The LEAP2 Response to Cancer-Related Anorexia-Cachexia Syndrome in Male Mice and Patients.

The hormone ghrelin serves a protective role in cancer-related anorexia-cachexia syndrome (CACS)-a condition in which plasma levels of ghrelin rise, its administration lessens CACS severity, and experimentally reduced signaling by its receptor (GHSR) worsens fat loss and anorexia and accelerates death. Yet, actions for the related hormone liver-expressed antimicrobial peptide-2 (LEAP2), which is an endogenous GHSR antagonist, are unexplored in CACS. Here, we found that plasma LEAP2 and LEAP2/ghrelin ratio were lower in Lewis lung carcinoma (LLC) and RM-9 prostate cancer CACS mouse models. Ghrelin deletion exaggerated losses of tumor-free body weight and fat mass, reduced food intake, reduced soleus muscle weight, and/or lowered grip strength in LLC or RM-9 tumor-bearing mice. LEAP2 deletion lessened reductions in tumor-free body weight and fat mass and increased food intake in LLC or RM-9 tumor-bearing mice. In a 55-subject cohort of patients with CACS or weight-stable cancer, the plasma LEAP2/total ghrelin ratio was negatively correlated with 6-month weight change preceding blood collection. These data demonstrate that ghrelin deletion exacerbates CACS in the LLC and RM-9 tumor-bearing mouse models while contrastingly, LEAP2 deletion reduces measures of CACS in these tumor-bearing mouse models. Further, they suggest that lower plasma LEAP2/ghrelin ratio protects against worsened CACS.

Muscle-derived IL-1ß regulates EcSOD expression via the NBR1-p62-Nrf2 pathway in muscle during cancer cachexia.

Oxidative stress contributes to the loss of skeletal muscle mass and function in cancer cachexia. However, this outcome may be mitigated by an improved endogenous antioxidant defence system. Here, using the well-established oxidative stress-inducing muscle atrophy model of Lewis lung carcinoma (LLC) in 13-week-old male C57BL/6J mice, we demonstrate that extracellular superoxide dismutase (EcSOD) levels increase in the cachexia-prone extensor digitorum longus muscle. LLC transplantation significantly increased interleukin-1ß (IL-1ß) expression and release from extensor digitorum longus muscle fibres. Moreover, IL-1ß treatment of C2C12 myotubes increased NBR1, p62 phosphorylation at Ser351, Nrf2 nuclear translocation and EcSOD protein expression. Additional studies in vivo indicated that intramuscular IL-1ß injection is sufficient to stimulate EcSOD expression, which is prevented by muscle-specific knockout of p62 and Nrf2 (i.e. in p62 skmKO and Nrf2 skmKO mice, respectively). Finally, since an increase in circulating IL-1ß may lead to unwanted outcomes, we demonstrate that targeting this pathway at p62 is sufficient to drive muscle EcSOD expression in an Nrf2-dependent manner. In summary, cancer cachexia increases EcSOD expression in extensor digitorum longus muscle via muscle-derived IL-1ß-induced upregulation of p62 phosphorylation and Nrf2 activation. These findings provide further mechanistic evidence for the therapeutic potential of p62 and Nrf2 to mitigate cancer cachexia-induced muscle atrophy. KEY POINTS: Oxidative stress plays an important role in muscle atrophy during cancer cachexia. EcSOD, which mitigates muscle loss during oxidative stress, is upregulated in 13-week-old male C57BL/6J mice of extensor digitorum longus muscles during cancer cachexia. Using mouse and cellular models, we demonstrate that cancer cachexia promotes muscle EcSOD protein expression via muscle-derived IL-1ß-dependent stimulation of the NBR1-p62-Nrf2 signalling pathway. These results provide further evidence for the potential therapeutic targeting of the NBR1-p62-Nrf2 signalling pathway downstream of IL-1ß to mitigate cancer cachexia-induced muscle atrophy.

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 in vitro 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.

Diet-induced obesity does not exacerbate cachexia in male mice bearing Lewis-lung carcinoma tumors.

