Cancer-associated Muscle Wasting Research Articles

Tumor-Derived Sarcopenia Factors Are Diverse in Different Tumor Types: A Pan-Cancer Analysis.

Cancer-associated muscle wasting is a widespread syndrome in people with cancer and is characterized by weight loss and muscle atrophy, leading to increased morbidity and mortality. However, the tumor-derived factors that affect the development of muscle wasting and the mechanism by which they act remain unknown. To address this knowledge gap, we aimed to delineate differences in tumor molecular characteristics (especially secretion characteristics) between patients with and without sarcopenia across 10 tumor types from The Cancer Genome Atlas (TCGA). We integrated radiological characteristics from CT scans of TCGA cancer patients, which allowed us to calculate skeletal muscle area (SMA) to confirm sarcopenia. We combined TCGA and GTEx (The Genotype-Tissue Expression) data to analyze upregulated secretory genes in 10 tumor types compared with normal tissues. Upregulated secretory genes in the tumor microenvironment and their relation to SMA were analyzed to identify potential muscle wasting biomarkers (560 samples). Meanwhile, their predictive values for patient survival was validated in 3530 samples in 10 tumor types. A total of 560 participants with transcriptomic data and SMA were included. Among those, 136 participants (24.28%) were defined as having sarcopenia based on SMA. Enrichment analysis for upregulated secretory genes in cancers revealed that pathways associated with muscle wasting were strongly enriched in tumor types with a higher prevalence of sarcopenia. A series of SMA-associated secretory protein-coding genes were identified in cancers, which showed distinct gene expression profiles according to tumor type, and could be used to predict prognosis in cancers (p value ≤ 0.002). Unfortunately, those genes were different and rarely overlapped across tumor types. Tumor secretome characteristics were closely related to sarcopenia. Highly expressed secretory mediators in the tumor microenvironment were associated with SMA and could affect the overall survival of cancer patients, which may provide a valuable starting point for the further understanding of the molecular basis of muscle wasting in cancers. More importantly, tumor-derived pro-sarcopenic factors differ across tumor types and genders, which implies that mechanisms of cancer-associated muscle wasting are complex and diverse across tumors, and may require individualized treatment approaches.

THU515 Vitamin D Binding Protein Induces Skeletal Muscle Atrophy And Contributes To Cancer-associated Muscle Wasting

Abstract Disclosure: T. Raiteri: None. S. Reano: None. A. Scircoli: None. I. Zaggia: None. F. Prodam: None. N. Filigheddu: None. Vitamin D binding protein (VDBP), encoded by the Gc gene, is a multifunctional serum glycoprotein synthesized by hepatocytes, whose primary function is the transport of vitamin D metabolites in the bloodstream. In addition, VDBP enhances the chemotactic activity of neutrophil chemoattractants and takes part in the actin-scavenger system by acting as a monomeric G-actin-binding protein. Several studies have reported a correlation between increased levels of VDBP and various pathologies often associated with muscle wasting, including different types of tumors. Given these findings, we hypothesized that VDBP may play a role in muscle atrophy and cancer-associated muscle wasting and investigated its direct effects on skeletal muscle homeostasis in vitro and in vivo. We showed that, in C2C12 myotubes, VDBP uptake through megalin-mediated endocytosis induced mitochondrial dysfunction and exacerbation of mitophagy that resulted in atrophy. Furthermore, VDBP treatment impaired myoblast differentiation and the formation and stability of acetylcholine receptor (AChR) clusters, an in vitro model of neuromuscular junctions (NMJ). The main molecular mechanism behind the atrophic effect of VDBP is the perturbation of intracellular actin dynamics due to VDBP's ability to bind G-actin. To assess if VDBP has a causative role in muscle homeostasis also in vivo, we experimentally induced the expression of VDBP in Gc knock-out mice (VDBP KO) by adeno-associated virus (AAV)-mediated gene delivery, and we observed that a relatively small amount of circulating VDBP was sufficient to affect muscle performances and reduce muscle mass. Moreover, coherently with the in vitro data, VDBP induced morphological abnormalities in the NMJ. Coherently with the upregulation of VDBP observed in cancer patients, we detected an increase of VDBP in two commonly used models of cancer cachexia, C26 colon carcinoma and Lewis Lung Carcinoma (LLC) tumor-bearing mice. To test the hypothesis that VDBP could play a role in cancer-associated muscle wasting, we induced cancer cachexia in VDBP KO mice by inoculating LLC cells. Tumor-bearing VDBP KO mice preserved their body weight and performances, and the muscle loss and NMJ dismantling were minimal compared to cachectic WT mice. Although the primary function of VDBP is the transport of vitamin D metabolites, we demonstrated that VDBP acts as a hormone per se, having a direct pro-atrophic activity on skeletal muscle. Altogether, our data suggest that VDBP is a new player in the onset of cancer-associated muscle wasting, thus representing a potential therapeutic target to treat cancer cachexia. Presentation: Thursday, June 15, 2023

