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We investigated the impact of tumor burden on muscle wasting in metastatic (m) and xenograft (x) models of colorectal cancer (CRC). Male Nod SCID γ and CD2F1 mice were injected subcutaneously or intrasplenically with HCT116 or C26 tumor cells, respectively. CRC tumors resulted in significant muscle wasting regardless of tumor type or model, although muscle loss was exacerbated in mHCT116 hosts. The mHCT116 model decreased ribosomal (r)RNA content and rDNA transcription, whereas the mC26 model showed no loss of rRNA and the upregulation of rDNA transcription. The xHCT116 model reduced mTOR, RPS6, and 4E-BP1 phosphorylation, whereas the mHCT116 model had a similar effect on RPS6 and 4E-BP1 without altering mTOR phosphorylation. The C26 models caused a reduction in 4E-BP1 phosphorylation independent of mTOR. Muscle interleukin (IL)-6 mRNA was elevated in all models except xHCT116, and the NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) mRNA was induced only in the mC26 model. IL-1β mRNA increased in all groups with greater expression in metastatic relative to the xenograft model regardless of tumor types. Our findings indicate that HCT116 tumor burden results in more drastic muscle wasting and anabolic deficits, whereas C26 tumor burden causes similar muscle wasting but exhibits a divergent proinflammatory phenotype. These results highlight potentially important divergence in the pathogenesis of muscle wasting among preclinical models of CRC and demonstrate that tumor burden plays a role in determining anabolic deficits and the expression of proinflammatory effectors of muscle wasting in a tumor-type-dependent manner.

Tumor- and bone-derived soluble factors have been proposed to participate in the alterations of skeletal muscle size and function in cachexia. We previously showed that mice bearing ovarian cancer (OvCa) exhibit cachexia associated with marked bone loss, whereas bone-targeting agents, such as bisphosphonates, are able to preserve muscle mass in animals exposed to anticancer drugs. De-identified CT images and plasma samples from female patients affected with OvCa were used for body composition assessment and quantification of circulating cross-linked C-telopeptide type I (CTX-I) and receptor activator of NF-kB ligand (RANKL), respectively. Female mice bearing ES-2 tumors were used to characterize cancer- and RANKL-associated effects on muscle and bone. Murine C2C12 and human HSMM myotube cultures were used to determine the OvCa- and RANKL-dependent effects on myofiber size. To the extent of isolating new regulators of bone and muscle in cachexia, here we demonstrate that subjects affected with OvCa display evidence of cachexia and increased bone turnover. Similarly, mice carrying OvCa present high RANKL levels. By using in vitro and in vivo experimental models, we found that elevated circulating RANKL is sufficient to cause skeletal muscle atrophy and bone resorption, whereas bone preservation by means of antiresorptive and anti-RANKL treatments concurrently benefit muscle mass and function in cancer cachexia. Altogether, our data contribute to identifying RANKL as a novel therapeutic target for the treatment of musculoskeletal complications associated with RANKL-expressing non-metastatic cancers. © 2021 American Society for Bone and Mineral Research (ASBMR).

Colorectal cancer (CRC) is frequently accompanied by cachexia, an uncured multi-systemic wasting syndrome that affects the majority of patients, especially when the disease recurs with liver metastases (LMs). Muscle and bone loss are amongst the most detrimental symptoms of cachexia and directly cause increased morbidity and mortality. We and others have shown that CRC also promotes metabolic and genomic perturbations of the liver, and further, that formation of LMs exacerbates muscle and bone wasting. These observations, along with evidence that liver-derived factors (i.e., hepatokines) may poorly influence musculoskeletal health, suggest an endocrine role of the liver in mediating cancer-induced cachexia. In the present study, we identified the hepatokine insulin-like growth factor binding protein 1 (IGFBP1) as a novel mediator of musculoskeletal wasting in CRC. Plasma from CRC patients and preclinical mouse models of CRC (C26, MC38, HCT116, ApcMin/+) were assessed for circulating IGFBP1 levels. AML12 hepatocytes were cultured with CRC cells (C26, MC38, HCT116) to assess tumor induced hepatic IGFBP1 production, while C2C12 myotubes and osteoclast precursor cells were cultured to examine the in vitro effects of IGFBP1 on myotube atrophy and osteoclastogenesis. 8-week-old male wild-type (WT) C57BL/6J and IGFBP1-KO mice were intrasplenically injected with MC38 tumor cells (mMC38) to mimic hepatic dissemination of cancer cells, while sham-operated animals received saline. Animals were assessed for muscle force 24-hours prior to euthanasia, and skeletal muscles and bone were collected for molecular and morphological analyses. CRC patients and CRC tumor-bearing mice demonstrated markedly elevated circulating plasma IGFBP1, also supported by increased liver IGFBP1 mRNA expression in C26, MC38 and HCT116 hosts. Follow-up in vitro studies demonstrated that co-culturing CRC cells (lacking IGFBP1 expression) with AML12 hepatocytes promotes IGFBP1 production, thereby suggesting that IGFBP1 is purely host-derived. Further, treatment with recombinant IGFBP1 was sufficient to stimulate osteoclastogenesis, while also promoting atrophy of C2C12 myotubes. Conversely, use of anti-IGFBP1 neutralizing antibodies prevented osteoclastogenesis and preserved C2C12 myotube size when exposed to plasma from mice bearing CRCs. Notably, WT mMC38 bearers displayed reductions in muscle mass and strength, as well as in trabecular bone volume fraction (BV/TV) and trabecular number (Tb.N). Conversely, IGFBP1-KO tumor hosts exhibited preserved skeletal muscle and bone mass. Altogether, our data implicate IGFBP1, an exquisitely liver-derived factor, as a novel mediator of musculoskeletal deficits in CRC cachexia, and supports novel strategies to counteract host-derived factors in the treatment of cancer-associated multi-organ complications. Citation Format: Joshua R. Huot, Fabrizio Pin, Andrea Bonetto. IGFBP1 mediates musculoskeletal defects in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3490.

