Cachectic Muscle Research Articles

Integrative study of skeletal muscle mitochondrial dysfunction in a murine pancreatic cancer-induced cachexia model

Pancreatic ductal adenocarcinoma (PDAC), the most common pancreatic cancer, is a deadly cancer, often diagnosed late and resistant to current therapies. PDAC patients are frequently affected by cachexia characterized by muscle mass and strength loss (sarcopenia) contributing to patient frailty and poor therapeutic response. This study assesses the mechanisms underlying mitochondrial remodeling in the cachectic skeletal muscle, through an integrative exploration combining functional, morphological, and omics-based evaluation of gastrocnemius muscle from KIC genetically engineered mice developing autochthonous pancreatic tumor and cachexia. Cachectic PDAC KIC mice exhibit severe sarcopenia with loss of muscle mass and strength associated with reduced muscle fiber’s size and induction of protein degradation processes. Mitochondria in PDAC atrophied muscles show reduced respiratory capacities and structural alterations, associated with deregulation of oxidative phosphorylation and mitochondrial dynamics pathways. Beyond the metabolic pathways known to be altered in sarcopenic muscle (carbohydrates, proteins, and redox), lipid and nucleic acid metabolisms are also affected. Although the number of mitochondria per cell is not altered, mitochondrial mass shows a twofold decrease and the mitochondrial DNA threefold, suggesting a defect in mitochondrial genome homeostasis. In conclusion, this work provides a framework to guide toward the most relevant targets in the clinic to limit PDAC-induced cachexia.

Integrative study of skeletal muscle mitochondrial dysfunction in a murine pancreatic cancer-induced cachexia model.

Pancreatic ductal adenocarcinoma (PDAC), the most common pancreatic cancer, is a deadly cancer, often diagnosed late and resistant to current therapies. PDAC patients are frequently affected by cachexia characterized by muscle mass and strength loss (sarcopenia) contributing to patient frailty and poor therapeutic response. This study assesses the mechanisms underlying mitochondrial remodeling in the cachectic skeletal muscle, through an integrative exploration combining functional, morphological, and omics-based evaluation of gastrocnemius muscle from KIC genetically engineered mice developing autochthonous pancreatic tumor and cachexia. Cachectic PDAC KIC mice exhibit severe sarcopenia with loss of muscle mass and strength associated with reduced muscle fiber's size and induction of protein degradation processes. Mitochondria in PDAC atrophied muscles show reduced respiratory capacities and structural alterations, associated with deregulation of oxidative phosphorylation and mitochondrial dynamics pathways. Beyond the metabolic pathways known to be altered in sarcopenic muscle (carbohydrates, proteins, and redox), lipid and nucleic acid metabolisms are also affected. Although the number of mitochondria per cell is not altered, mitochondrial mass shows a twofold decrease and the mitochondrial DNA threefold, suggesting a defect in mitochondrial genome homeostasis. In conclusion, this work provides a framework to guide toward the most relevant targets in the clinic to limit PDAC-induced cachexia.

Abstract B014: NF-kB Regulates Innate Immunity in the Muscle Microenvironment to Control Distinct States of Wasting in Pancreatic Cancer-Induced Cachexia

Abstract Cancer cachexia is a debilitating syndrome characterized by unintentional weight loss largely due to the depletion of adipose and skeletal muscle mass. Cachexia occurs in at least half of all patients with cancer, with increasing incidences in more advanced cases, and is estimated to be responsible for greater than 20% of all cancer related deaths. In pancreatic ductal adenocarcinoma (PDAC), which has a 5-year survival rate of 13%, the incidence of cachexia can be as high as 80% and is associated with poor disease outcomes. Mitigating the effects of cachexia has the potential to increase both quality of life and survival for patients with PDAC. However, there are currently no effective means of treating cachexia, thereby underscoring the need to further understand the pathology of this disease. Although cancer cachexia is classically characterized as a systemic inflammatory disorder, emerging evidence indicates that weight loss also associates with local tissue inflammation. We queried the regulation of this inflammation and its causality to cachexia by exploring skeletal muscle, whose atrophy strongly associates with poor outcomes. Using both a genetically engineered mouse model of PDAC- induced cachexia named KPP and patient samples, we show that cachectic muscle is marked by enhanced innate immunity. Accumulation of macrophages is regulated by an NF-kB activity that localizes to multiple cells within the muscle microenvironment. In addition, this accumulation of macrophages derives from an equal contribution of infiltrating monocytes and resident cells. Moreover, NF-kB activated cells and macrophages undergo a crosstalk; whereas NF-kB+ cells signal and recruit macrophages to inhibit muscle regeneration and promote atrophy, but interestingly at the same time can also protect myofibers, macrophages signal back to NF-kB+ cells to sustain an innate immune response in a feed-forward loop. Together, we propose that NF-kB functions in multiple cells in the muscle microenvironment to regulate innate immunity that both promotes and protects against muscle wasting in cancer. Citation Format: Denis C Guttridge, Benjamin R Pryce, Alexander Oles, Erin E Talbert, Katherine A Morgan, David A Mahvi, Michael C Ostrowski, Teresa A Zimmers, James G Tidball, David J Wang. NF-kB Regulates Innate Immunity in the Muscle Microenvironment to Control Distinct States of Wasting in Pancreatic Cancer-Induced Cachexia [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B014.