A TGF-β/KLF10 signaling axis regulates atrophy-associated genes to induce muscle wasting in pancreatic cancer

Cancer cachexia, and its associated complications, represent a large and currently untreatable roadblock to effective cancer management. Many potential therapies have been proposed and tested-including appetite stimulants, targeted cytokine blockers, and nutritional supplementation-yet highly effective therapies are lacking. Innovative approaches to treating cancer cachexia are needed. Members of the Kruppel-like factor (KLF) family play wide-ranging and important roles in the development, maintenance, and metabolism of skeletal muscle. Within the KLF family, we identified KLF10 upregulation in a multitude of wasting contexts-including in pancreatic, lung, and colon cancer mouse models as well as in human patients. We subsequently interrogated loss-of-function of KLF10 as a potential strategy to mitigate cancer associated muscle wasting. In vivo studies leveraging orthotopic implantation of pancreas cancer cells into wild-type and KLF10 KO mice revealed significant preservation of lean mass and robust suppression of pro-atrophy muscle-specific ubiquitin ligases Trim63 and Fbxo32, as well as other factors implicated in atrophy, calcium signaling, and autophagy. Bioinformatics analyses identified Transforming growth factor beta (TGF-β), a known inducer of KLF10 and cachexia promoting factor, as a key upstream regulator of KLF10. We provide direct in vivo evidence that KLF10 KO mice are resistant to the atrophic effects of TGF-β. ChIP-based binding studies demonstrated direct binding to Trim63, a known wasting-associated atrogene. Taken together, we report a critical role for the TGF-β/KLF10 axis in the etiology of pancreatic cancer-associated muscle wasting and highlight the utility of targeting KLF10 as a strategy to prevent muscle wasting and limit cancer-associated cachexia.

Metabolomic Methods to Predict Cancer-Associated Skeletal Muscle Wasting from Profiles of Urinary Metabolites

It is known that cachexia is a cancer-associated muscle wasting and reduction in the function status due to treatment especially chemotherapy and in life expectancy, to assess cachexia muscle loss there are many methods as Computed Tomography (CT) scan which has many disadvantages as high cost, invasive, time consuming and cannot detect early stages of muscle wasting [1]. This novel approach uses single-time point urinary metabolite profiles for determining whether the patient is subjected to muscle wasting by analysis of 93 random urine samples from patients with cancer using 2 successive CT images by assessing of lumbar skeletal muscle area by cubic centimeter to estimate the rate of muscle change per time for every patient. The average muscle change over time was -4.71% in 100 days in the muscle-losing group and 3.91% in 100 days in the control group. Bivariate statistics identified metabolites to muscle wasting including constituents and metabolites of muscle as creatine and creatinine, amino acids as serine, threonine, glutamic acid, isoleucine and valine. These results suggest that urine analysis of a single random urine sample is cheap, fast, and non-invasive tool to screen muscle loss and it is useful to clarify prediction of related metabolic conditions possible with methodology presented. The metabolomics methods used in this article are Univariate analysis T-test, correlation analysis, principal component analysis and heatmap clustering.

Abstract 3985: TGF-B/KLF10 signaling regulates muscle wasting in pancreatic cancer cachexia

Abstract Cancer cachexia, and its associated complications, represent a large and currently untreatable roadblock to better cancer therapy. Many potential therapies have been suggested and tested - including appetite stimulants, targeted cytokine blockers, and nutritional supplementation, but effective therapies have still not found a place in the clinic. Thus, a new approach to treating cancer cachexia is warranted. Members of the Kruppel-like factor family play wide-ranging and important roles in the development, maintenance, and metabolism of muscle. Upon review both our own and multiple publically available datasets, we identified KLF10 upregulation in a multitude of cachectic muscles - including in pancreatic, lung, and colon cancer mouse models and in human cachexia patients. Thus, in this study, we interrogated loss-of-function of KLF10 in the context of pancreatic cancer associated muscle wasting. Ex vivo experiments on satellite cells isolated from KLF10 Knockout (KO) mice showed resistance to the effect of KPC conditioned media on atrophy. In vivo studies using a KLF10 KO mouse model showed that upon orthotopic implantation of T4-KPC cells, the mice demonstrated a significant preservation of lean mass and significant alterations in the expression of muscle-specific ubiquitin ligases Trim63 and Fbxo32, as well as other genes implicated in atrophy, calcium signaling, and autophagy. Pathway analysis of RNA-seq data from these mice identified TGF-B, a known inducer of KLF10, as an important upstream regulator of the response. Further in vivo experimentation showed KLF10 KO mice to be resistant to the atrophic effects of TGF-B injections. In conclusion, we report a novel role of KLF10 in the manifestation of cachexia in pancreatic cancer. Citation Format: Daniel F. Gibbard, Aneesha Dasgupta, Rebecca E. Schmitt, Alexandra M. Ducharme, Paige C. Arneson, Elizabeth S. Bruinsma, John R. Hawse, Jason D. Doles. TGF-B/KLF10 signaling regulates muscle wasting in pancreatic cancer cachexia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3985.