Cachexia is a muscle-wasting syndrome commonly observed in patients with cancer, which can significantly worsen clinical outcomes. Because of a global rise in obesity, the coexistence of cachexia in obese individuals poses unique challenges, with the impact of excessive adiposity on cachexia severity and underlying pathophysiology not well defined. Understanding the interplay between cachexia and obesity is crucial for improving diagnosis and treatment strategies for these patients; therefore, the present study examined differences in cachexia between lean and obese mice bearing Lewis lung carcinoma (LLC) tumors. Nine-week-old, male C57Bl6J mice were placed on either a chow or a high-fat diet (HFD) for 9 wk. After the diet intervention, mice were inoculated with LLC or vehicle. Markers of cachexia, such as body and muscle loss, were noted in both chow and HFD groups with tumors. Tumor weight of HFD animals was greater than that of chow. LLC tumors reduced gastrocnemius, plantaris, and soleus mass, regardless of diet. The tibialis anterior and plantaris mass and cross-sectional area of type IIb/x fibers in the gastrocnemius were not different between HFD-chow, HFD-tumor, and chow-tumor. Using RNA sequencing (RNA-seq) of the plantaris muscle from chow-tumor and HFD-tumor groups, we identified ∼400 differentially expressed genes. Bioinformatic analysis identified changes in lipid metabolism, mitochondria, bioenergetics, and proteasome degradation. Atrophy was not greater despite larger tumor burden in animals fed an HFD, and RNA-seq data suggests that partial protection is mediated through differences in mitochondrial function and protein degradation, which may serve as future mechanistic targets.NEW & NOTEWORTHY This study provides timely information on the interaction between obesity and cancer cachexia. Lean and obese animals show signs of cachexia with reduced body weight, adipose tissue, and gastrocnemius muscle mass. There was not significant wasting in the tibialis anterior, plantaris, or fast twitch fibers in the gastrocnemius muscle of obese animals with tumors. RNA-seq analysis reveals that obese tumor bearing animals had differential expression of mitochondria- and degradation-related genes, which may direct future studies in mechanistic research.

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

BACKGROUND: 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. METHODS: Male C57BL/6 mice were given a purified high fat or standard diet for 16 weeks before being implanted with 106 Lewis lung carcinoma (LLC) cells. Mice were monitored for 25 days, and hindlimb muscles were collected for cachexia indices and mitochondrial assessment via western blotting, high-resolution respirometry and transmission electron microscopy (TEM). RESULTS: 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. CONCLUSIONS: 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.

Bckdk-Mediated Branch Chain Amino Acid Metabolism Reprogramming Contributes to Muscle Atrophy during Cancer Cachexia.

SCOPE: Branched chain amino acids (BCAAs) are essential amino acids and important nutrient signals for energy and protein supplementation. The study uses muscle-specific branched-chain α-keto acid dehydrogenase kinase (Bckdk) conditional knockout (cKO) mice to reveal the contribution of BCAA metabolic dysfunction to muscle wasting. METHOD AND RESULTS: Muscle-specific Bckdk-cKO mice are generated through crossbreeding of Bckdkf/f mice with Myf5Cre mice. Lewis lung cancer (LLC) tumor transplantation is used to establish the cancer cachexia model. The occurrence of cancer cachexia is accelerated in the muscle-specific Bckdk-cKO mice after bearing LLC tumor. Wasting skeletal muscle is characterized by increased protein ubiquitination degradation and impaired protein synthesis. The wasting muscle gastrocnemius is mechanized as a distinct BCAA metabolic dysfunction. Based on the atrophy phenotype resulting from BCAA metabolism dysfunction, the optimized BCAA supplementation improves the survival of cancer cachexia in muscle-specific Bckdk-cKO mice bearing LLC tumors, and improves the occurrence of cancer cachexia. The mechanism of BCAA supplementation on muscle mass preservation is based on the promotion of protein synthesis and the inhibition of protein ubiquitination degradation. CONCLUSIONS: Dysfunctional BCAA metabolism contributes to the inhibition of protein synthesis and increases protein degradation in the cancer cachexia model of muscle-specific Bckdk-cKO mice bearing LLC tumors. The reprogramming of BCAA catabolism exerts therapeutic effects by stimulating protein synthesis and inhibiting protein degradation in skeletal muscle.

Exosomal EIF5A derived from Lewis lung carcinoma induced adipocyte wasting in cancer cachexia.