Abstract B007: Optimization of an Orthotopic Mouse Model of Pancreatic Cancer to Simulate Liver Metastasis and Cancer Cachexia

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with a poor prognosis due to two key features. The first is metastasis at presentation, occurring in approximately 80% of PDAC patients, with 90% of these metastases occurring in the liver. The second is cachexia, which compromises treatment tolerance and reduces the quality of life for patients. Although various mouse models of PDAC exist, recapitulating both metastatic and cachectic features have been challenging. In this study, we optimized an orthotopic mouse model of PDAC by altering implantation sites, subcloning parental murine PDAC cells, adjusting the number of transplanted cells, and varying the age of recipient mice to study liver metastasis and cancer cachexia. These modifications significantly increased the latency of tumor development and the time course of the disease, concurrently increasing the rate of liver metastasis to approximately 70%. Furthermore, reliable cachexia endpoints were also achieved in both PDAC mice with and without metastases, mirroring the condition in patients. We also found that cachectic muscles from PDAC mice with metastasis exhibit a similar transcriptional profile to muscles derived from mice and patients without metastasis. Together, this model is likely to be advantageous in both advancing our understanding of the mechanisms of PDAC cachexia and in evaluating novel therapeutics. Citation Format: David J Wang, Victoria Spadafora, Benjamin R Pryce, Alexander Oles, Erin E Talbert, Micheal C Ostrowski, Denis C Guttridge. Optimization of an Orthotopic Mouse Model of Pancreatic Cancer to Simulate Liver Metastasis and Cancer Cachexia [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B007.

Decreased skeletal muscle intramyocellular lipid droplet-mitochondrial contact contributes to myosteatosis in cancer cachexia.

Cancer cachexia, the unintentional loss of lean mass, contributes to functional dependency, poor treatment outcomes, and decreased survival. Although its pathogenicity is multifactorial, metabolic dysfunction remains a hallmark of cachexia. However, significant knowledge gaps exist in understanding the role of skeletal muscle lipid metabolism and dynamics in this condition. We examined skeletal muscle metabolic dysfunction, intramyocellular lipid droplet (LD) content, LD morphology and subcellular distribution, and LD-mitochondrial interactions using the Lewis lung carcinoma (LLC) murine model of cachexia. C57/BL6 male mice (n = 20) were implanted with LLC cells (106) in the right flank or underwent PBS sham injections. Skeletal muscle was excised for transmission electron microscopy (TEM; soleus), oil red O/lipid staining [tibialis anterior (TA)], and protein (gastrocnemius). LLC mice had a greater number (232%; P = 0.006) and size (130%; P = 0.023) of intramyocellular LDs further supported by increased oil-red O positive (87%; P = 0.0109) and "very high" oil-red O positive (178%; P = 0.0002) fibers compared with controls and this was inversely correlated with fiber size (R2 = 0.5294; P < 0.0001). Morphological analyses of LDs show increased elongation and complexity [aspect ratio: intermyofibrillar (IMF) = 9%, P = 0.046) with decreases in circularity [circularity: subsarcolemmal (SS) = 6%, P = 0.042] or roundness (roundness: whole = 10%, P = 0.033; IMF = 8%, P = 0.038) as well as decreased LD-mitochondria touch (-15%; P = 0.006), contact length (-38%; P = 0.036), and relative contact (86%; P = 0.004). Furthermore, dysregulation in lipid metabolism (adiponectin, CPT1b) and LD-associated proteins, perilipin-2 and perilipin-5, in cachectic muscle (P < 0.05) were observed. Collectively, we provide evidence that skeletal muscle myosteatosis, altered LD morphology, and decreased LD-mitochondrial interactions occur in a preclinical model of cancer cachexia.NEW & NOTEWORTHY We sought to advance our understanding of skeletal muscle lipid metabolism and dynamics in cancer cachexia. Cachexia increased the number and size of intramyocellular lipid droplets (LDs). Furthermore, decreases in LD-mitochondrial touch, contact length, and relative contact along with increased LD shape complexity with decreases in circularity and roundness. Dysregulation in lipid metabolism and LD-associated proteins was also documented. Collectively, we show that myosteatosis, altered LD morphology, and decreased LD-mitochondrial interactions occur in cancer cachexia.