Background: Colorectal cancer (CRC) is frequently accompanied by cachexia, an uncured multi-organ wasting syndrome, debilitating musculoskeletal health, physical function, and overall survival. Fibroblast growth factor 21 (FGF21) is a modulator of musculoskeletal health and metabolism and has been associated with CRC risk and progression. Here, we investigated the effects of tumor derived FGF21 on musculoskeletal health in CRC. Methods: Plasma from CRC patients and preclinical models of CRC (C26, MC38) were assessed for circulating FGF21 levels, and CRC cell lines (C26, MC38, HCT116) were screened for FGF21 expression. Using CRISPR/Cas9 technology, FGF21 was deleted from MC38 cells (KO-MC38). 8-week-old male C57BL/6J mice were subcutaneously injected (1.0x106) with wild-type (WT-MC38) or FGF21-/- (KO-MC38) MC38 tumor cells, while experimental control animals received saline (n = 10-13/group). Animals underwent electrophysiological testing to obtain motor unit number estimation (MUNE) 48 hours prior to euthanasia and were assessed for maximum plantarflexion torque 24 hours prior to euthanasia. At the time of euthanasia, plasma was collected for assessment of systemic FGF21, while tumors and skeletal muscles (gastrocnemius, quadriceps, and tibialis anterior) were excised and weighed. Femur bones were processed for microcomputed tomography (µCT) imaging and analysis. Results: CRC patients and MC38 tumor hosts demonstrated elevated circulating plasma FGF21 (p Conclusion: Our data suggest that targeting CRC-derived FGF21 halts tumor growth, resulting in preservation of musculoskeletal health and function. Thus, counteracting tumor-derived FGF21 may serve as a therapeutic intervention against the progression of cancer and cancer-associated cachexia. Citation Format: Fabrizio Pin, Anika Shimonty, Joshua Robert Huot. Targeting FGF21 inhibits tumor growth and attenuates cachexia in experimental colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 365.

Previous studies proposed the Triggering Receptor Expressed on Myeloid Cells 2 (TREM2), a receptor expressed in myeloid cells including microglia in brain and osteoclasts in bone, as a link between brain and bone disease. The TREM2 R47H variant is a known risk factor for Alzheimer's disease (AD), the most common form of dementia. To investigate whether altered TREM2 signaling could contribute to bone and skeletal muscle loss, independently of central nervous system defects, we used mice globally hemizygous for the TREM2 R47H variant (TREM2

Background Advanced colorectal cancer (CRC) is often accompanied by the development of liver metastases, as well as cachexia, a multi‐organ co‐morbidity primarily affecting skeletal (SKM) and cardiac muscles. Activin receptor type 2B (ACVR2B) signalling is known to cause SKM wasting, and its inhibition restores SKM mass and prolongs survival in cancer. Using a recently generated mouse model, here we tested whether ACVR2B blockade could preserve multiple organs, including skeletal and cardiac muscle, in the presence of metastatic CRC. Methods NSG male mice (8 weeks old) were injected intrasplenically with HCT116 human CRC cells (mHCT116), while sham‐operated animals received saline (n = 5–10 per group). Sham and tumour‐bearing mice received weekly injections of ACVR2B/Fc, a synthetic peptide inhibitor of ACVR2B. Results mHCT116 hosts displayed losses in fat mass ( − 79%, P

Skeletal muscle repair is accomplished by satellite cells (MuSCs) in cooperation with interstitial stromal cells (ISCs), but the relationship between the function of these cells and the metabolic state of myofibers remains unclear. This study reports an altered proportion of MuSCs and ISCs (including adipogenesis-regulatory cells; Aregs) induced by the transgenic overexpression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) in the myofibers (MCK-PGC-1α mice). Although PGC-1α-driven increase of MuSCs does not accelerate muscle regeneration, myogenic progenitors isolated from MCK-PGC-1α mice and transplanted into intact and regenerating muscles are more prone to fuse with recipient myofibers than those derived from wild-type donors. Moreover, both young and aged MCK-PGC-1α animals exhibit reduced perilipin-positive areas when challenged with an adipogenic stimulus, demonstrating low propensity to accumulate adipocytes within the muscle. Overall, these results unveil that increased PGC-1α expression in the myofibers favors pro-myogenic and anti-adipogenic cell populations in the skeletal muscle. ispartof: ISCIENCE vol:25 issue:11 ispartof: location:United States status: published