Exosomal microRNAs in cancer-related sarcopenia: Tumor-derived exosomal microRNAs in muscle atrophy.

Cancer-associated sarcopenia is a complex metabolic syndrome marked by muscle mass wasting. Muscle wasting is a serious complication that is a primary contributor to cancer-related mortality. The underlying molecular mechanisms of cancer-associated sarcopenia have not been completely described to date. In general, evidence shows that the main pathophysiological alterations in sarcopenia are associated with the degradation of cellular components, an exceptional inflammatory secretome and mitochondrial dysfunction. Importantly, we highlight the prospect that several miRNAs carried by tumor-derived exosomes that have shown the ability to promote inflammatory secretion, activate catabolism, and even participate in the regulation of cellular degradation pathways can be delivered to and exert effects on muscle cells. In this review, we aim to describe the current knowledge about the functions of exosomal miRNAs in the induction of cancer-associated muscle wasting and propose potential treatment strategies.

Methylarginine metabolites are associated with attenuated muscle protein synthesis in cancer-associated muscle wasting

Cancer cachexia is characterized by reductions in peripheral lean muscle mass. Prior studies have primarily focused on increased protein breakdown as the driver of cancer-associated muscle wasting. Therapeutic interventions targeting catabolic pathways have, however, largely failed to preserve muscle mass in cachexia, suggesting that other mechanisms might be involved. In pursuit of novel pathways, we used untargeted metabolomics to search for metabolite signatures that may be linked with muscle atrophy. We injected 7-week-old C57/BL6 mice with LLC1 tumor cells or vehicle. After 21 days, tumor-bearing mice exhibited reduced body and muscle mass and impaired grip strength compared with controls, which was accompanied by lower synthesis rates of mixed muscle protein and the myofibrillar and sarcoplasmic muscle fractions. Reductions in protein synthesis were accompanied by mitochondrial enlargement and reduced coupling efficiency in tumor-bearing mice. To generate mechanistic insights into impaired protein synthesis, we performed untargeted metabolomic analyses of plasma and muscle and found increased concentrations of two methylarginines, asymmetric dimethylarginine (ADMA) and NG-monomethyl-l-arginine, in tumor-bearing mice compared with control mice. Compared with healthy controls, human cancer patients were also found to have higher levels of ADMA in the skeletal muscle. Treatment of C2C12 myotubes with ADMA impaired protein synthesis and reduced mitochondrial protein quality. These results suggest that increased levels of ADMA and mitochondrial changes may contribute to impaired muscle protein synthesis in cancer cachexia and could point to novel therapeutic targets by which to mitigate cancer cachexia.

The wasting-associated metabolite succinate disrupts myogenesis and impairs skeletal muscle regeneration.

Muscle wasting is a debilitating co-morbidity affecting most advanced cancer patients. Alongside enhanced muscle catabolism, defects in muscle repair/regeneration contribute to cancer-associated wasting. Among the factors implicated in suppression of muscle regeneration are cytokines that interfere with myogenic signal transduction pathways. Less understood is how other cancer/wasting-associated cues, such as metabolites, contribute to muscle dysfunction. This study investigates how the metabolite succinate affects myogenesis and muscle regeneration. We leveraged an established ectopic metabolite treatment (cell permeable dimethyl-succinate) strategy to evaluate the ability of intracellular succinate elevation to 1) affect myoblast homeostasis (proliferation, apoptosis), 2) disrupt protein dynamics and induce wasting-associated atrophy, and 3) modulate in vitro myogenesis. In vivo succinate supplementation experiments (2% succinate, 1% sucrose vehicle) were used to corroborate and extend in vitro observations. Metabolic profiling and functional metabolic studies were then performed to investigate the impact of succinate elevation on mitochondria function. We found that in vitro succinate supplementation elevated intracellular succinate about 2-fold, and did not have an impact on proliferation or apoptosis of C2C12 myoblasts. Elevated succinate had minor effects on protein homeostasis (~25% decrease in protein synthesis assessed by OPP staining), and no significant effect on myotube atrophy. Succinate elevation interfered with in vitro myoblast differentiation, characterized by significant decreases in late markers of myogenesis and fewer nuclei per myosin heavy chain positive structure (assessed by immunofluorescence staining). While mice orally administered succinate did not exhibit changes in overall body composition or whole muscle weights, these mice displayed smaller muscle myofiber diameters (~6% decrease in the mean of non-linear regression curves fit to the histograms of minimum feret diameter distribution), which was exacerbated when muscle regeneration was induced with barium chloride injury. Significant decreases in the mean of non-linear regression curves fit to the histograms of minimum feret diameter distributions were observed 7 days and 28 days post injury. Elevated numbers of myogenin positive cells (3-fold increase) supportive of the differentiation defects observed in vitro were observed 28 days post injury. Metabolic profiling and functional metabolic assessment of myoblasts revealed that succinate elevation caused both widespread metabolic changes and significantly lowered maximal cellular respiration (~35% decrease). This study broadens the repertoire of wasting-associated factors that can directly modulate muscle progenitor cell function and strengthens the hypothesis that metabolic derangements are significant contributors to impaired muscle regeneration, an important aspect of cancer-associated muscle wasting.