Preventing loss of sirt1 lowers mitochondrial oxidative stress and preserves C2C12 myotube diameter in an invitro model of cancer cachexia.

Cancer cachexia is a multifactorial syndrome associated with advanced cancer that contributes to mortality. Cachexia is characterized by loss of body weight and muscle atrophy. Increased skeletal muscle mitochondrial reactive oxygen species (ROS) is a contributing factor to loss of muscle mass in cachectic patients. Mice inoculated with Lewis lung carcinoma (LLC) cells lose weight, muscle mass, and have lower muscle sirtuin-1 (sirt1) expression. Nicotinic acid (NA) is a precursor to nicotinamide dinucleotide (NAD+) which is exhausted in cachectic muscle and is a direct activator of sirt1. Mice lost body and muscle weight and exhibited reduced skeletal muscle sirt1 expression after inoculation with LLC cells. C2C12 myotubes treated with LLC-conditioned media (LCM) had lower myotube diameter. We treated C2C12 myotubes with LCM for 24 h with or without NA for 24 h. C2C12 myotubes treated with NA maintained myotube diameter, sirt1 expression, and had lower mitochondrial superoxide. We then used a sirt1-specific small molecule activator SRT1720 to increase sirt1 activity. C2C12 myotubes treated with SRT1720 maintained myotube diameter, prevented loss of sirt1 expression, and attenuated mitochondrial superoxide production. Our data provides evidence that NA may be beneficial in combating cancer cachexia by maintaining sirt1 expression and decreasing mitochondrial superoxide production.

WITHDRAWN: P4HB Inhibits Cachectic Muscle Regeneration by Restricting Myoblast Migration

Cachexia is a prevalent syndrome in advanced cancer characterized by skeletal muscle atrophy. Impaired muscle regeneration exacerbates the loss of muscle mass. However, the mechanism underlying cachectic impairments in muscle regeneration remains unclear. Previous research revealed that P4HB accumulates in cachexia muscle. In this study, we explored the role of P4HB in muscle regeneration. We induced muscle injury in cachectic mice to demonstrate the impairment of muscle regeneration. Subsequently, we used inducible overexpression cell lines and muscle-specific knockout mice to elucidate the inhibitory role of P4HB in muscle regeneration. Using mass spectrometry, we identified MYH9 as a downstream molecule of P4HB, demonstrating that P4HB affects myoblast migration and thus impacts muscle regeneration. Our results suggest that the accumulation of P4HB in cachectic muscle contributes to regeneration impairment, indicating that P4HB is a potential therapeutic target for cachexia treatment.

Optimization of a mouse model of pancreatic cancer to simulate the human phenotypes of metastasis and cachexia

BackgroundPancreatic ductal adenocarcinoma (PDAC) presents with a high mortality rate. Two important features of PDAC contribute to this poor outcome. The first is metastasis which occurs in ~ 80% of PDAC patients. The second is cachexia, which compromises treatment tolerance for patients and reduces their quality of life. Although various mouse models of PDAC exist, recapitulating both metastatic and cachectic features have been challenging.MethodsHere, we optimize an orthotopic mouse model of PDAC by altering several conditions, including the subcloning of parental murine PDAC cells, implantation site, number of transplanted cells, and age of recipient mice. We perform spatial profiling to compare primary and metastatic immune microenvironments and RNA sequencing to gain insight into the mechanisms of muscle wasting in PDAC-induced cachexia, comparing non-metastatic to metastatic conditions.ResultsThese modifications extend the time course of the disease and concurrently increase the rate of metastasis to approximately 70%. Furthermore, reliable cachexia endpoints are achieved in both PDAC mice with and without metastases, which is reminiscent of patients. We also find that cachectic muscles from PDAC mice with metastasis exhibit a similar transcriptional profile to muscles derived from mice and patients without metastasis.ConclusionTogether, this model is likely to be advantageous in both advancing our understanding of the mechanism of PDAC cachexia, as well as in the evaluation of novel therapeutics.

