Cachectic Muscle Research Articles

2-Deoxy-D-glucose Alleviates Cancer Cachexia-Induced Muscle Wasting by Enhancing Ketone Metabolism and Inhibiting the Cori Cycle.

Cachexia is characterized by progressive weight loss accompanied by the loss of specific skeletal muscle and adipose tissue. Increased lactate production, either due to the Warburg effect from tumors or accelerated glycolysis effects from cachectic muscle, is the most dangerous factor for cancer cachexia. This study aimed to explore the efficiency of 2-deoxy-D-glucose (2-DG) in blocking Cori cycle activity and its therapeutic effect on cachexia-associated muscle wasting. A C26 adenocarcinoma xenograft model was used to study cancer cachectic metabolic derangements. Tumor-free lean mass, hindlimb muscle morphology, and fiber-type composition were measured after in vivo 2-DG administration. Activation of the ubiquitin-dependent proteasome pathway (UPS) and autophagic–lysosomal pathway (ALP) was further assessed. The cachectic skeletal muscles of tumor-bearing mice exhibited altered glucose and lipid metabolism, decreased carbohydrate utilization, and increased lipid β-oxidation. Significantly increased gluconeogenesis and decreased ketogenesis were observed in cachectic mouse livers. 2-DG significantly ameliorated cancer cachexia-associated muscle wasting and decreased cachectic-associated lean mass levels and fiber cross-sectional areas. 2-DG inhibited protein degradation-associated UPS and ALP, increased ketogenesis in the liver, and promoted ketone metabolism in skeletal muscle, thus enhancing mitochondrial bioenergetic capacity. 2-DG effectively prevents muscle wasting by increasing ATP synthesis efficiency via the ketone metabolic pathway and blocking the abnormal Cori cycle.

PGC1α overexpression preserves muscle mass and function in cisplatin-induced cachexia.

BackgroundChemotherapy induces a cachectic‐like phenotype, accompanied by skeletal muscle wasting, weakness and mitochondrial dysfunction. Peroxisome proliferator‐activated receptor‐gamma coactivator‐1 alpha (PGC1α), a regulator of mitochondrial biogenesis, is often reduced in cachectic skeletal muscle. Overexpression of PGC1α has yielded mixed beneficial results in cancer cachexia, yet investigations using such approach in a chemotherapy setting are limited. Utilizing transgenic mice, we assessed whether overexpression of PGC1α could combat the skeletal muscle consequences of cisplatin.MethodsYoung (2 month) and old (18 month) wild‐type (WT) and PGC1α transgenic male and female mice (Tg) were injected with cisplatin (C; 2.5 mg/kg) for 2 weeks, while control animals received saline (n = 5–9/group). Animals were assessed for muscle mass and force, motor unit connectivity, and expression of mitochondrial proteins.ResultsYoung WT + C mice displayed reduced gastrocnemius mass (male: −16%, P < 0.0001; female: −11%, P < 0.001), muscle force (−6%, P < 0.05, both sexes), and motor unit number estimation (MUNE; male: −53%, P < 0.01; female: −51%, P < 0.01). Old WT + C male and female mice exhibited gastrocnemius wasting (male: −22%, P < 0.05; female: −27%, P < 0.05), muscle weakness (male: −20%, P < 0.0001; female: −17%, P < 0.01), and loss of MUNE (male: −82%, P < 0.01; female: −62%, P < 0.05), suggesting exacerbated cachexia compared with younger animals. Overexpression of PGC1α had mild protective effects on muscle mass in young Tg + C male only (gastrocnemius: +10%, P < 0.05); however, force and MUNE were unchanged in both young Tg + C male and female, suggesting preservation of neuromuscular function. In older male, protective effects associated with PGC1α overexpression were heighted with Tg + C demonstrating preserved muscle mass (gastrocnemius: +34%, P < 0.001), muscle force (+13%, P < 0.01), and MUNE (+3‐fold, P < 0.05). Similarly, old female Tg + C did not exhibit muscle wasting or reductions in MUNE, and had preserved muscle force (+11%, P < 0.05) compared with female WT + C. Follow‐up molecular analysis demonstrated that aged WT animals were more susceptible to cisplatin‐induced loss of mitochondrial proteins, including PGC1α, OPA1, cytochrome‐C, and Cox IV.ConclusionsIn our study, the negative effects of cisplatin were heighted in aged animals, whereas overexpression of PGC1α was sufficient to combat the neuromuscular dysfunction caused by cisplatin, especially in older animals. Hence, our observations indicate that aged animals may be more susceptible to develop chemotherapy side toxicities and that mitochondria‐targeted strategies may serve as a tool to prevent chemotherapy‐induced muscle wasting and weakness.