Abstract A106: Key differences in muscular index and tumor burden reveal unique biology in ethnically diverse patients with pancreatic cancer

Abstract Introduction: The racial disparities in pancreatic cancer (PC), while not fully appreciated, are recognized amongst PC experts. African Americans are diagnosed more frequently, present with more advanced disease, and suffer from higher mortality rates than White patients. Overall, cancer cachexia, or cancer-associated muscle wasting, greatly contributes to both morbidity and mortality. While cachexia is experienced by more than 80% of patients with PC, PC-induced cachexia and how it contributes to a disparity amongst African Americans is under investigation. African Americans with PC present with increased muscle wasting (-29%) than their White counterparts with PC (-14%). Therefore, we hypothesize that an established radiographically obtained muscular index (a psoas index) uniquely corresponds to increased disease burden in African Americans with early-stage disease. Methods: African American and White patients with pancreatic ductal adenocarcinoma who underwent surgery at our institution between 2010 and 2017 were included in this retrospective study. A muscular index for each patient was measured from preoperative CT scans by dividing the average area of the psoas muscles at L3 by the L3 vertebral body area to normalize the measurement for patient size. Supplementary relevant clinical, pathologic, and laboratory values were used for comparison. Results: 15 African American and 15 White surgical patients with PC were matched by gender and history of neoadjuvant therapy; age was similar in both groups (65.7 vs. 64.3, p=0.6566). Gross tumor size was similar in African Americans and Whites (2.553 vs. 3.093, p=0.3097). Tumor size, however, inversely correlated to psoas index in African Americans (r=0.4330, p=0.0077), but not in Whites (r=0.002525, p=0.8588). African Americans with lower psoas indices generally had larger tumors. Similar to tumor size, the positive lymph node ratio (LNR) inversely correlated to psoas index in African Americans (r=0.3930, p=0.0124), but not in Whites (r=0.08673, p=0.2867). LNR was significantly greater in Whites than African Americans (0.0627 vs. 0.2253, p=0.0020). Overall survival was, however, similar in both groups (15.8 vs. 14.3, p=0.7117). Conclusion: A decreased psoas index in African American patients is associated with greater tumor size and an increased positive LNR. Notably, the most powerful outcome variable (LNR) did not correlate with psoas index in White patients even in the presence of increased nodal metastases. Most surprisingly, overall survival was similar in both patient groups. This suggests that a limited tumor burden does not provide a survival benefit for African Americans. We conclude that recognizing biologic variance among patients with ethnic diversity will allow better strategies to characterize metabolism, tumor microenvironment, and muscle architecture in PC. Together this will lead to improved overall survival. Citation Format: Miles E. Cameron, Patrick W. Underwood, Michael H. Gerber, Steven J. Hughes, Andrew R. Judge, Jennifer B. Permuth, Jose G. Trevino. Key differences in muscular index and tumor burden reveal unique biology in ethnically diverse patients with pancreatic cancer [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr A106.

A 96-well culture platform enables longitudinal analyses of engineered human skeletal muscle microtissue strength

Three-dimensional (3D) in vitro models of human skeletal muscle mimic aspects of native tissue structure and function, thereby providing a promising system for disease modeling, drug discovery or pre-clinical validation, and toxicity testing. Widespread adoption of this research approach is hindered by the lack of easy-to-use platforms that are simple to fabricate and that yield arrays of human skeletal muscle micro-tissues (hMMTs) in culture with reproducible physiological responses that can be assayed non-invasively. Here, we describe a design and methods to generate a reusable mold to fabricate a 96-well platform, referred to as MyoTACTIC, that enables bulk production of 3D hMMTs. All 96-wells and all well features are cast in a single step from the reusable mold. Non-invasive calcium transient and contractile force measurements are performed on hMMTs directly in MyoTACTIC, and unbiased force analysis occurs by a custom automated algorithm, allowing for longitudinal studies of function. Characterizations of MyoTACTIC and resulting hMMTs confirms the capability of the device to support formation of hMMTs that recapitulate biological responses. We show that hMMT contractile force mirrors expected responses to compounds shown by others to decrease (dexamethasone, cerivastatin) or increase (IGF-1) skeletal muscle strength. Since MyoTACTIC supports hMMT long-term culture, we evaluated direct influences of pancreatic cancer chemotherapeutics agents on contraction competent human skeletal muscle myotubes. A single application of a clinically relevant dose of Irinotecan decreased hMMT contractile force generation, while clear effects on myotube atrophy were observed histologically only at a higher dose. This suggests an off-target effect that may contribute to cancer associated muscle wasting, and highlights the value of the MyoTACTIC platform to non-invasively predict modulators of human skeletal muscle function.