Compound Z526 alleviates chemotherapy-induced cachectic muscle loss by ameliorating oxidative stress-driven protein metabolic imbalance and apoptosis

Chemotherapy is one of the primary and indispensable intervention against cancers though it is always accompanied by severe side effects especially cachexia. Cachexia is a fatal metabolic disorder syndrome, mainly characterized by muscle loss. Oxidative stress is the key factor that trigger cachectic muscle loss by inducing imbalance in protein metabolism and apoptosis. Here, we showed an oral compound (Z526) exhibited potent alleviating effects on C2C12 myotube atrophy induced by various chemotherapeutic agents in vitro as well as mice muscle loss and impaired grip force induced by oxaliplatin in vivo. Furthermore, Z526 also could ameliorate C2C12 myotube atrophy induced by the combination of chemotherapeutic agents with conditioned medium of various tumor cells in vitro as well as mice muscle atrophy of C26 tumor-bearing mice treated with oxaliplatin. The pharmacological effects of Z526 were based on its potency in reducing oxidative stress in cachectic myocytes and muscle tissues, which inhibited the activation of NF-κB and STAT3 to decrease Atrogin-1-mediated protein degradation, activated the AKT/mTOR signaling pathway to promote protein synthesis, regulated Bcl-2/BAX ratio to reduce Caspase-3-triggered apoptosis. Our work suggested Z526 to be an optional strategy for ameliorating cachexia muscle atrophy in the multimodality treatment of cancers.

Obesity worsens mitochondrial quality control and does not protect against skeletal muscle wasting in murine cancer cachexia.

More than 650 million people are obese (BMI>30) worldwide, which increases their risk for several metabolic diseases and cancer. While cachexia and obesity are at opposite ends of the weight spectrum, leading many to suggest a protective effect of obesity against cachexia, mechanistic support for obesity's benefit is lacking. Given that obesity and cachexia are both accompanied by metabolic dysregulation, we sought to investigate the impact of obesity on skeletal muscle mass loss and mitochondrial dysfunction in murine cancer cachexia. Male C57BL/6 mice were given a purified high fat or standard diet for 16weeks before being implanted with 106 Lewis lung carcinoma (LLC) cells. Mice were monitored for 25days, and hindlimb muscles were collected for cachexia indices and mitochondrial assessment via western blotting, high-resolution respirometry and transmission electron microscopy (TEM). Obese LLC mice experienced significant tumour-free body weight loss similar to lean (-12.8% vs. -11.8%, P=0.0001) but had reduced survival (33.3% vs. 6.67%, χ2 =10.04, P=0.0182). Obese LLC mice had reduced muscle weights (-24%, P<0.0354) and mCSA (-16%, P=0.0004) with similar activation of muscle p65 (P=0.0337), and p38 (P=0.0008). ADP-dependent coupled respiration was reduced in both Obese and Obese LLC muscle (-30%, P=0.0072) consistent with reductions in volitional cage activity (-39%, P<0.0001) and grip strength (-41%, P<0.0001). TEM revealed stepwise reductions in intermyofibrillar and subsarcolemmal mitochondrial size with Obese (IMF: -37%, P=0.0009; SS: -21%, P=0.0101) and LLC (IMF: -40%, P=0.0019; SS: -27%, P=0.0383) mice. Obese LLC mice had increased pAMPK (T172; P=0.0103) and reduced FIS1 (P=0.0029) and DRP1 (P<0.0001) mitochondrial fission proteins, which were each unchanged in Lean LLC. Further, mitochondrial TEM analysis revealed that Obese LLC mice had an accumulation of damaged and dysfunctional mitochondria (IMF: 357%, P=0.0395; SS: 138%, P=0.0174) in concert with an accumulation of p62 (P=0.0328) suggesting impaired autophagy and clearance of damaged mitochondria. Moreover, we observed increases in electron lucent vacuoles only in Obese LLC muscle (IMF: 421%, P=0.0260; SS: 392%, P=0.0192), further supporting an accumulation of damaged materials that cannot be properly cleared in the obese cachectic muscle. Taken together, these results demonstrate that obesity is not protective against cachexia and suggest exacerbated impairments to mitochondrial function and quality control with a particular disruption in the removal of damaged mitochondria. Our findings highlight the need for consideration of the severity of obesity and pre-existing metabolic conditions when determining the impact of weight status on cancer-induced cachexia and functional mitochondrial deficits.

FK506 bypasses the effect of erythroferrone in cancer cachexia skeletal muscle atrophy

Skeletal muscle atrophy is a hallmark of cachexia, a wasting condition typical of chronic pathologies, that still represents an unmet medical need. Bone morphogenetic protein (BMP)-Smad1/5/8 signaling alterations are emerging drivers of muscle catabolism, hence, characterizing these perturbations is pivotal to develop therapeutic approaches. We identified two promoters of "BMP resistance" in cancer cachexia, specifically the BMP scavenger erythroferrone (ERFE) and the intracellular inhibitor FKBP12. ERFE is upregulated in cachectic cancer patients' muscle biopsies and in murine cachexia models, where its expression is driven by STAT3. Moreover, the knock down of Erfe or Fkbp12 reduces muscle wasting in cachectic mice. To bypass the BMP resistance mediated by ERFE and release the brake on the signaling, we targeted FKBP12 with low-dose FK506. FK506 restores BMP-Smad1/5/8 signaling, rescuing myotube atrophy by inducing protein synthesis. In cachectic tumor-bearing mice, FK506 prevents muscle and body weight loss and protects from neuromuscular junction alteration, suggesting therapeutic potential for targeting the ERFE-FKBP12 axis.