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.

Diet-Induced Gene Expression Changes of Cachectic Muscle, Adipose, and Liver

ObjectivesCancer cachexia is a systemic disease characterized by muscle and adipose loss that cannot be reversed by increasing caloric intake. Our previous research has shown insulin resistance precedes cancer cachexia in the C26 mouse model of cachexia, and a diet high in linoleic acid, the essential omega-6 polyunsaturated fatty acid, attenuates the C26-induced insulin resistance. Therefore, to better understand how dietary linoleic acid is improving insulin sensitivity, we characterized gene expression changes in three major tissues responsible for controlling insulin sensitivity: skeletal muscle, adipose, and liver. MethodsMale CD2F1 (Charles River, MA) were randomized to semi-purified diet (24% fat by weight) containing fat prominently from lard, or containing fat prominently from safflower oil (a linoleic acid-rich oil). One week after diet randomization, mice were inoculated with colon-26 (C26) adenocarcinoma cells (1.0E6 cells). 13 days after inoculation mice were euthanized and gastrocnemius skeletal muscle, epididymal white adipose tissue, and liver tissue were collected for total transcriptome analysis using poly-A enriched next generation RNA-sequencing. Differentially expressed genes were selected based on p-values < 0.05. ResultsThere were no detectable differences in body weight or food intake between the two diets in mice with C26 tumors. Between the two diets 12 genes were differentially expressed in the muscle, while 57 genes were differentially expressed in the liver, and 314 genes were differentially expressed in adipose. ConclusionsA linoleic acid enriched diet had little effect on the skeletal muscle transcriptome but induced larger transcriptome changes in liver and adipose. This could suggest dietary linoleic acid increases insulin sensitivity through affecting metabolism in adipose and liver, rather than skeletal muscle. Determining these diet-induced transcriptome changes allows us to better target tissue-specific molecular mechanisms of linoleic acid in future research. Funding SourcesThis work was supported by the Pelotonia Fellowship Program, the Ohio State University Comprehensive Cancer Center, and the National Institutes of Health.

The Effect of Glucose Administration on Yes‐associated Protein (YAP) Activity in Healthy and Cachectic Mouse Muscles

Yes‐associated protein (YAP) is a downstream effector of Hippo signaling and regulates skeletal muscle size and homeostasis. The change of YAP activity has been reported in aging and dystrophic muscles. Our preliminary data showed that tumor burden alters the YAP activity in skeletal muscle. Recent studies showed that glucose metabolism also regulates YAP activity in cancer cells; however, it is unknown whether glucose administration stimulates YAP activity in skeletal muscle. The purpose of this study was to determine whether the YAP activity would change after acute glucose administration in healthy and cachectic mouse muscles. We hypothesized that glucose could stimulate YAP activity, at least in healthy muscle. Approximately 4‐month‐old wild‐type (WT, n=10) and ApcMin/+ mice (Min, n=10) were used in this study. Each group of mice was assigned to either control (Con, n=5 per group) or glucose administration (Glu n=5 per group) treatment. At first, all the mice fasted for four hours. Glucose dissolved in phosphate‐buffered saline (PBS, 2 mg/kg) was administered intraperitoneally to Glu groups. Con groups received PBS injections. Thirty minutes after glucose administration, the mice were euthanized, and plantaris muscles (PLAN) were removed. The time to sacrifice was based on the peak insulin secretion during a glucose tolerance test. To ensure injection accuracy, the glucose levels were measured using a handheld glucometer before glucose injection and at death. After total protein was extracted, routine Western blotting was performed using 50~100 µg of the total protein. Each phosphorylated protein was normalized to its respective total protein. Unpaired t‐test (WT vs. Min) and a two‐way ANOVA (genotype x glucose treatment) were used for statistical analysis. The levels of significance were set at 0.05. The body weight of Min mice was smaller than that of WT mice (19.8±1.2 mg vs. 17.3±0.5 mg for WT and Min, respectively, p=0.05). Likewise, the PLAN muscle mass of Min mice was smaller than that of WT mice (19.8±1.2 mg vs. 17.3±0.5 mg for WT and Min, respectively, p=0.05). There was no significant difference in fasting glucose levels between WT and Min mice, and glucose administration increased plasma glucose levels regardless of genotype (main effect of glucose administration, p&lt;0.05). The activity levels of glycogen synthase kinase 3 beta (GSK3β) were comparable at the baseline (1.1±0.1 vs. 1.1±0.2 for WT and Min, respectively) and after glucose administration (1.1±0.2 vs. 1.1±0.4 for WT and Min, respectively). Min mice showed a reduction in total ribosomal protein S6 level after fasting (1.0±0.2 vs. 0.4±0.2 for WT and Min, respectively, p&lt;0.05) and in response to glucose administration (2.7±1.5 vs. 0.6±0.4 for WT and Min, respectively, p&lt;0.05). Interestingly, the basal YAP levels were comparable between WT and Min mice (1.0±0.1 vs. 1.1±0.1 for WT and Min, respectively), and the glucose administration did not alter the YAP levels (1.1±0.1 vs. 1.1±0.2 for WT and Min, respectively). These data suggest that YAP activity is independent of acute glucose administration in healthy and cachectic mouse skeletal muscles.