Background and Hypothesis: We and others have shown that chemotherapy promotes skeletal muscle wasting and weakness (i.e., cachexia) by disrupting mitochondrial homeostasis and causing oxidative stress. Peroxisome proliferative-activated receptor gamma coactivator 1-alpha (PGC1α) is a pivotal regulator of mitochondrial biogenesis and is involved in reducing oxidative damage in skeletal muscle. Hence, in the present study we investigated whether overexpression of skeletal muscle PGC1α (mPGC1α) was sufficient to preserve skeletal muscle mass and function in young and old mice treated with cisplatin. Experimental Design or Project Methods: Young (2-month; n = 5) and old (18-month; n = 5-8) male wild type (WT) or mPGC1α transgenic mice were treated with cisplatin (2.5mg/kg), while age-matched WT mice received vehicle for 2 weeks. Animals were assessed for muscle force and motor unit number estimation (MUNE). Skeletal muscles were weighed and processed for molecular analyses, including assessment of mitochondrial protein content. Results: Young WT mice exposed to cisplatin showed evidence of cachexia, as indicated by reduced gastrocnemius size (-16%), plantarflexion force (-8%) and MUNE (-56%), whereas mPGC1α mice were only partially protected. Interestingly, despite exacerbated cachexia in aged WT mice treated with chemotherapy, as demonstrated by markedly decreased gastrocnemius size (-22%), plantarflexion force (-18%) and MUNE (-80%) compared to untreated WT, muscle mass, strength and innervation were fully preserved in age-matched mPGC1α mice. Follow-up molecular analyses revealed that WT animals exposed to chemotherapy present loss of muscle mitochondrial proteins PGC1α, OPA1 and CytochromeC, whereas their levels in mPGC1α mice were robustly increased. Conclusion and Potential Impact: Altogether, our data suggest that PGC1α plays a pivotal role in preserving skeletal muscle mass and function, usually impaired by anticancer treatments. These findings enforce developing mitochondria-targeting therapeutics to combat the negative consequences that chemotherapy has on skeletal muscle.

Background: Developing effective treatments for musculoskeletal complications in cancer patients requires understanding metabolic effects of cancer on bone, and particularly osteocytes, the most abundant bone cell and key regulator of bone remodeling. However, little is known regarding how cancer impacts normal osteocyte energy metabolic pathways, such as glycolysis. Given that changes in metabolism are important regulators of cellular function, it is essential to determine how osteocyte metabolism is disrupted by cancer and how this may impact skeletal and whole-body health. Methods: Mice inoculated with saline (N=5) or C26 cells (N=6) were sacrificed after 2 weeks. Bones were harvested for metabolic profiling by GC-MS, gene expression by RT-PCR and bone morphology by µCT. Differentiated IDG-SW3 osteocyte-like cells were cocultured with C26 cells for 12-24hrs and metabolites and gene expression analyzed by GC-MS and RT-PCR. Results: Trabecular bone mass was significantly decreased in the C26 mice. GC-MS analysis revealed decreased glucose in C26 mice tibiae, but no change in lactate. The bone resorption promoting gene Rankl was upregulated, whereas the inhibitor Opg was unchanged. Bone mineralization regulators Mepe and Phex were decreased. In vitro metabolic studies revealed increased glucose and lactate in IDG-SW3 cell lysate; culture media glucose levels were decreased whereas lactate was increased in the co-cultures with C26 cells. RT-PCR demonstrated increases in the glycolysis promoter Hif1α in addition to glycolysis pathway genes including Glut1, Hk2, Slc16a3 and Pdk1. Rankl was also increased in the IDG-SW3 cells co-cultured with the C26 cells whereas Opg, Phex, and Mepe were downregulated. Conclusion: Glycolysis is upregulated in mouse bone and in vitro IDG-SW3 cells exposed to cancer. Our study provides novel understanding for how cancer affects bone metabolism. Integrating these results with whole body metabolism will aid in the development of novel therapeutic strategies to target musculoskeletal and systemic complications of cancer.

PURPOSE OF REVIEW: While the function of osteocytes under physiologic conditions is well defined, their role and involvement in cancer disease remains relatively unexplored, especially in a context of non-bone metastatic cancer. This review will focus on describing the more advanced knowledge regarding the interactions between osteocytes and cancer. RECENT FINDINGS: We will discuss the involvement of osteocytes in the onset and progression of osteosarcoma, with the common bone cancers, as well as the interaction that is established between osteocytes and multiple myeloma. Mechanisms responsible for cancer dissemination to bone, as frequently occurs with advanced breast and prostate cancers will be reviewed. While a role for osteocytes in the stimulation and proliferation of cancer cells has been reported, protective effects of osteocytes against bone colonization have been described as well, thus increasing ambiguity regarding the role of osteocytes in cancer progression and dissemination. Lastly, supporting the idea that skeletal defects can occur also in the absence of direct cancer dissemination or osteolytic lesions directly adjacent to the bone, our recent findings will be presented showing that in the absence of bone metastases, the bone microenvironment and, particularly, osteocytes, can manifest a clear and dramatic response to the distant, non-metastatic tumor. SUMMARY: Our observations support new studies to clarify whether treatments designed to preserve the osteocytes can be combined with traditional anticancer therapies, even when bone is not directly affected by tumor growth.

Cancer cachexia is a frequently neglected debilitating syndrome that, beyond representing a primary cause of death and cancer therapy failure, negatively impacts on patients’ quality of life. Given the complexity of its multisystemic pathogenesis, affecting several organs beyond the skeletal muscle, defining an effective therapeutic approach has failed so far. Revamped attention of the scientific community working on cancer cachexia has focused on mitochondrial alterations occurring in the skeletal muscle as potential triggers of the complex metabolic derangements, eventually leading to hypercatabolism and tissue wasting. Mitochondrial dysfunction may be simplistically viewed as a cause of energy failure, thus inducing protein catabolism as a compensatory mechanism; however, other peculiar cachexia features may depend on mitochondria. On the one side, chemotherapy also impacts on muscle mitochondrial function while, on the other side, muscle-impaired regeneration may result from insufficient energy production from damaged mitochondria. Boosting mitochondrial function could thus improve the energetic status and chemotherapy tolerance, and relieve the myogenic process in cancer cachexia. In the present work, a focused review of the available literature on mitochondrial dysfunction in cancer cachexia is presented along with preliminary data dissecting the potential role of stimulating mitochondrial biogenesis via PGC-1α overexpression in distinct aspects of cancer-induced muscle wasting.