Protective effects of hachimijiogan (HJG), a Japanese Kampo medicine, on cancer cachectic muscle wasting in mice.

Myogenesis is required to generate skeletal muscle tissue and to maintain skeletal muscle mass. Decreased myogenesis under various pathogenic conditions results in muscular atrophy. Through a small screening of Japanese traditional (Kampo) medicines, hachimijiogan (HJG) was shown to promote the myogenic differentiation of C2C12 myoblasts through the upregulation of myogenin. In tumor-bearing cancer-cachectic mice, HJG was also found to have a protective effect against cancer-cachectic muscle wasting. This effect was significant when HJG was administered in combination with aerobic exercise by treadmill running. Moreover, HJG ameliorated the cellular atrophy of C2C12 myotubes induced by treatment with conditioned medium derived from a colon-26 cancer cell culture. In addition, HJG suppressed H2O2-dependent myotube atrophy, suggesting that HJG could reverse the atrophic phenotypes by eliminating reactive oxygen species.

Activin A-Expressing Polymorphonuclear Myeloid-Derived Suppressor Cells Infiltrate Skeletal and Cardiac Muscle and Promote Cancer Cachexia.

Cachexia is a major cause of death in cancer and leads to wasting of cardiac and skeletal muscle, as well as adipose tissue. Various cellular and soluble mediators have been postulated in driving cachexia; however, the specific mechanisms behind this muscle wasting remain poorly understood. In this study, we found polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) to be critical for the development of cancer-associated cachexia. Significant expansion of PMN-MDSCs was observed in the cardiac and skeletal muscles of cachectic murine models. Importantly, the depletion of this cell subset, using depleting anti-Ly6G Abs, attenuated this cachectic phenotype. To elucidate the mechanistic involvement of PMN-MDSCs in cachexia, we examined major mediators, that is, IL-6, TNF-α, and arginase 1. By employing a PMN-MDSC-specific Cre-recombinase mouse model, we showed that PMN-MDSCs were not maintained by IL-6 signaling. In addition, PMN-MDSC-mediated cardiac and skeletal muscle loss was not abrogated by deficiency in TNF-α or arginase 1. Alternatively, we found PMN-MDSCs to be critical producers of activin A in cachexia, which was noticeably elevated in cachectic murine serum. Moreover, inhibition of the activin A signaling pathway completely protected against cardiac and skeletal muscle loss. Collectively, we demonstrate that PMN-MDSCs are active producers of activin A, which in turn induces cachectic muscle loss. Targeting this immune/hormonal axis will allow the development of novel therapeutic interventions for patients afflicted with this debilitating syndrome.

Adenosine monophosphate-activated protein kinase is elevated in human cachectic muscle and prevents cancer-induced metabolic dysfunction in mice.

