervention (nutraceuticals plus exercise) may be a more effective treatment for CC. This review focuses on the current state of the field on the role of inflammation and OS in the pathogenesis of muscle atrophy during CC, and how nutraceuticals and physical activity may act synergistically to limit muscle wasting and dysfunction.

Trabectedin and Lurbinectedin Extend Survival of Mice Bearing C26 Colon Adenocarcinoma, without Affecting Tumor Growth or Cachexia.

Trabectedin (ET743) and lurbinectedin (PM01183) limit the production of inflammatory cytokines that are elevated during cancer cachexia. Mice carrying C26 colon adenocarcinoma display cachexia (i.e., premature death and body wasting with muscle, fat and cardiac tissue depletion), high levels of inflammatory cytokines and subsequent splenomegaly. We tested whether such drugs protected these mice from cachexia. Ten-week-old mice were inoculated with C26 cells and three days later randomized to receive intravenously vehicle or 0.05 mg/kg ET743 or 0.07 mg/kg PM01183, three times a week for three weeks. ET743 or PM01183 extended the lifespan of C26-mice by 30% or 85%, respectively, without affecting tumor growth or food intake. Within 13 days from C26 implant, both drugs did not protect fat, muscle and heart from cachexia. Since PM01183 extended the animal survival more than ET743, we analyzed PM01183 further. In tibialis anterior of C26-mice, but not in atrophying myotubes, PM01183 restrained the NF-κB/PAX7/myogenin axis, possibly reducing the pro-inflammatory milieu, and failed to limit the C/EBPß/atrogin-1 axis. Inflammation-mediated splenomegaly of C26-mice was inhibited by PM01183 for as long as the treatment lasted, without reducing IL-6, M-CSF or IL-1ß in plasma. ET743 and PM01183 extend the survival of C26-bearing mice unchanging tumor growth or cachexia but possibly restrain muscle-related inflammation and C26-induced splenomegaly.

Group I Paks support muscle regeneration and counteract cancer-associated muscle atrophy.

BACKGROUND: Skeletal muscle is characterized by an efficient regeneration potential that is often impaired during myopathies. Understanding the molecular players involved in muscle homeostasis and regeneration could help to find new therapies against muscle degenerative disorders. Previous studies revealed that the Ser/Thr kinase p21 protein-activated kinase 1 (Pak1) was specifically down-regulated in the atrophying gastrocnemius of Yoshida hepatoma-bearing rats. In this study, we evaluated the role of group I Paks during cancer-related atrophy and muscle regeneration. METHODS: We examined Pak1 expression levels in the mouse Tibialis Anterior muscles during cancer cachexia induced by grafting colon adenocarcinoma C26 cells and in vitro by dexamethasone treatment. We investigated whether the overexpression of Pak1 counteracts muscle wasting in C26-bearing mice and in vitro also during interleukin-6 (IL6)-induced or dexamethasone-induced C2C12 atrophy. Moreover, we analysed the involvement of group I Paks on myogenic differentiation in vivo and in vitro using the group I chemical inhibitor IPA-3. RESULTS: We found that Pak1 expression levels are reduced during cancer-induced cachexia in the Tibialis Anterior muscles of colon adenocarcinoma C26-bearing mice and in vitro during dexamethasone-induced myotube atrophy. Electroporation of muscles of C26-bearing mice with plasmids directing the synthesis of PAK1 preserves fiber size in cachectic muscles by restraining the expression of atrogin-1 and MuRF1 and possibly by inducing myogenin expression. Consistently, the overexpression of PAK1 reduces the dexamethasone-induced expression of MuRF1 in myotubes and increases the phospho-FOXO3/FOXO3 ratio. Interestingly, the ectopic expression of PAK1 counteracts atrophy in vitro by restraining the IL6-Stat3 signalling pathway measured in luciferase-based assays and by reducing rates of protein degradation in atrophying myotubes exposed to IL6. On the other hand, we observed that the inhibition of group I Paks has no effect on myotube atrophy in vitro and is associated with impaired muscle regeneration in vivo and in vitro. In fact, we found that mice treated with the group I inhibitor IPA-3 display a delayed recovery from cardiotoxin-induced muscle injury. This is consistent with in vitro experiments showing that IPA-3 impairs myogenin expression and myotube formation in vessel-associated myogenic progenitors, C2C12 myoblasts, and satellite cells. Finally, we observed that IPA-3 reduces p38α/ß phosphorylation that is required to proceed through various stages of satellite cells differentiation: activation, asymmetric division, and ultimately myotube formation. CONCLUSIONS: Our data provide novel evidence that is consistent with group I Paks playing a central role in the regulation of muscle homeostasis, atrophy and myogenesis.