Cachexia is frequently accompanied by severe metabolic derangements, although the mechanisms responsible for this debilitating condition remain unclear. Pyruvate dehydrogenase kinase (PDK)4, a critical regulator of cellular energetic metabolism, was found elevated in experimental models of cancer, starvation, diabetes, and sepsis. Here we aimed to investigate the link between PDK4 and the changes in muscle size in cancer cachexia. High PDK4 and abnormal energetic metabolism were found in the skeletal muscle of colon-26 tumor hosts, as well as in mice fed a diet enriched in Pirinixic acid, previously shown to increase PDK4 levels. Viral-mediated PDK4 overexpression in myotube cultures was sufficient to promote myofiber shrinkage, consistent with enhanced protein catabolism and mitochondrial abnormalities. On the contrary, blockade of PDK4 was sufficient to restore myotube size in C2C12 cultures exposed to tumor media. Our data support, for the first time, a direct role for PDK4 in promoting cancer-associated muscle metabolic alterations and skeletal muscle atrophy.-Pin, F., Novinger, L. J., Huot, J. R., Harris, R. A., Couch, M. E., O'Connell, T. M., Bonetto, A. PDK4 drives metabolic alterations and muscle atrophy in cancer cachexia.

Cancer cachexia is characterized by loss of muscle mass and function. Increased protein catabolism, inflammation, impaired anabolism, and mitochondrial function markedly contribute to the pathogenesis of this syndrome. Physical activity has been suggested as a useful tool to prevent or at least delay the onset and progression of cancer-induced muscle wasting. Two main types of exercise can be adopted, namely, resistance and endurance training. The present study is aimed to investigate the effectiveness of a combined (resistance + endurance) exercise protocol in preventing/reverting cancer-induced muscle wasting.Mice bearing the C26 colon carcinoma have been used as a model of cancer cachexia. They have been exposed to combined exercise training during 6 wk (4 before tumor implantation, 2 during tumor growth). Climbing a 1-m ladder inclined at 85° has been used for resistance training, while aerobic (endurance) exercise has been carried out on the same day using a motorized wheel.In C26-bearing mice, both muscle mass and strength are improved by combined training, while just the latter increased in exercised healthy animals. Such a pattern is associated with modulations of two markers of autophagy, namely, LC3B-I/II ratio, increased in sedentary tumor hosts and reduced in exercised C26-bearing mice, and p62, steadily increased in both sedentary and trained tumor-bearing animals. Finally, combined training is not able to modify PGC-1α protein levels, but it improves succinate dehydrogenase activity, both reduced in the muscle of the C26 hosts.The data reported in the present study show that combined training improves muscle mass and function in the C26 hosts, likely modulating autophagy and improving mitochondrial function; these observations suggest that combined exercise might become part of a multimodal approach to treat cancer cachexia.

Background Cancer cachexia is a metabolic disorder involving perturbed energy balance and altered mitochondrial function. Chemotherapy is a primary treatment option for many types of cancer, but there is substantial evidence that some chemotherapeutic agents can also lead to the development and progression of cachexia. In this study, we apply a comprehensive and systems level metabolomics approach to characterize the metabolic perturbations in murine models of cancer‐induced and chemotherapy‐induced cachexia. Knowledge of the unique pathways through which cancer and chemotherapy drive cachexia is necessary in order to develop effective treatments. Methods The murine Colon26 (C26) adenocarcinoma xenograft model was used to study the metabolic derangements associated with cancer‐induced cachexia. In vivo administration of Folfiri (5‐fluorouracil, irinotecan, and leucovorin) was used to model chemotherapy‐induced cachexia. Comprehensive metabolic profiling was carried out using both nuclear magnetic resonance‐based and mass spectrometry‐based platforms. Analyses included plasma, muscle, and liver tissue to provide a systems level profiling. Results The study involved four groups of CD2F1 male mice (n = 4–5), including vehicle treated (V), C26 tumour hosts (CC), Folfiri treated (F), and C26 tumour hosts treated with Folfiri (CCF). Significant weight loss including skeletal muscle was observed for each of the experimental groups with the tumour hosts showing the most dramatic change (−3.74 g vs. initial body weight in the CC group). Skeletal muscle loss was evident in all experimental groups compared with V, with the CCF combination resulting in the most severe depletion of quadriceps mass (−38% vs. V; P

Cancer cachexia is a multifactorial syndrome characterized by anorexia, body wasting, and muscle and adipose tissue loss, impairing patient's tolerance to anticancer treatments and survival. The aim of the present study was to compare the effects induced in mice by tumor growth alone (C26) or in combination with chemotherapy [C26 oxaliplatin and 5-fluorouracil (oxfu)] and to evaluate the potential of moderate exercise. Oxfu administration to C26 mice exacerbated muscle wasting and triggered autophagy or mitophagy, decreased protein synthesis, and induced mitochondrial alterations. Exercise in C26 oxfu mice counteracted the loss of muscle mass and strength, partially rescuing autophagy and mitochondrial function. Nevertheless, exercise worsened survival in C26 oxfu mice in late stages of cachexia. In summary, chemotherapy further impinges on cancer-induced alterations, worsening muscle wasting. An ideal multifactorial and early intervention to prevent cancer cachexia could take advantage of exercise, improving patient's energy metabolism, mobility, and quality of life.-Ballaro, R., Beltra, M., De Lucia, S., Pin, F., Ranjbar, K., Hulmi, J. J., Costelli, P., Penna, F. Moderate exercise in mice improves cancer plus chemotherapy-induced muscle wasting and mitochondrial alterations.