Cancer cachexia is a systemic inflammation-driven syndrome, characterized by muscle atrophy and adipose tissue wasting, with progressive weight loss leading to serious impairment of physiological function. Extracellular vesicles (EVs) derived from cancer cells play a significant role in adipocyte lipolysis, yet the mechanism remain uneclucidated. In this study, EVs derived from Lewis lung carcinoma (LLC) cells were extracted and characterized. 3T3-L1 and HIB1B adipocytes were cultured with conditioned medium or EVs from LLC, and LLC cells were used to establish a cancer cachexia mouse model. EVs derived from LLC cells were taken up by 3T3-L1 and HIB1B adipocytes, and derived exosomal EIF5A protein-induced lipolysis of adipocytes. High level of EIF5A was expressed in EVs from LLC cells, exosomal EIF5A is linked to lipid metabolism. Elevated expression of EIF5A is associated with shorter overall survival in lung cancer patients. Western blots, glycerol release and Oil red O staining assays were used to evaluate lipolysis of adipocytes. The reduction of lipolysis in 3T3-L1 and HIB1B adipocytes is achieved through silencing EIF5A or treating with pharmacologic inhibitor GC7 in vitro, and suppressing the expression of EIF5A in LLC cells by infected with shRNA or GC7 treatment partly alleviated white and brown adipose tissue lipolysis in vivo. Mechanistically, EIF5A directly binds with G protein-coupled bile acid receptor 1 (GPBAR1) mRNA to promote its translation and then activates cAMP response element binding protein (CREB) signaling pathway to induce lipolysis. This study demonstrates that exosomal EIF5A from LLC cells, with hypusinated EIF5A, has a lipolytic effect on adipocyte and adipose tissues in cancer cachexia model. Exosomal EIF5A could be involved in lipolysis and these findings indicate that a novel regulator and potential target for cachexia treatment.

Muscle p62 stimulates the expression of antioxidant proteins alleviating cancer cachexia.

Oxidative stress plays an important role in skeletal muscle atrophy during cancer cachexia, and more glycolytic muscles are preferentially affected. Sequestosome1/SQSTM1 (i.e., p62), particularly when phosphorylated at Ser 349 (Ser 351 in mice), competitively binds to the Kelch-like ECH-associated protein 1 (Keap1) activating Nuclear factor erythroid 2-related factor 2 (Nrf2). Nrf2 then stimulates the transcription of antioxidant/electrophile-responsive elements in target genes. However, a potential role for p62 in the protection of muscle wasting in cachexia remains to be determined. Here, using the well-established cachexia-inducing model of Lewis Lung Carcinoma (LLC) in mice we demonstrate higher expression of antioxidant proteins (i.e., NQO1, HO-1, GSTM1, CuZnSOD, MnSOD, and EcSOD) in the more oxidative and cachexia resistant soleus muscle than in the more glycolytic and cachexia prone extensor digitorum longus muscle. This was accompanied by higher p62 (total and phosphorylated) and nuclear Nrf2 levels in the soleus, which were paralleled by higher expression of proteins known to either phosphorylate or promote p62 phosphorylation (i.e., NBR1, CK1, PKCδ, and TAK1). Muscle-specific p62 gain-of-function (i.e., in p62 mTg mice) activated Nrf2 nuclear translocation and increased the expression of multiple antioxidant proteins (i.e., CuZnSOD, MnSOD, EcSOD, NQO1, and GSTM1) in glycolytic muscles. Interestingly, skeletal muscle Nrf2 haplodeficiency blunted the increases of most of these proteins (i.e., CuZnSOD, EcSOD, and NQO1) suggesting that muscle p62 stimulates antioxidant protein expression also via additional, yet to be determined mechanisms. Of note, p62 gain-of-function mitigated glycolytic muscle wasting in LLC-affected mice. Collectively, our findings identify skeletal muscle p62 as a potential therapeutic target for cancer cachexia.

The time-course of cancer cachexia onset reveals biphasic transcriptional disruptions in female skeletal muscle distinct from males.