Abstract 4004: Unique patient signatures underlie health disparities in ethnically diverse patients with pancreatic cancer

Abstract Introduction: Health disparities in pancreatic cancer (PC) exist but how the underlying biology contributes to this is poorly described. Black Americans have a higher prevalence, present with more advanced disease, and suffer from higher mortality rates than other ethnic groups. Cancer cachexia, or cancer-associated muscle wasting, affects more than 80% of patients with PC. Unique muscular characteristics exist in different ethnic groups which might be contributing to pancreatic cancer health disparities. We hypothesize that Black patients with PC have a distinct muscular profile and are more affected by cancer cachexia than Whites and that unique genomic variations might contribute to this phenomenon in PC tumors between Black and White patients. Methods: Black (including African Americans and Afro-Caribbeans) and non-Hispanic White surgically resected patients with PC between 2010 and 2017 were case matched by age, gender, and tumor grade. Muscular indices were measured from CT scans by dividing the area of the psoas muscles by the L3 vertebral body area to normalize for patient size. Whole-exome sequencing was performed on DNA from tumor and adjacent benign parenchyma. Results: Healthy Blacks have a greater psoas index than Whites (0.8995 vs. 0.6988, p=0.004). When comparing patients with PC, Blacks and Whites were found to have similar psoas indices. Black patients had a greater percent decrease in psoas index than White patients (-25.29% vs. -12.37%, p=0.04). Tumor size inversely correlated to psoas index in Blacks (r=0.4330, p=0.007), but not in Whites (r=0.0025, p=0.9). Similar to tumor size, the positive lymph node ratio (LNR) inversely correlated to psoas index in Black patients (r=0.3930, p=0.01), but not in White patients (r=0.0867, p=0.2). LNR was significantly greater in Whites than Blacks (0.0627 vs. 0.2253, p=0.002), but overall survival was similar in both groups (15.8 vs. 14.3, p=0.7). In whole-exome sequencing, there were 22 mutations, of which several harbored implications in chemoresistance (ABCF1, ANAPC1), metastasis (CYP2A7, CYP21A2), and pathways of tumorigenesis (PHACTR4, PPP1R12B), and had 100% penetrance in Blacks but not in Whites. Seven mutations were present only in tumors of Whites but not Blacks. Conclusion: Black Americans with PC suffer more muscle loss in the early stages of disease than Whites. A decreased psoas index in Black patients is associated with worse prognostic factors. In our genetic analysis, we identified 27 novel mutations not before reported in the literature. We conclude that recognizing biological variance in patients may underlie health disparities and allow investigations toward defining specific targets in a diverse group of patients with PC. Citation Format: Miles E. Cameron, Patrick W. Underwood, Jinping Lai, Jennifer B. Permuth, Andrew R. Judge, Jose G. Trevino. Unique patient signatures underlie health disparities in ethnically diverse patients with pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4004.

Novel molecular targets of muscle wasting in cancer patients.

Cancer-associated muscle wasting affects many patients and leads to reduced patient function, decreased quality of life and poor responses to surgical and oncological treatments. Despite advancements in the understanding of its pathophysiology, no current treatment or accepted strategy for successful management exists. In this review, we provide an update on potential novel therapeutic targets in cancer cachexia. Recent research has focused on molecular mechanisms underlying cancer-associated muscle wasting, allowing identification of potential therapeutic targets and the development of several promising drugs. However, due to the multifactorial and patient-specific pathogenesis of cachexia, the demonstration of a measurable and meaningful clinical effect in randomized controlled trials has proven difficult. Potential novel targets such as circulating macrophage inhibitory cytokine 1/growth differentiation factor 15 and ZRT/IRT-like protein 14 have shown relevance in animal models, but their therapeutic manipulation has yet to be translated to patients. Increasing evidence has suggested that a single therapy may not be successful and a targeted, multimodal approach is required. The management of cancer-associated muscle wasting is complex. Future clinical trials should focus on early multimodal therapeutic interventions involving targeted therapies, with careful deliberation of chosen nutritional and functional outcomes.

Opposing effects of 25-hydroxy- and 1α,25-dihydroxy-vitamin D3 on pro-cachectic cytokine-and cancer conditioned medium-induced atrophy in C2C12 myotubes.