Colorectal cancer (CRC) is a leading cause of cancer-related death, and the prevalence of CRC in young adults is on the rise, making this a largescale clinical concern. Advanced CRC patients often present with liver metastases (LM) and an increased incidence of cachexia, i.e., musculoskeletal wasting. Despite its high incidence in CRC patients, cachexia remains an unresolved issue, and animal models for the study of CRC cachexia, in particular, metastatic CRC cachexia, remain limited, therefore, we aimed to establish a new model of metastatic CRC cachexia. C57BL/6 male mice (8 weeks old) were subcutaneously (MC38) or intrasplenically injected (mMC38) with MC38 murine CRC cells to disseminate LM, while experimental controls received saline (n = 5–8/group). The growth of subcutaneous MC38 tumors was accompanied by a reduction in skeletal muscle mass (−16%, quadriceps muscle), plantarflexion force (−22%) and extensor digitorum longus (EDL) contractility (−20%) compared to experimental controls. Meanwhile, the formation of MC38 LM (mMC38) led to heighted reductions in skeletal muscle mass (−30%, quadriceps), plantarflexion force (−28%) and EDL contractility (−35%) compared to sham-operated controls, suggesting exacerbated cachexia associated with LM. Moreover, both MC38 and mMC38 tumor hosts demonstrated a marked loss of bone indicated by reductions in trabecular (Tb.BV/TV: −49% in MC38, and −46% in mMC38) and cortical (C.BV/TV: −12% in MC38, and −8% in mMC38) bone. Cell culture experiments revealed that MC38 tumor-derived factors directly promote myotube wasting (−18%) and STAT3 phosphorylation (+5-fold), while the pharmacologic blockade of STAT3 signaling was sufficient to preserve myotube atrophy in the presence of MC38 cells (+21%). Overall, these results reinforce the notion that the formation of LM heightens cachexia in an experimental model of CRC.

Muscle wasting in cancer is associated with deficits in protein synthesis, yet, the mechanisms underlying this anabolic impairment remain poorly understood. The capacity for protein synthesis is mainly determined by the abundance of muscle ribosomes, which is in turn regulated by transcription of the ribosomal (r)RNA genes (rDNA). In this study, we investigated whether muscle loss in a preclinical model of ovarian cancer is associated with a reduction in ribosomal capacity and was a consequence of impaired rDNA transcription. Tumor bearing resulted in a significant loss in gastrocnemius muscle weight and protein synthesis capacity, and was consistent with a significant reduction in rDNA transcription and ribosomal capacity. Despite the induction of the ribophagy receptor NUFIP1 mRNA and the loss of NUFIP1 protein, in vitro studies revealed that while inhibition of autophagy rescued NUFIP1, it did not prevent the loss of rRNA. Electrophoretic analysis of rRNA fragmentation from both in vivo and in vitro models showed no evidence of endonucleolytic cleavage, suggesting that rRNA degradation may not play a major role in modulating muscle ribosome abundance. Our results indicate that in this model of ovarian cancer-induced cachexia, the ability of skeletal muscle to synthesize protein is compromised by a reduction in rDNA transcription and consequently a lower ribosomal capacity. Thus, impaired ribosomal production appears to play a key role in the anabolic deficits associated with muscle wasting in cancer cachexia.

Advanced colorectal cancer (CRC) is often accompanied by development of liver metastases (LMs) and skeletal muscle wasting (i.e., cachexia). Despite plaguing the majority of CRC patients, cachexia remains unresolved. By using mice injected with Colon-26 mouse tumors, either subcutaneously (s.c.; C26) or intrasplenically to mimic hepatic dissemination of cancer cells (mC26), here we aimed to further characterize functional, molecular, and metabolic effects on skeletal muscle and examine whether LMs exacerbate CRC-induced cachexia. C26-derived LMs were associated with progressive loss of body weight, as well as with significant reductions in skeletal muscle size and strength, in line with reduced phosphorylation of markers of protein anabolism and enhanced protein catabolism. mC26 hosts showed prevalence of fibers with glycolytic metabolism and enhanced lipid accumulation, consistent with abnormalities of mitochondrial homeostasis and energy metabolism. In a comparison with mice bearing s.c. C26, cachexia appeared exacerbated in the mC26 hosts, as also supported by differentially expressed pathways within skeletal muscle. Overall, our model recapitulates the cachectic phenotype of metastatic CRC and reveals that formation of LMs resulting from CRC exacerbate cancer-induced skeletal muscle wasting by promoting differential gene expression signatures.

Purpose of review Cancer patients undergoing chemotherapy often experience very debilitating side effects, including unintentional weight loss, nausea, and vomiting. Changes in body composition, specifically lean body mass (LBM), are known to have important implications for anticancer drug toxicity and cancer prognosis. Currently, chemotherapy dosing is based on calculation of body surface area, although this approximation does not take into consideration the variability in lean and adipose tissue mass. Recent findings Patients with depletion of muscle mass present higher chemotherapy-related toxicity, whereas patients with larger amounts of LBM show fewer toxicities and better outcomes. Commonly used chemotherapy regimens promote changes in body composition, primarily by affecting skeletal muscle, as well as fat and bone mass. Experimental evidence has shown that pro-atrophy mechanisms, abnormal mitochondrial metabolism, and reduced protein anabolism are primarily implicated in muscle depletion. Muscle-targeted pro-anabolic strategies have proven successful in preserving lean tissue in the occurrence of cancer or following chemotherapy. Summary Muscle wasting often occurs as a consequence of anticancer treatments and is indicative of worse outcomes and poor quality of life in cancer patients. Accurate assessment of body composition and preservation of muscle mass may reduce chemotherapy toxicity and improve the overall survival.