BACKGROUND: Cancer-cachexia (CC) is a debilitating condition affecting up to 80% of cancer patients and contributing to 40% of cancer-related deaths. While evidence suggests biological sex differences in the development of CC, assessments of the female transcriptome in CC are lacking, and direct comparisons between sexes are scarce. This study aimed to define the time course of Lewis lung carcinoma (LLC)-induced CC in females using transcriptomics, while directly comparing biological sex differences. RESULTS: We found the global gene expression of the gastrocnemius muscle of female mice revealed biphasic transcriptomic alterations, with one at 1 week following tumor allograft and another during the later stages of cachexia development. The early phase was associated with the upregulation of extracellular-matrix pathways, while the later phase was characterized by the downregulation of oxidative phosphorylation, electron transport chain, and TCA cycle. When DEGs were compared to a known list of mitochondrial genes (MitoCarta), ~ 47% of these genes were differently expressed in females exhibiting global cachexia, suggesting transcriptional changes to mitochondrial gene expression happens concomitantly to functional impairments previously published. In contrast, the JAK-STAT pathway was upregulated in both the early and late stages of CC. Additionally, we observed a consistent downregulation of Type-II Interferon signaling genes in females, which was associated with protection in skeletal muscle atrophy despite systemic cachexia. Upregulation of Interferon signaling was noted in the gastrocnemius muscle of cachectic and atrophic male mice. Comparison of female tumor-bearing mice with males revealed ~ 70% of DEGs were distinct between sexes in cachectic animals, demonstrating dimorphic mechanisms of CC. CONCLUSION: Our findings suggest biphasic disruptions in the transcriptome of female LLC tumor-bearing mice: an early phase associated with ECM remodeling and a late phase, accompanied by the onset of systemic cachexia, affecting overall muscle energy metabolism. Notably, ~ 2/3 of DEGs in CC are biologically sex-specific, providing evidence of dimorphic mechanisms of cachexia between sexes. Downregulation of Type-II Interferon signaling genes appears specific to CC development in females, suggesting a new biological sex-specific marker of CC not reliant on the loss of muscle mass, that might represent a protective mechanism against muscle loss in CC in female mice.

Eicosapentaenoic Acid-Enriched Phospholipids Alleviate Skeletal Muscle Atrophy in Lewis Lung Carcinoma Mouse Model.

SCOPE: Skeletal muscle atrophy is a critical feature of cancer-associated cachexia (CAC) and it is responsible for poor quality of life and high mortality in cancer patients. The previous study demonstrates that eicosapentaenoic acid-enriched phospholipids (EPA-PL) prevent body weight loss in a mouse model of CAC. However, the role of EPA-PL on cancer-induced skeletal muscle atrophy remains unclear. METHODS AND RESULTS: In the present study, a Lewis lung carcinoma (LLC) mouse model is established, then the effect and underlying mechanism of EPA-PL on skeletal muscle atrophy in LLC-bearing mice are investigated. The results reveal that EPA-PL treatment significantly attenuates skeletal muscle atrophy in LLC-bearing mice, as evidenced by suppressing the reductions of skeletal muscle mass, myofiber cross-sectional area, and grip strength. Besides, the study finds that EPA-PL alleviated cancer-induced skeletal muscle atrophy via balancing muscle protein degradation and synthesis, inhibiting type I oxidative muscle fibers atrophy, and promoting mitochondrial function. Furthermore, the results also indicate that EPA-PL may counteract skeletal muscle atrophy in LLC mouse model via a sirtuin 1-dependent mechanism. CONCLUSION: These findings provide evidence that EPA-PL may be beneficial as a nutritional supplement for prevention and treatment of cancer-induced skeletal muscle atrophy.

Development of skeletal muscle fibrosis in a rodent model of cancer cachexia.

Cachexia is characterized by losses in lean body mass and its progression results in worsened quality of life and exacerbated outcomes in cancer patients. However, the role and impact of fibrosis during the early stages and development of cachexia in under-investigated. The purpose of this study was to determine if fibrosis occurs during cachexia development, and to evaluate this in both sexes. Female and male C57BL6/J mice were injected with phosphate-buffered saline or Lewis Lung Carcinoma (LLC) at 8-week of age, and tumors were allowed to develop for 1, 2, 3, or 4 weeks. 3wk and 4wk female tumor-bearing mice displayed a dichotomy in tumor growth and were reassigned to high tumor (HT) and low tumor (LT) groups. In vitro analyses were also performed on cocultured C2C12 and 3T3 cells exposed to LLC conditioned media. Immunohistochemistry and quantitative polymerase chain reaction (qPCR) analysis were used to investigate fibrosis and fibrosis-related signaling in skeletal muscle. Collagen deposition in skeletal muscle was increased in the 1wk, LT, and HT groups in female mice. However, collagen deposition was only increased in the 4wk group in male mice. In general, female mice displayed earlier alterations in extracellular matrix (ECM)-related genes beginning at 1wk post-LLC injection. Whereas this was not seen in males. While overall tumor burden is tightly correlated to cachexia development in both sexes, fibrotic development is not. Male mice did not exhibit early-stage alterations in ECM-related genes contrary to what was noted in female mice.