Decoding the role of inflammation-related microRNAs in cancer cachexia: a study using HPV16-transgenic mice and in silico approaches

Cachexia is associated with poor prognosis in cancer patients, and inflammation is one of its main drive factors. MicroRNAs have recently emerged as important players in cancer cachexia and are involved in reciprocal regulation networks with pro-inflammatory signaling pathways. We hypothesize that inflammation-driven cancer cachexia is regulated by specific microRNAs. The aim of this study is to explore the expression and role of inflammation-related microRNAs in muscle wasting. HPV16-transgenic mice develop systemic inflammation and muscle wasting and are a model for cancer cachexia. We employed gastrocnemius muscle samples from these mice to study the expression of microRNAs. Bioinformatic tools were then used to explore their potential role in muscle wasting. Among the microRNAs studied, miR-223-3p (p = 0.004), let-7b-5p (p = 0.034), miR-21a-5p (p = 0.034), miR-150-5p (p = 0.027), and miR-155-5p (p = 0.011) were significantly upregulated in muscles from cachectic mice. In silico analysis showed that these microRNAs participate in several processes related to muscle wasting, including muscle structure development and regulation of the MAPK pathway. When analyzing protein-protein interactions (PPI)-networks, two major clusters and the top 10 hubs were obtained. From the top 10, Kras (p = 0.050) and Ccdn1 (p = 0.009) were downregulated in cachectic muscles, as well as Map2k3 (p = 0.007). These results show that miR-223-3p, let-7b-5p, miR-21a-5p, miR-150-5p, and miR-155-5p, play a role in muscle wasting in HPV16 transgenic mice, possible through regulating the MAPK cascades. Future experimental studies are required to validate our in silico analysis, and to explore the usefulness of these microRNAs and MAPK signaling as new potential biomarkers or therapy targets for cancer cachexia.

Cathepsin K activity controls cachexia-induced muscle atrophy via the modulation of IRS1 ubiquitination.

BackgroundCachexia is a complicated metabolic disorder that is characterize by progressive atrophy of skeletal muscle. Cathepsin K (CTSK) is a widely expressed cysteine protease that has garnered attention because of its enzymatic and non‐enzymatic functions in signalling in various pathological conditions. Here, we examined whether CTSK participates in cancer‐induced skeletal muscle loss and dysfunction, focusing on protein metabolic imbalance.MethodsMale 9‐week‐old wild‐type (CTSK+/+, n = 10) and CTSK‐knockout (CTSK−/−, n = 10) mice were injected subcutaneously with Lewis lung carcinoma cells (LLC; 5 × 105) or saline, respectively. The mice were then subjected to muscle mass and muscle function measurements. HE staining, immunostaining, quantitative polymerase chain reaction, enzyme‐linked immunosorbent assay, and western blotting were used to explore the CTSK expression and IRS1/Akt pathway in the gastrocnemius muscle at various time points. In vitro measurements included CTSK expression, IRS1/Akt pathway‐related target molecule expressions, and the diameter of C2C12 myotubes with or without LLC‐conditioned medium (LCM). An IRS1 ubiquitin assay, and truncation, co‐immunoprecipitation, and co‐localization experiments were also performed.ResultsCTSK+/+ cachectic animals exhibited loss of skeletal muscle mass (muscle weight loss of 15%, n = 10, P < 0.01), muscle dysfunction (grip strength loss > 15%, n = 10, P < 0.01), and fibre area (average area reduction > 30%, n = 5, P < 0.01). Compared with that of non‐cachectic CTSK+/+ mice, the skeletal muscle of cachectic CTSK+/+ mice exhibited greater degradation of insulin receptor substrate 1 (IRS1, P < 0.01). In this setting, cachectic muscles exhibited decreases in the phosphorylation levels of protein kinase B (Akt308, P < 0.01; Akt473, P < 0.05) and anabolic‐related proteins (the mammalian target of rapamycin, P < 0.01) and increased levels of catabolism‐related proteins (muscle RING‐finger protein‐1, P < 0.01; MAFbx1, P < 0.01) in CTSK+/+ mice (n = 3). Although there was no difference in LLC tumour growth (n = 10, P = 0.44), CTSK deletion mitigated the IRS1 degradation, loss of the skeletal muscle mass (n = 10, P < 0.01), and dysfunction (n = 10, P < 0.01). In vitro, CTSK silencing prevented the IRS1 ubiquitination and loss of the myotube myosin heavy chain content (P < 0.01) induced by LCM, and these changes were accelerated by CTSK overexpression even without LCM. Immunoprecipitation showed that CTSK selectively acted on IRS1 in the region of amino acids 268 to 574. The results of co‐transfection of IRS1‐N‐FLAG or IRS1‐C‐FLAG with CTSK suggested that CTSK selectively cleaves IRS1 and causes ubiquitination‐related degradation of IRS1.ConclusionsThese results demonstrate that CTSK plays a novel role in IRS1 ubiquitination in LLC‐induced muscle wasting, and suggest that CTSK could be an effective therapeutic target for cancer‐related cachexia.