Loss of skeletal muscle is one of the main features of cancer cachexia. Vitamin D (VD) deficiency is associated with impairment of muscle mass and performance and is highly prevalent in cachectic patients; therefore, VD supplementation has been proposed to counteract cancer cachexia-associated muscle loss. However, in both cachectic cancer patients and tumour-bearing animals, VD supplementation led to disappointing results, urging the need for a better understanding of VD activity on skeletal muscle. Cancer-associated muscle wasting was reproduced in vitro by treating C2C12 myotubes with cancer cell conditioned medium, a combination of TNF-α and IFNγ or IL-6 pro-cachectic cytokines. The biological effects and mechanisms of action of 1,25-dihydroxy VD (1,25 VD) and its precursor 25-hydroxy VD (25 VD) on myotubes were explored. We demonstrated that only 25 VD was able to protect from atrophy by activating Akt signalling, inducing protein synthesis, and stimulating the autophagic flux, while 1,25 VD had an atrophic activity per se, increasing FoxO3 levels, inducing the expression of atrogenes, and blocking the autophagic flux. Furthermore, we showed that the contrasting activities of these VD metabolites on C2C12 myotubes depend on a differential induction of VD-24-hydroxylase and transformation of VD metabolites in pro-atrophic 24-hydroxylated products, as silencing of VD-24-hydroxylase reduced the atrophic activity of 1,25 VD. Altogether these data might explain the lack of efficacy of VD treatment in vivo for the protection of muscle mass in cancer.

Cancer cachexia is defined by an ongoing loss of skeletal muscle mass.

Since 2007, a quantitative, specific and precise approach to the detection of muscle loss has become accessible with the advent of image-based assessments. Computed tomography images acquired as part of standard cancer care are the serendipitous substrate for these analyses. Three radiologically-determined abnormalities, sarcopenia (severe muscle depletion), catabolic loss of muscle over time, and reduced muscle radiation attenuation associate with progressive functional impairment, treatment-related complications, reduced quality of life, and mortality. Fundamental understanding of muscle wasting in cancer cachexia has been developed on a base of clinical and experimental studies, which have identified alterations in muscle protein synthesis, autophagy and ubiquitin-mediated proteolysis as key contributors to muscle loss. The etiology of cancer-associated muscle wasting is multifactorial. Tumor metabolism captures energy fuels and amino acids, and a suite of tumor-derived molecules elicits catabolic pathways at the tissue level in muscle. Endocrine, neural and inflammatory derangements add further catabolic drive. Antineoplastic agents make a substantial contribution to muscle wasting by directly action on muscle cells, as well as secondarily via their systemic side effects. Encouraging data is emerging as to the potential reversibility of muscle loss and/or reduced muscle radiation attenuation through modulation of specific mechanisms. In the first line, pain and symptom management is a key element of the prevention of catabolic loss of muscle. Intake of intake of high-quality proteins and ω-3 polyunsaturated fatty acids support retention or gain of muscle mass. While there is no approved drug therapy for the indication of cancer-associated muscle wasting, there is preliminary evidence for robust gain of skeletal muscle mass in research studies of new therapeutics including inhibitors of mitogen-activated protein kinase kinases and ghrelin receptor agonists.

Cancer Cachexia

One negative side effect of cancer that dramatically affects cancer prognosis is the development of cancer cachexia. Cancer cachexia is defined as cancer-associated muscle wasting. This is thought to be at least partially mediated by increased energy expenditure and is responsible for the death of 20-40% of all cancer patients. Although the liver is known to be the predominant regulator of whole body metabolism, there is little known about its relationship to the development of cancer cachexia. PURPOSE: The purpose of this exploratory study was to investigate alterations in liver metabolism by examining measures of glycogen storage throughout the progression of Lewis Lung Carcinoma (LLC) induced cancer cachexia. METHODS: C57BL/6J mice were injected with 1X106 LLC Cells in the left posterior leg, and the control group with phosphate buffered saline (PBS). The experimental groups included PBS, 1wk, 2wk, 3wk, and 4wk of cancer progression with 10-16 in each group. Sections of liver (n=~8/group) were cut and periodic acid-Schiff (PAS) stain for glycogen was completed. Images were analyzed for total area of stain as well as intensity of stain using NIS-Elements imaging software. A Welch’s one-way analysis of variance was used to determine differences between groups, a Tukey post hoc was used to determine differences between means. Significance was denoted at p<0.05. RESULTS: 4wk animals had ~30% larger livers compared to all other groups with no other differences detected (~1000mg compared to ~1300mg, p<0.05). However, there were no statistical differences detected between groups on either PAS area stained (~50% across groups, p>0.05) or intensity of stain (Arbitrary Intensity Unit ~30 across groups, p>0.05). Furthermore, neither glycogen area nor intensity correlated with liver size (r = 0.12 and r = 0.16 respectively). CONCLUSION: Differences in liver sizes are not attributable to glycogen storage. Though there were no differences in glycogen content, the increase in liver size suggests disruption of other processes in the liver. For future projects, we will further investigate mechanisms for liver hypertrophy in order to determine the relationship between the liver and cancer cachexia progression. This study was supported by The Arkansas Bioscience Institute and National Institutes of Health R15AR069913.