Effects of S-pindolol in mouse pancreatic and lung cancer cachexia models.

BACKGROUND: It is known that S-pindolol attenuates muscle loss in animal models of cancer cachexia and sarcopenia. In cancer cachexia, it also significantly reduced mortality and improved cardiac function, which is strongly compromised in cachectic animals. METHODS: Here, we tested 3 mg/kg/day of S-pindolol in two murine cancer cachexia models: pancreatic cancer cachexia (KPC) and Lewis lung carcinoma (LLC). RESULTS: Treatment of mice with 3 mg/kg/day of S-pindolol in KPC or LLC cancer cachexia models significantly attenuated the loss of body weight, including lean mass and muscle weights, leading to improved grip strength compared with placebo-treated mice. In the KPC model, treated mice lost less than half of the total weight lost by placebo (-0.9 ± 1.0 vs. -2.2 ± 1.4 g for S-pindolol and placebo, respectively, P < 0.05) and around a third of the lean mass lost by tumour-bearing controls (-0.4 ± 1.0 vs. -1.5 ± 1.5 g for S-pindolol and placebo, respectively, P < 0.05), whereas loss of fat mass was similar. In the LLC model, the gastrocnemius weight was higher in sham (108 ± 16 mg) and S-pindolol tumour-bearing (94 ± 15 mg) mice than that in placebo (83 ± 12 mg), whereas the soleus weight was only significantly higher in the S-pindolol-treated group (7.9 ± 1.7 mg) than that in placebo (6.5 ± 0.9). Grip strength was significantly improved by S-pindolol treatment (110.8 ± 16.2 vs. 93.9 ± 17.1 g for S-pindolol and placebo, respectively). A higher grip strength was observed in all groups; whereas S-pindolol-treated mice improved by 32.7 ± 18.5 g, tumour-bearing mice only show minimal improvements (7.3 ± 19.4 g, P < 0.01). CONCLUSIONS: S-pindolol is an important candidate for clinical development in the treatment of cancer cachexia that strongly attenuates loss of body weight and lean body mass. This was also seen in the weight of individual muscles and resulted in higher grip strength.

EXT418, a novel long-acting ghrelin, mitigates Lewis lung carcinoma induced cachexia in mice.

BACKGROUND: Ghrelin is a potential therapy for cachexia due to its orexigenic properties and anabolic effects on muscle and fat. However, its clinical use is limited by the short half-life of active (acylated) ghrelin (~11 min in humans). EXT418 is a novel long-acting, constitutively active ghrelin analog created by covalently linking it to a vitamin D derivative. Here, we evaluated the effects and mechanisms of action of EXT418 on Lewis lung carcinoma (LLC)-induced cachexia in mice. METHODS: Male C57BL/6J mice (5- to 7-month-old) were implanted with 1 × 106 heat-killed (HK) or live LLC cells. When the tumour was palpable, mice were injected with vehicle (T + V) or EXT418 daily (T + 418 Daily, 0.25 mg/kg/day) or every other day (T + 418 EOD, 0.5 mg/kg/EOD) for up to 14 days, whereas HK-treated mice were given vehicle (HK + V). Subsets of T + 418 Daily or EOD-treated mice were pair-fed to the T + V group. Body composition and grip strength were evaluated before tumour implantation and at the end of the experiment. Molecular markers were probed in muscles upon termination. RESULTS: In tumour-bearing mice, administration of EXT418 daily or EOD partially prevented weight loss (T + V vs. T + 418 Daily, P = 0.030; and vs. T + 418 EOD, P = 0.020). Similar effects were observed in whole body fat and lean body mass. Grip strength in tumour-bearing mice was improved by EXT418 daily (P = 0.010) or EOD (P = 0.008) administration compared with vehicle-treated mice. These effects of EXT418 on weight and grip strength were partially independent of food intake. EXT418 daily administration also improved type IIA (P = 0.015), IIB (P = 0.037) and IIX (P = 0.050) fibre cross-sectional area (CSA) in tibialis anterior (TA) and EXT418 EOD improved CSA of IIB fibres in red gastrocnemius (GAS; P = 0.005). In skeletal muscles, tumour-induced increases in atrogenes Fbxo32 and Trim63 were ameliorated by EXT418 treatments (TA and GAS/plantaris, PL), which were independent of food intake. EXT418 administration decreased expression of the mitophagy marker Bnip3 (GAS/PL; P ≤ 0.010). Similar effects of EXT418 EOD were observed in p62 (GAS/PL; P = 0.039). In addition, EXT418 treatments ameliorated the tumour-induced elevation in muscle Il6 transcript levels (TA and GAS/PL), independently of food intake. Il-6 transcript levels in adipose tissue and circulating IL-10 were elevated in response to the tumour but these increases were not significant with EXT418 administration. Tumour mass was not altered by EXT418. CONCLUSIONS: EXT418 mitigates LLC-induced cachexia by attenuating skeletal muscle inflammation, proteolysis, and mitophagy, without affecting tumour mass and partially independent of food intake.