Calpain inhibitors inhibit mitochondrial calpain activity to ameliorate apoptosis of cocultured myoblast.

Cancer cachexia is a fatal syndrome associated with muscle regeneration disability. Tumor factors induce the apoptosis of myoblasts to impair the regeneration of skeletal muscle. Cancer cachectic myoblast apoptosis is associated with mitochondria injury. It has been reported that activated mitochondrial calpain caused mitochondria injury in mouse cardiomyocytes and pulmonary smooth muscle. We wondered if mitochondrial calpains exist in skeletal myoblast and their potential role in myoblast apoptosis of cancer cachexia. We used a transwell to build a novel myoblast-carcinoma cell coculture model to simulate the cancer cachexia environment in vitro. Calpain inhibitors, calpastatin (CAST) and calpeptin (CAPT), were used during coculture. We found for the first time that two calpains (calpain-1 and calpain-2) and CAST were present in the mitochondria of myoblast. The activation of mitochondrial calpain decreased mitochondrial complex I activity, promoted mitochondrial permeability transition pore opening, and impaired mitochondrial membrane potential in myoblast during coculture, which induced myoblasts apoptosis. CAST and CAPT protected myoblasts from apoptosis by inhibiting mitochondrial calpain activity, which may attenuate or even reverse cancer cachectic muscle atrophy by improving muscle regeneration ability. Our study provides a new perspective for understanding the mechanism of cancer cachexia, and will further contribute to treat cancer cachexia by focusing on the mitochondrial calpain activity.

Aerobic Exercise Training and In Vivo Akt Activation Counteract Cancer Cachexia by Inducing a Hypertrophic Profile through eIF-2α Modulation.

Simple SummaryChronic disease-related muscle atrophy is a serious public health problem since it reduces mobility and contributes to increases in hospitalization costs. Unfortunately, there is no approved treatment for muscle wasting at present. Thus, an understanding of the mechanisms underlying the control of muscle mass and function under chronic diseases can pave the way for the discovery of innovative therapeutic strategies to counteract muscle wasting. Since numerous types of cancer induce cachexia, which has no cure nor an effective treatment, the main proposal here was to study the effects of AET in cancer cachexia, and to investigate, through in vivo manipulation of the Akt/mTORC1 pathway, whether the cachectic muscle still presents conditions to respond adaptively to hypertrophic stimuli. Our results could provide a basis for innovative research lines to better understand muscle plasticity and to investigate potential therapeutic approaches necessary to prevent muscle loss.Cancer cachexia is a multifactorial and devastating syndrome characterized by severe skeletal muscle mass loss and dysfunction. As cachexia still has neither a cure nor an effective treatment, better understanding of skeletal muscle plasticity in the context of cancer is of great importance. Although aerobic exercise training (AET) has been shown as an important complementary therapy for chronic diseases and associated comorbidities, the impact of AET on skeletal muscle mass maintenance during cancer progression has not been well documented yet. Here, we show that previous AET induced a protective mechanism against tumor-induced muscle wasting by modulating the Akt/mTORC1 signaling and eukaryotic initiation factors, specifically eIF2-α. Thereafter, it was determined whether the in vivo Akt activation would induce a hypertrophic profile in cachectic muscles. As observed for the first time, Akt-induced hypertrophy was able and sufficient to either prevent or revert cancer cachexia by modulating both Akt/mTORC1 pathway and the eIF-2α activation, and induced a better muscle functionality. These findings provide evidence that skeletal muscle tissue still preserves hypertrophic potential to be stimulated by either AET or gene therapy to counteract cancer cachexia.