Metabolic dysfunction and cachexia are associated with poor cancer prognosis. With no pharmacological treatments, it is crucial to define the molecular mechanisms causing cancer-induced metabolic dysfunction and cachexia. Adenosine monophosphate-activated protein kinase (AMPK) connects metabolic and muscle mass regulation. As AMPK could be a potential treatment target, it is important to determine the function for AMPK in cancer-associated metabolic dysfunction and cachexia. We therefore established AMPK's roles in cancer-associated metabolic dysfunction, insulin resistance and cachexia. In vastus lateralis muscle biopsies from n=26 patients with non-small cell lung cancer (NSCLC), AMPK signalling and protein content were examined by immunoblotting. To determine the role of muscle AMPK, male mice overexpressing a dominant-negative AMPKα2 (kinase-dead [KiDe]) specifically in striated muscle were inoculated with Lewis lung carcinoma (LLC) cells (wild type [WT]: n=27, WT+LLC: n=34, mAMPK-KiDe: n=23, mAMPK-KiDe+LLC: n=38). Moreover, male LLC-tumour-bearing mice were treated with (n=10)/without (n=9) 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) to activate AMPK for 13days. Littermate mice were used as controls. Metabolic phenotyping of mice was performed via indirect calorimetry, body composition analyses, glucose and insulin tolerance tests, tissue-specific 2-[3H]deoxy-d-glucose (2-DG) uptake and immunoblotting. Patients with NSCLC presented increased muscle protein content of AMPK subunits α1, α2, β2, γ1 and γ3 ranging from +27% to +79% compared with control subjects. In patients with NSCLC, AMPK subunit protein content correlated with weight loss (α1, α2, β2 and γ1), fat-free mass (α1, β2 and γ1) and fat mass (α1 and γ1). Tumour-bearing mAMPK-KiDe mice presented increased fat loss and glucose and insulin intolerance. LLC in mAMPK-KiDe mice displayed lower insulin-stimulated 2-DG uptake in skeletal muscle (quadriceps: -35%, soleus: -49%, extensor digitorum longus: -48%) and the heart (-29%) than that in non-tumour-bearing mice. In skeletal muscle, mAMPK-KiDe abrogated the tumour-induced increase in insulin-stimulated TBC1D4thr642 phosphorylation. The protein content of TBC1D4 (+26%), pyruvate dehydrogenase (PDH; +94%), PDH kinases (+45% to +100%) and glycogen synthase (+48%) was increased in skeletal muscle of tumour-bearing mice in an AMPK-dependent manner. Lastly, chronic AICAR treatment elevated hexokinase II protein content and normalized phosphorylation of p70S6Kthr389 (mTORC1 substrate) and ACCser212 (AMPK substrate) and rescued cancer-induced insulin intolerance. Protein contents of AMPK subunits were upregulated in skeletal muscle of patients with NSCLC. AMPK activation seemed protectively inferred by AMPK-deficient mice developing metabolic dysfunction in response to cancer, including AMPK-dependent regulation of multiple proteins crucial for glucose metabolism. These observations highlight the potential for targeting AMPK to counter cancer-associated metabolic dysfunction and possibly cachexia.

Molecular mechanisms of cancer cachexia-related loss of skeletal muscle mass: data analysis from preclinical and clinical studies.

Cancer cachexia is a systemic hypoanabolic and catabolic syndrome that diminishes the quality of life of cancer patients, decreases the efficiency of therapeutic strategies and ultimately contributes to decrease their lifespan. The depletion of skeletal muscle compartment, which represents the primary site of protein loss during cancer cachexia, is of very poor prognostic in cancer patients. In this review, we provide an extensive and comparative analysis of the molecular mechanisms involved in the regulation of skeletal muscle mass in human cachectic cancer patients and in animal models of cancer cachexia. We summarize data from preclinical and clinical studies investigating how the protein turnover is regulated in cachectic skeletal muscle and question to what extent the transcriptional and translational capacities, as well as the proteolytic capacity (ubiquitin-proteasome system, autophagy-lysosome system and calpains) of skeletal muscle are involved in the cachectic syndrome in human and animals. We also wonder how regulatory mechanisms such as insulin/IGF1-AKT-mTOR pathway, endoplasmic reticulum stress and unfolded protein response, oxidative stress, inflammation (cytokines and downstream IL1ß/TNFα-NF-κB and IL6-JAK-STAT3 pathways), TGF-ß signalling pathways (myostatin/activin A-SMAD2/3 and BMP-SMAD1/5/8 pathways), as well as glucocorticoid signalling, modulate skeletal muscle proteostasis in cachectic cancer patients and animals. Finally, a brief description of the effects of various therapeutic strategies in preclinical models is also provided. Differences in the molecular and biochemical responses of skeletal muscle to cancer cachexia between human and animals (protein turnover rates, regulation of ubiquitin-proteasome system and myostatin/activin A-SMAD2/3 signalling pathways) are highlighted and discussed. Identifying the various and intertwined mechanisms that are deregulated during cancer cachexia and understanding why they are decontrolled will provide therapeutic targets for the treatment of skeletal muscle wasting in cancer patients.

Activation of β-catenin in mesenchymal progenitors leads to muscle mass loss.

Loss of muscle mass is a common manifestation of chronic disease. We find the canonical Wnt pathway to be activated in mesenchymal progenitors (MPs) from cancer-induced cachectic mouse muscle. Next, we induce β-catenin transcriptional activity in murine MPs. As a result, we observe expansion of MPs in the absence of tissue damage, as well as rapid loss of muscle mass. Because MPs are present throughout the organism, we use spatially restricted CRE activation and show that the induction of tissue-resident MP activation is sufficient to induce muscle atrophy. We further identify increased expression of stromal NOGGIN and ACTIVIN-A as key drivers of atrophic processes in myofibers, and we verify their expression by MPs in cachectic muscle. Finally, we show that blocking ACTIVIN-A rescues the mass loss phenotype triggered by β-catenin activation in MPs, confirming its key functional role and strengthening the rationale for targeting this pathway in chronic disease.