Understanding the Role of Exercise in Cancer Cachexia Therapy.

Cachexia, the unintentional loss of body weight, is prevalent in many cancer types, and the associated skeletal muscle mass depletion increases patient morbidity and mortality. While anorexia can be present, cachexia is not reversible with nutritional therapies alone. Pharmacological agents have been proposed to treat this condition, but there are currently no approved treatments. Nonetheless, the hallmark characteristics associated with cancer cachexia remain viable foundations for future therapies. Regular physical activity holds a promising future as a nonpharmacological alternative to improve patient survival through cachexia prevention. Evidence suggests exercise training is beneficial during cancer treatment and survival. However, the mechanistic examination of cachectic skeletal muscle's response to exercise is both needed and justified. The primary objective of this review is to discuss the role of exercise for the prevention and treatment of cancer-associated muscle wasting. Initially, we provide an overview of systemic alterations induced by cancer and their role in the regulation of wasting processes during cachexia progression. We then discuss how exercise could alter disrupted regulatory pathways related to growth and metabolism during cancer-induced muscle atrophy. Last, we outline current exercise prescription guidelines and how exercise could be a potential behavioral therapy to curtail cachexia development in cancer patients.

Cancer cachexia-when proteasomal inhibition is not enough.

Cachexia is a life threatening syndrome associated with several diseases, such as end‐stage heart failure, end‐stage renal disease, chronic obstructive pulmonary disease, chronic inflammation (i.e. rheumatoid arthritis), acquired immune deficiency syndrome, and cancer.1, 2 Cachexia is found in 31–87% of cancer patients especially in advanced disease stages.3 It is characterized by progressive weight loss, metabolic alterations, fatigue, and persistent reduction of body cell mass in response to a malignant tumour.2, 4, 5, 6 The incidence of cachexia in cancer patients is dependent on the type and site of the tumour. While patients with non‐Hodgkin's lymphoma, breast cancer, and sarcomas show low incidences, rates up to 83% in pancreatic cancer patients, and over 85% in patients with gastric cancer have been found. Additionally, around 60% of small‐cell and non‐small‐cell lung cancer patients develop cachexia.7, 8, 9 Cancer cachexia affects the function of several organs such as muscle, adipose tissue, liver, brain, immune system, and heart, collectively decreasing patients' quality of life and worsening their prognosis. Therefore, cachexia must be considered as a true multi‐organ syndrome.10 Because cancer cachexia leads to a decrease in physical performance and quality of life,11 and is associated with poor survival (accounting for more than 20% of cancer deaths,7, 12, 13, 14, 15) it is of major clinical relevance. Even more so since cachectic patients show lower response rates to chemotherapy7 and a reduced tolerance to anticancer treatment.16 Despite its importance, weight loss in cancer patients is rarely recognized, assessed,17 or treated actively.18, 19 Thus, cancer cachexia represents an important underappreciated clinical syndrome.

Study Design and Rationale for the Phase 3 Clinical Development Program of Enobosarm, a Selective Androgen Receptor Modulator, for the Prevention and Treatment of Muscle Wasting in Cancer Patients (POWER Trials).

Muscle wasting in cancer is a common and often occult condition that can occur prior to overt signs of weight loss and before a clinical diagnosis of cachexia can be made. Muscle wasting in cancer is an important and independent predictor of progressive functional impairment, decreased quality of life, and increased mortality. Although several therapeutic agents are currently in development for the treatment of muscle wasting or cachexia in cancer, the majority of these agents do not directly inhibit muscle loss. Selective androgen receptor modulators (SARMs) have the potential to increase lean body mass (LBM) and hence muscle mass, without the untoward side effects seen with traditional anabolic agents. Enobosarm, a nonsteroidal SARM, is an agent in clinical development for prevention and treatment of muscle wasting in patients with cancer (POWER 1 and 2 trials). The POWER trials are two identically designed randomized, double-blind, placebo-controlled, multicenter, and multinational phase 3 trials to assess the efficacy of enobosarm for the prevention and treatment of muscle wasting in subjects initiating first-line chemotherapy for non-small-cell lung cancer (NSCLC). To assess enobosarm’s effect on both prevention and treatment of muscle wasting, no minimum weight loss is required. These pivotal trials have pioneered the methodological and regulatory fields exploring a therapeutic agent for cancer-associated muscle wasting, a process hereby described. In each POWER trial, subjects will receive placebo (n = 150) or enobosarm 3 mg (n = 150) orally once daily for 147 days. Physical function, assessed as stair climb power (SCP), and LBM, assessed by dual-energy X-ray absorptiometry (DXA), are the co-primary efficacy endpoints in both trials assessed at day 84. Based on extensive feedback from the US Food and Drug Administration (FDA), the co-primary endpoints will be analyzed as a responder analysis. To be considered a physical function responder, a subject must have ≥10 % improvement in physical function compared to baseline. To meet the definition of response on LBM, a subject must have demonstrated no loss of LBM compared with baseline. Secondary endpoints include durability of response assessed at day 147 in those responding at day 84. A combined overall survival analysis for both studies is considered a key secondary safety endpoint. The POWER trials design was established with extensive clinical input and collaboration with regulatory agencies. The efficacy endpoints are a result of this feedback and discussion of the threshold for clinical benefit in patients at risk for muscle wasting. Full results from these studies will soon be published and will further guide the development of future anabolic trials. Clinical Trial ID: NCT01355484. https://clinicaltrials.gov/ct2/show/NCT01355484, NCT01355497. https://clinicaltrials.gov/ct2/show/NCT01355497?term=g300505&rank=1.