Decreased miR-497-5p Suppresses IL-6 Induced Atrophy in Muscle Cells.

Interleukin-6 (IL-6) is a pro-inflammatory cytokine associated with skeletal muscle wasting in cancer cachexia. The control of gene expression by microRNAs (miRNAs) in muscle wasting involves the regulation of thousands of target transcripts. However, the miRNA-target networks associated with IL6-induced muscle atrophy remain to be characterized. Here, we show that IL-6 promotes the atrophy of C2C12 myotubes and changes the expression of 20 miRNAs (5 up-regulated and 15 down-regulated). Gene Ontology analysis of predicted miRNAs targets revealed post-transcriptional regulation of genes involved in cell differentiation, apoptosis, migration, and catabolic processes. Next, we performed a meta-analysis of miRNA-published data that identified miR-497-5p, a down-regulated miRNAs induced by IL-6, also down-regulated in other muscle-wasting conditions. We used miR-497-5p mimics and inhibitors to explore the function of miR-497-5p in C2C12 myoblasts and myotubes. We found that miR-497-5p can regulate the expression of the cell cycle genes CcnD2 and CcnE1 without affecting the rate of myoblast cellular proliferation. Notably, miR-497-5p mimics induced myotube atrophy and reduced Insr expression. Treatment with miR-497-5p inhibitors did not change the diameter of the myotubes but increased the expression of its target genes Insr and Igf1r. These genes are known to regulate skeletal muscle regeneration and hypertrophy via insulin-like growth factor pathway and were up-regulated in cachectic muscle samples. Our miRNA-regulated network analysis revealed a potential role for miR-497-5p during IL6-induced muscle cell atrophy and suggests that miR-497-5p is likely involved in a compensatory mechanism of muscle atrophy in response to IL-6.

Cachectic muscle wasting in acute myeloid leukaemia: a sleeping giant with dire clinical consequences.

Acute myeloid leukaemia (AML) is a haematological malignancy with poor survival odds, particularly in the older (>65 years) population, in whom it is most prevalent. Treatment consists of induction and consolidation chemotherapy to remit the cancer followed by potentially curative haematopoietic cell transplantation. These intense treatments are debilitating and increase the risk of mortality. Patient stratification is used to mitigate this risk and considers a variety of factors, including body mass, to determine whether a patient is suitable for any or all treatment options. Skeletal muscle mass, the primary constituent of the body lean mass, may be a better predictor of patient suitability for, and outcomes of, AML treatment. Yet skeletal muscle is compromised by a variety of factors associated with AML and its clinical treatment consistent with cachexia, a life‐threatening body wasting syndrome. Cachectic muscle wasting is associated with both cancer and anticancer chemotherapy. Although not traditionally associated with haematological cancers, cachexia is observed in AML and can have dire consequences. In this review, we discuss the importance of addressing skeletal muscle mass and cachexia within the AML clinical landscape in view of improving survivability of this disease.

Activation of Akt-mTORC1 signalling reverts cancer-dependent muscle wasting.

BackgroundCancer‐related muscle wasting occurs in most cancer patients. An important regulator of adult muscle mass and function is the Akt–mTORC1 pathway. While Akt–mTORC1 signalling is important for adult muscle homeostasis, it is also a major target of numerous cancer treatments. Which role Akt–mTORC1 signalling plays during cancer cachexia in muscle is currently not known. Here, we aimed to determine how activation or inactivation of the pathway affects skeletal muscle during cancer cachexia.MethodsWe used inducible, muscle‐specific Raptor ko (mTORC1) mice to determine the effect of reduced mTOR signalling during cancer cachexia. On the contrary, in order to understand if skeletal muscles maintain their anabolic capacity and if activation of Akt–mTORC1 signalling can reverse cancer cachexia, we generated mice in which we can inducibly activate Akt specifically in skeletal muscles.ResultsWe found that mTORC1 signalling is impaired during cancer cachexia, using the Lewis lung carcinoma and C26 colon cancer model, and is accompanied by a reduction in protein synthesis rates of 57% (P < 0.01). Further reduction of mTOR signalling, as seen in Raptor ko animals, leads to a 1.5‐fold increase in autophagic flux (P > 0.001), but does not further increase muscle wasting. On the other hand, activation of Akt–mTORC1 signalling in already cachectic animals completely reverses the 15–20% loss in muscle mass and force (P < 0.001). Interestingly, Akt activation only in skeletal muscle completely normalizes the transcriptional deregulation observed in cachectic muscle, despite having no effect on tumour size or spleen mass. In addition to stimulating muscle growth, it is also sufficient to prevent the increase in protein degradation normally observed in muscles from tumour‐bearing animals.ConclusionsHere, we show that activation of Akt–mTORC1 signalling is sufficient to completely revert cancer‐dependent muscle wasting. Intriguingly, these results show that skeletal muscle maintains its anabolic capacities also during cancer cachexia, possibly giving a rationale behind some of the beneficial effects observed in exercise in cancer patients.