Ginsenoside Rd ameliorates muscle wasting by suppressing the signal transducer and activator of transcription 3 pathway.

BACKGROUND: The effects of some drugs, aging, cancers, and other diseases can cause muscle wasting. Currently, there are no effective drugs for treating muscle wasting. In this study, the effects of ginsenoside Rd (GRd) on muscle wasting were studied. METHODS: Tumour necrosis factor-alpha (TNF-α)/interferon-gamma (IFN-γ)-induced myotube atrophy in mouse C2C12 and human skeletal myoblasts (HSkM) was evaluated based on cell thickness. Atrophy-related signalling, reactive oxygen species (ROS) level, mitochondrial membrane potential, and mitochondrial number were assessed. GRd (10 mg/kg body weight) was orally administered to aged mice (23-24 months old) and tumour-bearing (Lewis lung carcinoma [LLC1] or CT26) mice for 5 weeks and 16 days, respectively. Body weight, grip strength, inverted hanging time, and muscle weight were assessed. Histological analysis was also performed to assess the effects of GRd. The evolutionary chemical binding similarity (ECBS) approach, molecular docking, Biacore assay, and signal transducer and activator of transcription (STAT) 3 reporter assay were used to identify targets of GRd. RESULTS: GRd significantly induced hypertrophy in the C2C12 and HSkM myotubes (average diameter 50.8 ± 2.6% and 49.9% ± 3.7% higher at 100 nM, vs. control, P ≤ 0.001). GRd treatment ameliorated aging- and cancer-induced (LLC1 or CT26) muscle atrophy in mice, which was evidenced by significant increases in grip strength, hanging time, muscle mass, and muscle tissue cross-sectional area (1.3-fold to 4.6-fold, vs. vehicle, P ≤ 0.05; P ≤ 0.01; P ≤ 0.001). STAT3 was found to be a possible target of GRd by the ECBS approach and molecular docking assay. Validation of direct interaction between GRd and STAT3 was confirmed through Biacore analysis. GRd also inhibited STAT3 phosphorylation and STAT3 reporter activity, which led to the inhibition of STAT3 nuclear translocation and the suppression of downstream targets of STAT3, such as atrogin-1, muscle-specific RING finger protein (MuRF-1), and myostatin (MSTN) (29.0 ± 11.2% to 84.3 ± 30.5%, vs. vehicle, P ≤ 0.05; P ≤ 0.01; P ≤ 0.001). Additionally, GRd scavenged ROS (91.7 ± 1.4% reduction at 1 nM, vs. vehicle, P ≤ 0.001), inhibited TNF-α-induced dysregulation of ROS level, and improved mitochondrial integrity (P ≤ 0.05; P ≤ 0.01; P ≤ 0.001). CONCLUSIONS: GRd ameliorates aging- and cancer-induced muscle wasting. Our findings suggest that GRd may be a novel therapeutic agent or adjuvant for reversing muscle wasting.

Inhibition of epidermal growth factor receptor suppresses parathyroid hormone-related protein expression in tumours and ameliorates cancer-associated cachexia.