Protein therapy of skeletal muscle atrophy and mechanism by angiogenic factor AGGF1.

Skeletal muscle atrophy is a common condition without a pharmacologic therapy. AGGF1 encodes an angiogenic factor that regulates cell differentiation, proliferation, migration, apoptosis, autophagy and endoplasmic reticulum stress, promotes vasculogenesis and angiogenesis and successfully treats cardiovascular diseases. Here, we report the important role of AGGF1 in the pathogenesis of skeletal muscle atrophy and attenuation of muscle atrophy by AGGF1. In vivo studies were carried out in impaired leg muscles from patients with lumbar disc herniation, two mouse models for skeletal muscle atrophy (denervation and cancer cachexia) and heterozygous Aggf1+/- mice. Mouse muscle atrophy phenotypes were characterized by body weight and myotube cross-sectional areas (CSA) using H&E staining and immunostaining for dystrophin. Molecular mechanistic studies include co-immunoprecipitation (Co-IP), western blotting, quantitative real-time PCR analysis and immunostaining analysis. Heterozygous Aggf1+/- mice showed exacerbated phenotypes of reduced muscle mass, myotube CSA, MyHC (myosin heavy chain) and α-actin, increased inflammation (macrophage infiltration), apoptosis and fibrosis after denervation and cachexia. Intramuscular and intraperitoneal injection of recombinant AGGF1 protein attenuates atrophy phenotypes in mice with denervation (gastrocnemius weight 81.3±5.7mg vs. 67.3±5.1mg for AGGF1 vs. buffer; P<0.05) and cachexia (133.7±4.7 vs. 124.3±3.2; P<0.05). AGGF1 expression undergoes remodelling and is up-regulated in gastrocnemius and soleus muscles from atrophy mice and impaired leg muscles from patients with lumbar disc herniation by 50-60% (P<0.01). Mechanistically, AGGF1 interacts with TWEAK (tumour necrosis factor-like weak inducer of apoptosis), which reduces interaction between TWEAK and its receptor Fn14 (fibroblast growth factor-inducing protein 14). This leads to inhibition of Fn14-induced NF-kappa B (NF-κB) p65 phosphorylation, which reduces expression of muscle-specific E3 ubiquitin ligase MuRF1 (muscle RING finger 1), resulting in increased MyHC and α-actin and partial reversal of atrophy phenotypes. Autophagy is reduced in Aggf1+/- mice due to inhibition of JNK (c-Jun N-terminal kinase) activation in denervated and cachectic muscles, and AGGF1 treatment enhances autophagy in two atrophy models by activating JNK. In impaired leg muscles of patients with lumbar disc herniation, MuRF1 is up-regulated and MyHC and α-actin are down-regulated; these effects are reversed by AGGF1 by 50% (P<0.01). These results indicate that AGGF1 is a novel regulator for the pathogenesis of skeletal muscle atrophy and attenuates skeletal muscle atrophy by promoting autophagy and inhibiting MuRF1 expression through a molecular signalling pathway of AGGF1-TWEAK/Fn14-NF-κB. More importantly, the results indicate that AGGF1 protein therapy may be a novel approach to treat patients with skeletal muscle atrophy.

GDF15 neutralization restores muscle function and physical performance in a mouse model of cancer cachexia

Cancer cachexia is a disorder characterized by involuntary weight loss and impaired physical performance. Decline in physical performance of patients with cachexia is associated with poor quality of life, and currently there are no effective pharmacological interventions that restore physical performance. Here we examine the effect of GDF15 neutralization in a mouse model of cancer-induced cachexia (TOV21G) that manifests weight loss and muscle function impairments. With comprehensive assessments, our results demonstrate that cachectic mice treated with the anti-GDF15 antibody mAB2 exhibit body weight gain with near-complete restoration of muscle mass and markedly improved muscle function and physical performance. Mechanistically, the improvements induced by GDF15 neutralization are primarily attributed to increased caloric intake, while altered gene expression in cachectic muscles is restored in caloric-intake-dependent and -independent manners. The findings indicate potential of GDF15 neutralization as an effective therapy to enhance physical performance of patients with cachexia.

Creatine modulates cellular energy metabolism and protects against cancer cachexia-associated muscle wasting.