Inhibition of heat shock protein (HSP) 90 reverses signal transducer and activator of transcription (STAT) 3-mediated muscle wasting in cancer cachexia mice.

Cancer cachexia is a common cause of death among cancer patients with no currently effective treatment available. In animal models, aberrant activation of STAT3 in skeletal muscle contributes to muscle wasting. However, clinically the factors regulating STAT3 activation and the molecular mechanisms involved remain incompletely understood. The expression of HSP90 and the activation of STAT3 were detected in muscle from the patients with cancer cachexia or the tumour-bearing cachectic mice. HSP90 inhibitors, including 17DMAG (alvespimycin) and PU-H71, were administered to cachexic mice and cachexia parameters, weight loss, food intake, survival rate, body composition, serum metabolites, muscle wasting pathology and catabolic activation were analysed. The co-culture of C2C12 myotube cells with C26 conditioned media was performed to investigate the pathological mechanism involved in catabolic muscle wasting. The roles of HSP90, STAT3 and FOXO1 in myotube atrophy were explored via overexpression or knockdown. An enhanced interaction between activated STAT3 and HSP90 in the skeletal muscle of cancer cachexia patients, is a crucial for the development of cachectic muscle wasting. HSP90 inhibitors 17DMAG and PU-H71 alleviated the muscle wasting in C26 and models or the myotube atrophy of C2C12 cells induced by C26 conditional medium. Prolonged STAT3 activation transactivated FOXO1 by binding directly to its promoter and triggered the muscle wasting in a FOXO1-dependent manner in muscle cells. The HSP90/STAT3/FOXO1 axis plays a critical role in cachectic muscle wasting, which might be a potential therapeutic target for the treatment of cancer cachexia.

Drosophila Larval Models of Invasive Tumorigenesis for In Vivo Studies on Tumour/Peripheral Host Tissue Interactions during Cancer Cachexia.

Cancer cachexia is a common deleterious paraneoplastic syndrome that represents an area of unmet clinical need, partly due to its poorly understood aetiology and complex multifactorial nature. We have interrogated multiple genetically defined larval Drosophila models of tumourigenesis against key features of human cancer cachexia. Our results indicate that cachectic tissue wasting is dependent on the genetic characteristics of the tumour and demonstrate that host malnutrition or tumour burden are not sufficient to drive wasting. We show that JAK/STAT and TNF-α/Egr signalling are elevated in cachectic muscle and promote tissue wasting. Furthermore, we introduce a dual driver system that allows independent genetic manipulation of tumour and host skeletal muscle. Overall, we present a novel Drosophila larval paradigm to study tumour/host tissue crosstalk in vivo, which may contribute to future research in cancer cachexia and impact the design of therapeutic approaches for this pathology.

Hippo Signaling Response To Eccentric Muscle Contractions In Cachectic Mouse Muscle

Hippo Signaling Response To Eccentric Muscle Contractions In Cachectic Mouse Muscle