BACKGROUND: Lung cancer is the primary cause of cancer deaths worldwide. Activation of epidermal growth factor receptor (EGFR) leads to lung cancer progression and poor prognosis while involuntary weight loss remains a major problem. Tumour-derived parathyroid hormone-related protein (PTHrP) emerged as a potential mediator of cachexia. Here, we investigated the modulatory role of EGFR signalling in PTHrP (encoded by Pthlh) gene expression and the impact of this relationship on cancer cachexia. METHODS: Global gene expression profiles of Lewis lung carcinoma (LLC) cells were analysed. Pthlh mRNA levels were measured by qRT-PCR in LLC cells treated with EGFR ligands and tyrosine kinase inhibitors (TKIs). LLC tumour-bearing mice received EGFR TKI erlotinib for 7 days via intraperitoneal injection or oral gavage. Tumour Pthlh mRNA, weight of fat/muscle tissue, and grip strength were assessed. RNA-seq data from The Cancer Genome Atlas and gene expression analysis tools were used to characterize expression profiles of PTHLH and EGFR along with correlation analysis of PTHLH with EGFR and transforming growth factor alpha (TGFA) in human lung cancer and head and neck squamous carcinoma (HNSC). Survival of lung squamous cell carcinoma (LUSC) and lung adenocarcinoma (LUAD) patients with EGFR gene alterations was analysed in regard to PTHLH expression. RESULTS: Expression of EGFR ligands, EGFR itself, and PTHrP co-clusters in LLC cells. Activation of EGFR signalling with its ligands significantly increases (3.8-fold, P < 0.0005) while EGFR TKIs significantly decrease (90%, P < 0.0005) Pthlh mRNA levels in LLC cells. Pthlh mRNA drops 65-75% (P < 0.0005) in tumours upon treatment of LLC tumour-bearing mice with erlotinib while their muscle mass and grip strength increase (9.2% P < 0.05, 23% P < 0.005, respectively) compared with tumour-bearing control mice. PTHLH is overexpressed in tumours of LUSC (45.8-fold, P < 0.05) and HNSC (17.5-fold, P < 0.05) compared with normal tissue. PTHLH expression correlates with EGFR and its ligand TGFA in both cancers (LUSC: n = 745, R = 0.32, P < 0.0001 and R = 0.51, P < 0.0001; HNSC: n = 545, R = 0.34, P < 0.001 and R = 0.50, P < 0.001, respectively). High PTHLH mRNA associates with poor overall survival in LUAD patients with activating EGFR mutations (n = 40, log-rank test, P = 0.0451). CONCLUSIONS: Epidermal growth factor receptor signalling regulates expression of cachexia mediator PTHrP. EGFR inhibition reduces PTHrP expression in LLC tumours and ameliorates cachexia in LLC tumour-bearing mice.

C/EBPß promotes the expression of atrophy-inducing factors by tumours and is a central regulator of cancer cachexia.

BACKGROUND: CCAAT/enhancer-binding protein ß (C/EBPß) is a transcription factor whose high expression in human cancers is associated with tumour aggressiveness and poor outcomes. Most advanced cancer patients will develop cachexia, characterized by loss of skeletal muscle mass. In response to secreted factors from cachexia-inducing tumours, C/EBPß is stimulated in muscle, leading to both myofibre atrophy and the inhibition of muscle regeneration. Involved in the regulation of immune responses, C/EBPß induces the expression of many secreted factors, including cytokines. Because tumour-secreted factors drive cachexia and aggressive tumours have higher expression of C/EBPß, we examined a potential role for C/EBPß in the expression of tumour-derived cachexia-inducing factors. METHODS: We used gain-of-function and loss-of-function approaches in vitro and in vivo to evaluate the role of tumour C/EBPß expression on the secretion of cachexia-inducing factors. RESULTS: We report that C/EBPß overexpression up-regulates the expression of 260 secreted protein genes, resulting in a secretome that inhibits myogenic differentiation (-31%, P < 0.05) and myotube maturation [-38% (fusion index) and -25% (myotube diameter), P < 0.05]. We find that knockdown of C/EBPß in cachexia-inducing Lewis lung carcinoma cells restores myogenic differentiation (+25%, P < 0.0001) and myotube diameter (+90%, P < 0.0001) in conditioned medium experiments and, in vivo, prevents muscle wasting (-51% for small myofibres vs. controls, P < 0.01; +140% for large myofibres, P < 0.01). Conversely, overexpression of C/EBPß in non-cachectic tumours converts their secretome into a cachexia-inducing one, resulting in reduced myotube diameter (-41%, P < 0.0001, EL4 model) and inhibition of differentiation in culture (-26%, P < 0.01, EL4 model) and muscle wasting in vivo (+98% small fibres, P < 0.001; -76% large fibres, P < 0.001). Comparison of the differently expressed transcripts coding for secreted proteins in C/EBPß-overexpressing myoblasts with the secretome from 27 different types of human cancers revealed ~18% similarity between C/EBPß-regulated secreted proteins and those secreted by highly cachectic tumours (brain, pancreatic, and stomach cancers). At the protein level, we identified 16 novel secreted factors that are present in human cancer secretomes and are up-regulated by C/EBPß. Of these, we tested the effect of three factors (SERPINF1, TNFRSF11B, and CD93) on myotubes and found that all had atrophic potential (-33 to -36% for myotube diameter, P < 0.01). CONCLUSIONS: We find that C/EBPß is necessary and sufficient to induce the secretion of cachexia-inducing factors by cancer cells and loss of C/EBPß in tumours attenuates muscle atrophy in an animal model of cancer cachexia. Our findings establish C/EBPß as a central regulator of cancer cachexia and an important therapeutic target.