BACKGROUND Cachexia frequently occurs in women with advanced ovarian cancer (OC), along with enhanced inflammation. Despite being responsible for one third of all cancer deaths, cachexia is generally under-studied in OC due to a limited number of pre-clinical animal models. We aimed to address this gap by characterizing the cachectic phenotype in a mouse model of OC. METHODS Nod SCID gamma mice (n = 6-10) were injected intraperitoneally with 1 × 107 ES-2 human OC cells to mimic disseminated abdominal disease. Muscle size and strength, as well as bone morphometry, were assessed. Tumour-derived effects on muscle fibres were investigated in C2C12 myotube cultures. IL-6 levels were detected in serum and ascites from tumour hosts, as well as in tumour sections. RESULTS In about 2 weeks, ES-2 cells developed abdominal tumours infiltrating omentum, mesentery, and adjacent organs. The ES-2 tumours caused severe cachexia with marked loss of body weight (-12%, P < 0.01) and ascites accumulation in the peritoneal cavity (4.7 ± 1.5 mL). Skeletal muscles appeared markedly smaller in the tumour-bearing mice (approximately -35%, P < 0.001). Muscle loss was accompanied by fibre atrophy, consistent with reduced muscle cross-sectional area (-34%, P < 0.01) and muscle weakness (-50%, P < 0.001). Body composition assessment by dual-energy X-ray absorptiometry revealed decreased bone mineral density (-8%, P < 0.01) and bone mineral content (-19%, P < 0.01), also consistent with reduced trabecular bone in both femurs and vertebrae, as suggested by micro-CT imaging of bone morphometry. In the ES-2 mouse model, cachexia was also associated with high tumour-derived IL-6 levels in plasma and ascites (26.3 and 279.6 pg/mL, respectively) and with elevated phospho-STAT3 (+274%, P < 0.001), reduced phospho-AKT (-44%, P < 0.001) and decreased mitochondrial proteins, as well as with increased protein ubiquitination (+42%, P < 0.001) and expression of ubiquitin ligases in the skeletal muscle of tumour hosts. Similarly, ES-2 conditioned medium directly induced fibre atrophy in C2C12 mouse myotubes (-16%, P < 0.001), consistent with elevated phospho-STAT3 (+1.4-fold, P < 0.001) and altered mitochondrial homoeostasis and metabolism, while inhibition of the IL-6/STAT3 signalling by means of INCB018424 was sufficient to restore the myotubes size. CONCLUSIONS Our results suggest that the development of ES-2 OC promotes muscle atrophy in both in vivo and in vitro conditions, accompanied by loss of bone mass, enhanced muscle protein catabolism, abnormal mitochondrial homoeostasis, and elevated IL-6 levels. Therefore, this represents an appropriate model for the study of OC cachexia. Our model will aid in identifying molecular mediators that could be effectively targeted in order to improve muscle wasting associated with OC.

Background Cancer cachexia is characterized by muscle depletion and exercise intolerance caused by an imbalance between protein synthesis and degradation and by impaired myogenesis. Myofibre metabolic efficiency is crucial so as to assure optimal muscle function. Some drugs are able to reprogram cell metabolism and, in some cases, to enhance metabolic efficiency. Based on these premises, we chose to investigate the ability of the metabolic modulator trimetazidine (TMZ) to counteract skeletal muscle dysfunctions and wasting occurring in cancer cachexia. Methods For this purpose, we used mice bearing the C26 colon carcinoma as a model of cancer cachexia. Mice received 5 mg/kg TMZ (i.p.) once a day for 12 consecutive days. A forelimb grip strength test was performed and tibialis anterior, and gastrocnemius muscles were excised for analysis. Ex vivo measurement of skeletal muscle contractile properties was also performed. Results Our data showed that TMZ induces some effects typically achieved through exercise, among which is grip strength increase, an enhanced fast-to slow myofibre phenotype shift, reduced glycaemia, PGC1α up-regulation, oxidative metabolism, and mitochondrial biogenesis. TMZ also partially restores the myofibre cross-sectional area in C26-bearing mice, while modulation of autophagy and apoptosis were excluded as mediators of TMZ effects. Conclusions In conclusion, our data show that TMZ acts like an ‘exercise mimetic’ and is able to enhance some mechanisms of adaptation to stress in cancer cachexia. This makes the modulation of the metabolism, and in particular TMZ, a suitable candidate for a therapeutic rehabilitative protocol design, particularly considering that TMZ has already been approved for clinical use.

Chemotherapy promotes the development of cachexia, a debilitating condition characterized by muscle and fat loss. ACVR2B/Fc, an inhibitor of the Activin Receptor 2B signaling, has been shown to preserve muscle mass and prolong survival in tumor hosts, and to increase bone mass in models of osteogenesis imperfecta and muscular dystrophy. We compared the effects of ACVR2B/Fc on muscle and bone mass in mice exposed to Folfiri. In addition to impairing muscle mass and function, Folfiri had severe negative effects on bone, as shown by reduced trabecular bone volume fraction (BV/TV), thickness (Tb.Th), number (Tb.N), connectivity density (Conn.Dn), and by increased separation (Tb.Sp) in trabecular bone of the femur and vertebra. ACVR2B/Fc prevented the loss of muscle mass and strength, and the loss of trabecular bone in femurs and vertebrae following Folfiri administration. Neither Folfiri nor ACVR2B/Fc had effects on femoral cortical bone, as shown by unchanged cortical bone volume fraction (Ct.BV/TV), thickness (Ct.Th) and porosity. Our results suggest that Folfiri is responsible for concomitant muscle and bone degeneration, and that ACVR2B/Fc prevents these derangements. Future studies are required to determine if the same protective effects are observed in combination with other anticancer regimens or in the presence of cancer.