Exercise Protects Against Cancer-Induced Gastrocnemius Metabolic Alterations

Cancer cachexia is a complex metabolic wasting disease. Currently, there are no clear diagnostic criteria, its effects are irreversible, and it cannot be treated. However, exercise has been shown to be beneficial for wasting disorders. PURPOSE: To determine how cancer cachexia alters muscle metabolism and whether aerobic exercise can protect against cancer-cachexia mediated alterations. METHODS: Male ApcMin mice (Apc) and litter-matched non-carrier mice (WT) underwent a 10-week voluntary wheel run (Ex) exercise regimen or remained sedentary (SED). At 15 weeks, gastrocnemius tissues were analyzed for metabolite signatures via non-targeted GC/MS metabolomics. RESULTS: Analysis identified 135 metabolites in the gastrocnemius of cachectic (SED + Apc) mice with 3 metabolites significantly (P < 0.05) increased compared to SED + WT mice: hydroxyproline, ornithine, and lysine. One metabolite was significantly (P < .01) decreased in SED + Apc compared to SED + WT mice: 1-5-1-5-anhydroglucitol. Variable Importance in Projection (VIP) analysis and pathway analysis revealed that the urea cycle, Warburg metabolism, inositol phosphate metabolism, and arginine and proline biosynthesis were affected by cancer cachexia. Following exercise, Ex+Apc gastrocnemius ornithine and hydroxyproline were significantly decreased (P < 0.01) compared to SED + Apc. Conversely, Ex+Apc gastrocnemius 1-5-anhydroglucitol was significantly (P < 0.01) increased compared to SED + Apc. CONCLUSION: These data indicate that cancer cachexia significantly disrupts arginine and proline metabolism, inositol phosphate metabolism, and the urea cycle in cachectic skeletal muscle. Interestingly, aerobic exercise appears to abolish many metabolic perturbations imposed by cancer cachexia. Hydroxyproline (elevated in SED + Apc) has been shown to be increased during times of metabolic stress, however exercise reversed this signal. 1-5-anhydroglucitol (decreased in SED + Apc) is a marker of glycemic control and was restored to basal levels with exercise. Such data may provide a valuable steppingstone in understanding the metabolic consequence of cancer cachexia as well as the identification of metabolic biomarkers. Furthermore, these data shed important light on the mechanisms of the benefits of exercise in cancer survivors.

Amiloride ameliorates muscle wasting in cancer cachexia through inhibiting tumor-derived exosome release

BackgroundCancer cachexia (CAC) reduces patient survival and quality of life. Developments of efficient therapeutic strategies are required for the CAC treatments. This long-term process could be shortened by the drug-repositioning approach which exploits old drugs approved for non-cachexia disease. Amiloride, a diuretic drug, is clinically used for treatments of hypertension and edema due to heart failure. Here, we explored the effects of the amiloride treatment for ameliorating muscle wasting in murine models of cancer cachexia.MethodsThe CT26 and LLC tumor cells were subcutaneously injected into mice to induce colon cancer cachexia and lung cancer cachexia, respectively. Amiloride was intraperitoneally injected daily once tumors were formed. Cachexia features of the CT26 model and the LLC model were separately characterized by phenotypic, histopathologic and biochemical analyses. Plasma exosomes and muscle atrophy-related proteins were quantitatively analyzed. Integrative NMR-based metabolomic and transcriptomic analyses were conducted to identify significantly altered metabolic pathways and distinctly changed metabolism-related biological processes in gastrocnemius.ResultsThe CT26 and LLC cachexia models displayed prominent cachexia features including decreases in body weight, skeletal muscle, adipose tissue, and muscle strength. The amiloride treatment in tumor-bearing mice distinctly alleviated muscle atrophy and relieved cachexia-related features without affecting tumor growth. Both the CT26 and LLC cachexia mice showed increased plasma exosome densities which were largely derived from tumors. Significantly, the amiloride treatment inhibited tumor-derived exosome release, which did not obviously affect exosome secretion from non-neoplastic tissues or induce observable systemic toxicities in normal healthy mice. Integrative-omics revealed significant metabolic impairments in cachectic gastrocnemius, including promoted muscular catabolism, inhibited muscular protein synthesis, blocked glycolysis, and impeded ketone body oxidation. The amiloride treatment evidently improved the metabolic impairments in cachectic gastrocnemius.ConclusionsAmiloride ameliorates cachectic muscle wasting and alleviates cancer cachexia progression through inhibiting tumor-derived exosome release. Our results are beneficial to understanding the underlying molecular mechanisms, shedding light on the potentials of amiloride in cachexia therapy.

Abstract 2656: Muscle wasting in head and neck carcinomas: Investigations on the underlying transcriptional reprogrammation