Cancer cachexia is a multifactorial syndrome defined by progressive loss of body weight with specific depletion of skeletal muscle and adipose tissue. Since there are no FDA-approved drugs that are available, nutritional intervention is recommended as a supporting therapy. Creatine supplementation has an ergogenic effect in various types of sports training, but the regulatory effects of creatine supplementation in cancer cachexia remain unknown. In this study, we investigated the impact of creatine supplementation on cachectic weight loss and muscle loss protection in a tumor-bearing cachectic mouse model, and the underlying molecular mechanism of body weight protection was further assessed. We observed decreased serum creatine levels in patients with cancer cachexia, and the creatine content in skeletal muscle was also significantly decreased in cachectic skeletal muscle in the C26 tumor-bearing mouse model. Creatine supplementation protected against cancer cachexia-associated body weight loss and muscle wasting and induced greater improvements in grip strength. Mechanistically, creatine treatment altered the dysfunction and morphological abnormalities of mitochondria, thus protecting against cachectic muscle wasting by inhibiting the abnormal overactivation of the ubiquitin proteasome system (UPS) and autophagic lysosomal system (ALS). In addition, electron microscopy revealed that creatine supplementation alleviated the observed increase in the percentage of damaged mitochondria in C26 mice, indicating that nutritional intervention with creatine supplementation effectively counteracts mitochondrial dysfunction to mitigate muscle loss in cancer cachexia. These results uncover a previously uncharacterized role for creatine in cachectic muscle wasting by modulating cellular energy metabolism to reduce the level of muscle cell atrophy.

Abstract A062: Skeletal Muscle Selective Autophagy Receptors are induced PDAC Cachexia

Abstract Cancer-induced cachexia is a hypermetabolic condition characterized by the unintentional wasting of muscle and adipose tissue, affecting over 80% of patients with pancreatic ductal adenocarcinoma (PDAC). Muscle wasting during cachexia is due to increased skeletal muscle protein degradation via ubiquitin-proteasome and autophagy-lysosome pathways. Autophagy-lysosome degradation requires delivery of cargo to the lysosome for destruction and recycling. Macroautophagy is the most prevalent component of autophagy, encompassing bulk and selective autophagy, and it requires the de novo synthesis of an autophagosome. Bulk autophagy randomly engulfs portions of the cytoplasm. Selective autophagy is mediated through selective autophagy receptors (SAR), which bind and couple cargo to the autophagosome via the general autophagy ligands LC3B and GABARAP. While an increase in the general autophagy machinery is well described in cachectic muscle, much less is known about how complexes and organelles are selectively targeted for degradation. Methods: Here, 12-week-old male C57BL/6J mice were orthotopically implanted with 1x105 KPC cells; controls underwent sham surgery. Half of the tumor-bearing mice were treated with 120 mg/kg gemcitabine and 10 mg/kg nab-paclitaxel (GemNP) at 4 and 10 days. For endpoint analysis, mice were euthanized at 14 days when KPC mice had significant body weight, muscle mass and muscle protein loss compared to SHAM controls. In our in vitro model of PDAC cachexia, KPC-conditioned media (CM) induces C2C12 myotube wasting; thus, myotubes were treated with 50% KPC-CM or control for 48hrs. Results: GemNP reduced end tumor mass by nearly 25% and prevented body weight and muscle loss. mRNAseq of gastrocnemius muscle demonstrated induction of ribosomal component gene expression, while deep proteomics revealed reduction of 30 ribosomal component proteins in KPC mice, consistent with ongoing destruction of ribosomes. Gene expression of general autophagy ligands, LC3B (2.9-fold) and GABARAP (1.6-fold), were increased in KPC mice. Gene expression for SARs associated with ribophagy (NUFIP1, 2.6-fold) and reticulophagy (Fam134b, 11.3-fold) were increased in KPC. FAM134b protein (1.8-fold) was also increased in KPC mice. SARs for lipophagy (PNPLA2, 3.2- and PNPLA8, 1.8-fold) and mitophagy (BNIP3, 4.5-fold) were induced in KPC versus SHAM, while SARs for glycophagy and ferritinophagy were similar. SQSTM1 (3.9-fold) and NBR1 (1.9-fold), general SARs for multiple organelles including aggrephagy, lysophagy, proteaphagy and pexophagy, were increased in KPC. This activation of SARs was due to tumor-induced wasting and not chemotherapy as SAR gene expression in KPC-GemNP mice was similar to SHAM controls. Finally, we also observe markers of SAR-mediated autophagy in our in vitro model of PDAC cachexia. Conclusion: These data indicate that muscle wasting in PDAC cachexia is through activation of selective autophagy of ribosomes, mitochondria, lipid droplets, endoplasmic reticulum, protein aggregates, lysosomes, peroxisomes, and proteasomes. Citation Format: Brittany R. Counts, Ashok Narasimhan, Tara S. Umberger, Emma H. Doud, Amber L. Mosley, Teresa A. Zimmers. Skeletal Muscle Selective Autophagy Receptors are induced PDAC Cachexia [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A062.