// Andrea Camperi 1, 5, * , Fabrizio Pin 1, 2, * , Domiziana Costamagna 1, 2, 8 , Fabio Penna 1, 2 , Maria Lopez Menduina 1, 3 , Zaira Aversa 4 , Teresa Zimmers 5 , Roberto Verzaro 6 , Raffaella Fittipaldi 7 , Giuseppina Caretti 7 , Francesco Maria Baccino 1 , Maurizio Muscaritoli 4 , Paola Costelli 1, 2 1 Department of Clinical and Biological Sciences, University of Turin, Turin, Italy 2 Interuniversity Institute of Myology, Italy 3 Department of Physiology, Complutense University of Madrid, Madrid, Spain 4 Department of Clinical Medicine, Sapienza University of Rome, Rome, Italy 5 Indiana University School of Medicine - IUPUI, Indianapolis, IN, USA 6 Department of Surgery, M.G. Vannini Hospital, Rome, Italy 7 Department of Biosciences, University of Milan, Milan, Italy 8 Current address: Translational Cardiomyology Laboratory, Stem Cell Biology and Embryology, Department of Development and Regeneration, University Hospital Gasthuisberg, Leuven, Belgium * Andrea Camperi and Fabrizio Pin equally contributed to the present study Correspondence to: Paola Costelli, email: paola.costelli@unito.it Keywords: muscle wasting, regeneration, vitamin D receptor, myogenin, circulating vitamin D Received: July 28, 2016 Accepted: January 27, 2017 Published: February 21, 2017 ABSTRACT Low circulating levels of vitamin D were associated with decreased muscle strength and physical performance. Along this line, the present study was aimed to investigate: i) the therapeutic potential of vitamin D in cancer-induced muscle wasting; ii) the mechanisms by which vitamin D affects muscle phenotype in tumor-bearing animals. Rats bearing the AH130 hepatoma showed decreased circulating vitamin D compared to control rats, while muscle vitamin D receptor (VDR) mRNA was up-regulated. Both circulating vitamin D and muscle VDR expression increased after vitamin D administration, without exerting appreciable effects on body weight and muscle mass. The effects of vitamin D on muscle cells were studied in C2C12 myocytes. Vitamin D-treated myoblasts did not differentiate properly, fusing only partially and forming multinucleated structures with aberrant shape and low myosin heavy chain content. Vitamin D treatment resulted in VDR overexpression and myogenin down-regulation. Silencing VDR expression in C2C12 cultures abrogated the inhibition of differentiation exerted by vitamin D treatment. These data suggest that VDR overexpression in tumor-bearing animals contributes to muscle wasting by impairing muscle regenerative program. In this regard, attention should be paid when considering vitamin D supplementation to patients affected by chronic pathologies where muscle regeneration may be involved.

Colorectal cancer (CRC) is often accompanied by formation of liver metastases (LM) and skeletal muscle wasting, i.e. cachexia. Despite affecting the majority of CRC patients, cachexia remains underserved, understudied and uncured. Animal models for the study of CRC-induced cachexia, in particular models containing LM, are sparse; therefore, we aimed to characterize two new models of CRC cachexia. Male NSG mice were injected subcutaneously (HCT116) or intrasplenically (mHCT116) with human HCT116 CRC tumor cells to disseminate LM, whereas experimental controls received saline (n=5-8/group). Tumor growth was accompanied by loss of skeletal muscle mass (HCT116: −20%; mHCT116: −31%; quadriceps muscle) and strength (HCT116: −20%; mHCT116: −27%), with worsened loss of skeletal muscle mass in mHCT116 compared with HCT116 (gastrocnemius: −19%; tibialis anterior: −22%; quadriceps: −21%). Molecular analyses revealed elevated protein ubiquitination in HCT116, whereas mHCT116 also displayed elevated Murf1 and atrogin-1 expression, along with reduced mitochondrial proteins PGC1α, OPA1, mitofusin 2 and cytochrome C. Further, elevated IL6 levels were found in the blood of mHCT116 hosts, which was associated with higher phosphorylation of STAT3 in skeletal muscle. To clarify whether STAT3 was a main player in muscle wasting in this model, HCT116 cells were co-cultured with C2C12 myotubes. Marked myotube atrophy (–53%) was observed, along with elevated phospho-STAT3 levels (+149%). Conversely, inhibition of STAT3 signaling by means of a JAK/STAT3 inhibitor was sufficient to rescue myotube atrophy induced by HCT116 cells (+55%). Overall, our results indicate that the formation of LM exacerbates cachectic phenotype and associated skeletal muscle molecular alterations in HCT116 tumor hosts., Summary: Colorectal cancer-derived liver metastases exacerbate cachexia, in line with activation of STAT3 signaling. STAT3 inhibition may improve cancer-associated muscle wasting.