Abstract Introduction: Cancer cachexia is a multifactorial syndrome associated with a skeletal muscle atrophy that affects at least 55% of patients bearing Head and Neck Squamous Cell Carcinomas (HNSCCs). To our knowledge, molecular mechanisms of muscle alterations have never been investigated on clinical muscle specimens from HNSCC patients. The aim of this study was to perform transcriptome profiling by bulk RNAseq on muscle fragments from HNSCC patients with and without cachexia and from nude mice bearing human HNSCC xenografts. Methods: Our study was made in three parts: 1) In Vitro investigations on human myoblasts co-cultivated with HNSCC cells. 2) In Situ investigations on muscles collected from nude mice xenografted with HNSSCs cells (FADU). 3) Exploration of clinical muscle samples collected from HNSCC patients. RNA sequencing was used to assess gene expression changes and gene function enrichment related to cachexia, in myoblasts co-cultivated with malignant cells as well as skeletal muscle samples collected from mice xenografted with HNSCC cells (FADU) or HNSCC patients. Results: In vitro experimentations showed significant impact of malignant cells on myoblast proliferation and differentiation. In nude mice, FADU xenografts resulted in distant loss of muscle mass at various anatomic locations. RNA sequencing of muscle samples from cachectic patients showed significant differences in gene expression compared with control muscles samples from tumor-free donors. Commonly modified pathways included immune and inflammatory response, mitochondrial metabolism and striated muscle differentiation. The most remarkable finding was the up-regulation of the BIRC3 transcript which encodes the anti-apoptotic protein c-IAP2. This alteration was observed both in human cachectic muscles and murine muscle fragments from tumor-bearing mice. Conclusions: Although the mechanism of BIRC3 up-regulation in cachectic muscle cells is not well understood, this finding is likely to be important for patient management in a context of promising therapeutic results obtained using Smac mimetics. Because these compounds are inhibitors of the c-IAP protein family, it will be important in the future to monitor their positive or negative effects on muscle alterations in cachectic HNSCC patients. Citation Format: Héla Hachicha, Iryna Pirozhkova, Aurore Gelin, François Bidault, Valérie Rouffiac, Thibault Dayris, Thierry Ragot, Ingrid Breuskin, Caroline Even, Philippe Gorphe, Pierre Busson. Muscle wasting in head and neck carcinomas: Investigations on the underlying transcriptional reprogrammation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2656.

An integrative transcriptome study reveals Ddit4/Redd1 as a key regulator of cancer cachexia in rodent models

Cancer cachexia is a multifactorial metabolic syndrome that causes up to 20% of cancer-related deaths. Muscle atrophy, the hallmark of cancer cachexia, strongly impairs the quality of life of cancer patients; however, the underlying pathological process is still poorly understood. Investigation of the disease pathogenesis largely relies on cachectic mouse models. In our study, the transcriptome of the cachectic gastrocnemius muscle in the C26 xenograft model was integrated and compared with that of 5 more different datasets. The bioinformatic analysis revealed pivotal gene ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of the disease, and the key genes were validated. Construction of the protein-protein interaction network and the comparison of pathways enriched in cancer cachexia with 5 other muscle atrophy models revealed Ddit4 (DNA damage-inducible transcript 4), as a key protein in cancer cachexia. The higher expression of Ddit4 in cachectic muscle was further validated in animal models and cachectic cancer patients. Further study revealed that p38 induced the expression of Ddit4, which in turn inhibited the mTOR pathway in atrophic cells.

Preclinical Investigation of Alpinetin in the Treatment of Cancer-Induced Cachexia via Activating PPARγ.

The ongoing loss of skeletal muscle is a central event of cancer cachexia, and its consequences include adverse effects on patient’s quality of life and survival. Alpinetin (Alp), a natural plant-derived flavonoid obtained from Alpinia katsumadai Hayata, has been reported to possess potent anti-inflammatory and antitumor activities. This study aimed to explore the therapeutic effect and underlying mechanism of Alp in the prevention of cancer cachexia. We found that Alp (25–100 μM) dose-dependently attenuated Lewis lung carcinoma–conditioned medium-induced C2C12 myotube atrophy and reduced expression of the E3 ligases Atrogin-1 and MuRF1. Moreover, Alp administration markedly improved vital features of cancer cachexia in vivo with visible reduction of the loss of tumor-free body weight and wasting of multiple tissues, including skeletal muscle, epididymal fat, and decreased expression of Atrogin-1 and MuRF1 in cachectic muscle. Alp suppressed the elevated spleen weight and serum concentrations of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6. Further, Alp treatment remained protective against cancer cachexia in the advanced stage of tumor growth. Molecular docking results suggested that Alp was docked into the active site of PPARγ with the docking score of –7.6 kcal/mol, forming a hydrogen bond interaction with PPARγ protein amino acid residue HIS449 with a bond length of 3.3 Å. Mechanism analysis revealed that Alp activated PPARγ, resulting in the downregulated phosphorylation of NF-κB and STAT3 in vitro and in vivo. PPARγ inhibition induced by GW9662 notably attenuated the improvement of Alp on the above cachexia phenomenon, indicating that PPARγ activation mediated the therapeutic effect of Alp. These findings suggested that Alp might be a potential therapeutic candidate against cancer cachexia.