The relationship between tumour necrosis, circulating IL-6 concentrations, and inflammatory responses in patients undergoing curative resection for colorectal cancer.

404 Background: Cancer-associated inflammation, in the form of systemic and local inflammation, and tumour necrosis are known to have prognostic value in colorectal cancer (CRC). In addition, recent work has reported a direct relationship between the systemic inflammatory response and loss of skeletal muscle in patients with CRC. However, the inter-relationships between these inflammatory responses, tumour necrosis, metabolic upset and circulating biochemical mediators are unclear in CRC. Interleukin-6 and its downstream signalling cascades have been implicated in both cancer-associated inflammation and cancer-associated muscle wasting. The aim of the present study was to examine whether circulating IL-6 concentrations may link tumour necrosis, local and systemic inflammatory responses, and metabolic upset in patients undergoing curative resection for colorectal cancer. Methods: The study included 118 patients undergoing surgery for CRC between 2004 and 2009. Data were collected from pre-operative blood tests. Routine pathology specimens were scored for Klintrup criteria and tumour necrosis. Results: Tumour necrosis was associated with increased T-stage (p&lt;0.01), reduced inflammatory cell infiltrate (p&lt;0.05), increased IL-6 (p&lt;0.001), IL-10 (p&lt;0.01), and VEGF (p&lt;0.001) and with markers of the systemic inflammatory response: mGPS (p&lt;0.001), anaemia (p&lt;0.05); increased white cell (p&lt;0.001), neutrophil (p&lt;0.05) and platelet (p&lt;0.001) counts. Circulating IL-6 was associated with increased IL-10 (p&lt;0.01), VEGF (p&lt;0.001), increased mGPS (p&lt;0.001), increased white cell (p&lt;0.01) and platelet (p&lt;0.01) counts and low skeletal muscle index (p&lt;0.01). On Spearman rank correlation there were significant associations between circulating concentrations of IL-6 and IL-10 (rs= 0.39, p&lt;0.001) and CRP (r= 0.42, p&lt;0.001). Conclusions: Interleukin-6 appears to be associated with systemic inflammation, tumour necrosis, and sarcopenia in colorectal cancer. However, the lack of an association between IL-6 and the local inflammatory response suggests a more complex relationship with the tumour inflammatory cell infiltrate.

Learning to predict cancer-associated skeletal muscle wasting from 1H-NMR profiles of urinary metabolites

Cancer-associated muscle wasting is associated with reduction in functional status, in response to treatment and in life expectancy. Methods currently used to assess muscle loss involve diagnostic imaging techniques such as computed tomography (CT), which are costly, inconvenient, invasive, time consuming and have limited ability to detect early or slowly evolving wasting. We present a novel approach using single time-point urinary metabolite profiles to determine whether a patient is experiencing muscle wasting. We analyzed 93 random urine samples from patients with cancer using 1H-NMR. Using two successive CT images we assessed their lumbar skeletal muscle area (cm2) to estimate the rate of muscle change (% loss or gain over time) for each patient. The average muscle change over time was −4.71%/100 days in the muscle-losing group and +3.91%/100 days in the comparator group. Bivariate statistics identified metabolites related with muscle loss, including constituents and metabolites of muscle (creatine, creatinine, 3-OH-isovalerate), amino acids (Leu, Ile, Val, Ala, Thr, Tyr, Gln, Ser) and intermediary metabolites. We also applied machine-learning techniques to identify patterns of urinary metabolites that identify which patients are likely to lose muscle mass. We evaluated the predictive performance of 8 machine-learning approaches using fivefold cross validation and permutation testing, and found that SVM provided the best generalization accuracy (82.2%). These results suggest that 1H-NMR analysis of a single random urine sample may be a fast, cheap, safe and inexpensive tool to screen and monitor muscle loss, and that useful classifiers for predicting related metabolic conditions are possible with the methodology presented.