Some chemotherapeutic agents have been shown to lead to the severe wasting syndrome known as cachexia resulting in dramatic losses of both skeletal muscle and adipose tissue. Previous studies have shown that chemotherapy-induced cachexia is characterized by unique metabolic alterations. Recent results from our laboratory and others have shown that the use of ACVR2B/Fc, a soluble form of the activin receptor 2B (ACVR2B), can mitigate muscle wasting induced by chemotherapy, although the underlying mechanisms responsible for such protective effects are unclear. In order to understand the biochemical mechanisms through which ACVR2B/Fc functions, we employed a comprehensive, multi-platform metabolomics approach. Using both nuclear magnetic resonance (NMR) and mass-spectrometry (MS), we profiled the metabolome of both serum and muscle tissue from four groups of mice including (1) vehicle, (2) the chemotherapeutic agent, Folfiri, (3) ACVR2B/Fc alone, and (4) combined treatment with both Folfiri and ACVR2B/Fc. The metabolic profiles demonstrated large effects with Folfiri treatment and much weaker effects with ACVR2B/Fc treatment. Interestingly, a number of significant effects were observed in the co-treatment group, with the addition of ACVR2B/Fc providing some level of rescue to the perturbations induced by Folfiri alone. The most prominent of these were a normalization of systemic glucose and lipid metabolism. Identification of these pathways provides important insights into the mechanism by which ACVR2B/Fc protects against chemotherapy-induced cachexia.

Background: SerpinB3 (SB3) is a hypoxia and hypoxia-inducible factor (HIF)-2&alpha, dependent cysteine-protease inhibitor up-regulated in hepatocellular carcinoma (HCC), released by cancer cells and able to stimulate proliferation and epithelial-to-mesenchymal-transition. Methods: In the study we employed transgenic and knock out SerpinB3 mice, liver cancer cell line, human HCC specimens, and mice receiving diethyl-nitrosamine (DEN) administration plus choline-deficient L-amino acid refined (CDAA) diet (DEN/CDAA protocol). Results: We provide detailed and mechanistic evidence that SB3 can act as a paracrine mediator able to affect the behavior of surrounding cells by differentially up-regulating, in normoxic conditions, HIF-1&alpha, and HIF-2&alpha, SB3 acts by (i) up-regulating HIF-1&alpha, transcription, facilitating cell survival in a harsh microenvironment and promoting angiogenesis, (ii) increasing HIF-2&alpha, stabilization via direct/selective NEDDylation, promoting proliferation of liver cancer cells, and favoring HCC progression. Moreover (iii) the highest levels of NEDD8-E1 activating enzyme (NAE1) mRNA were detected in a subclass of HCC patients expressing the highest levels of HIF-2&alpha, transcripts, (iv) mice undergoing DEN/CDAA carcinogenic protocol showed a positive correlation between SB3 and HIF-2&alpha, transcripts with the highest levels of NAE1 mRNA detected in nodules expressing the highest levels of HIF-2&alpha, transcripts. Conclusions: These data outline either HIF-2&alpha, and NEDDylation as two novel putative therapeutic targets to interfere with the procarcinogenic role of SerpinB3 in the development of HCC.

Background The loss of muscle mass (sarcopenia) and the associated reduced muscle strength are key limiting factors for elderly people’s quality of life. Improving muscle performance does not necessarily correlate with increasing muscle mass. In fact, particularly in the elderly, the main explanation for muscle weakness is a reduction of muscle quality rather than a loss of muscle mass, and the main goal to be achieved is to increase muscle strength. The effectiveness of Trimetazidine (TMZ) in preventing muscle functional impairment during ageing was assessed in our laboratory. Methods Aged mice received TMZ or vehicle for 12 consecutive days. Muscle function was evaluated at the end of the treatment by a grip test as well as by an inverted screen test at 0, 5, 7 and 12 days of TMZ treatment. After sacrifice, muscles were stored for myofiber cross-sectional area assessment and myosin heavy chain expression evaluation by western blotting. Results Chronic TMZ treatment does not affect the mass of both gastrocnemius and tibialis anterior muscles, while it significantly increases muscle strength. Indeed, both latency to fall and grip force are markedly enhanced in TMZ-treated versus untreated mice. In addition,TMZ administration results in higher expression of slow myosin heavy chain isoform and increased number of small-sized myofibers. Conclusions We report here some data showing that the modulation of skeletal muscle metabolism by TMZ increases muscle strength in aged mice. Reprogramming metabolism might therefore be a strategy worth to be further investigated in view of improving muscle performance in the elderly.

Cancer cachexia is a devastating metabolic syndrome characterized by systemic inflammation and massive muscle and adipose tissue wasting. Although it is responsible for approximately one-third of cancer deaths, no effective therapies are available and the underlying mechanisms have not been fully elucidated. We previously identified the bromodomain and extra-terminal domain (BET) protein BRD4 as an epigenetic regulator of muscle mass. Here we show that the pan-BET inhibitor (+)-JQ1 protects tumor-bearing mice from body weight loss and muscle and adipose tissue wasting. Remarkably, in C26-tumor-bearing mice (+)-JQ1 administration dramatically prolongs survival, without directly affecting tumor growth. By ChIP-seq and ChIP analyses, we unveil that BET proteins directly promote the muscle atrophy program during cachexia. In addition, BET proteins are required to coordinate an IL6-dependent AMPK nuclear signaling pathway converging on FoxO3 transcription factor. Overall, these findings indicate that BET proteins may represent a promising therapeutic target in the management of cancer cachexia., Cachexia is a metabolic syndrome leading to muscle and adipose tissue loss in majority of cancer patients. Here the authors show that, in a mouse model, BET inhibitor JQ1 counteracts muscle and adipose tissue wasting tempering cachexia and prolonging survival through a mechanism unrelated to tumour growth.