Early-Onset Physical Inactivity and Metabolic Dysfunction in Tumor-bearing Mice Is Associated with Accelerated Cachexia.

METHODS: Male C57BL/6J mice (12 wk of age) were injected with 1 × 106 LLC cells or phosphate-buffered saline (PBS) subcutaneously in the right flank, and tissue was collected 26-28 d after cell injection. Tumor volume was measured every 5 d throughout the study to calculate the tumor growth rate. Fifteen days after tumor inoculation, a subset of PBS (n = 11) and LLC (n = 16) mice were individually housed in metabolic Comprehensive Laboratory Animal Monitoring System cages for 5 d. RESULTS: LLC mice exhibited greater body weight loss (-5.1%), decreased muscle mass (-7%), decreased fat mass (-22%), and increased plasma interleukin-6 (212%) compared with PBS mice. Before the onset of cachexia, total cage activity was decreased in tumor-bearing mice. Cage activity was negatively associated with tumor mass and positively associated with hindlimb muscle mass. In addition, LLC mice had greater lipid oxidation than PBS mice. CONCLUSIONS: LLC mice exhibit early-onset physical inactivity and altered systemic lipid oxidation, which are associated with the eventual development of cachexia.

REDD1 deletion attenuates cancer cachexia in mice.

Cancer cachexia is a wasting disorder associated with advanced cancer that contributes to mortality. Cachexia is characterized by involuntary loss of body weight and muscle weakness that affects physical function. Regulated in DNA damage and development 1 (REDD1) is a stress-response protein that is transcriptionally upregulated in muscle during wasting conditions and inhibits mechanistic target of rapamycin complex 1 (mTORC1). C2C12 myotubes treated with Lewis lung carcinoma (LLC)-conditioned media increased REDD1 mRNA expression and decreased myotube diameter. To investigate the role of REDD1 in cancer cachexia, we inoculated 12-wk-old male wild-type or global REDD1 knockout (REDD1 KO) mice with LLC cells and euthanized 28 days later. Wild-type mice had increased skeletal muscle REDD1 expression, and REDD1 deletion prevented loss of body weight and lean tissue mass but not fat mass. We found that REDD1 deletion attenuated loss of individual muscle weights and loss of myofiber cross-sectional area. We measured markers of the Akt/mTORC1 pathway and found that, unlike wild-type mice, phosphorylation of both Akt and 4E-BP1 was maintained in the muscle of REDD1 KO mice after LLC inoculation, suggesting that loss of REDD1 is beneficial in maintaining mTORC1 activity in mice with cancer cachexia. We measured Foxo3a phosphorylation as a marker of the ubiquitin proteasome pathway and autophagy and found that REDD1 deletion prevented dephosphorylation of Foxo3a in muscles from cachectic mice. Our data provide evidence that REDD1 plays an important role in cancer cachexia through the regulation of both protein synthesis and protein degradation pathways.NEW & NOTEWORTHY Cancer cachexia is a debilitating and lethal consequence of many advanced cancers. REDD1, a negative regulator of mTORC1 activity, is an emerging target in cachexia. Our data show that skeletal muscle REDD1 expression is increased in LLC-induced cancer cachexia. Mice lacking REDD1 have attenuated skeletal muscle atrophy that is likely due to maintaining both protein synthesis and inhibiting protein degradation.

Growth hormone secretagogue receptor-1a mediates ghrelin's effects on attenuating tumour-induced loss of muscle strength but not muscle mass.

BACKGROUND: Ghrelin may ameliorate cancer cachexia (CC) by preventing anorexia, muscle, and fat loss. However, the mechanisms mediating these effects are not fully understood. This study characterizes the pathways involved in muscle mass and strength loss in the Lewis lung carcinoma (LLC)-induced cachexia model, and the effects of ghrelin in mice with or without its only known receptor: the growth hormone secretagogue receptor-1a ((GHSR-1a), Ghsr+/+ and Ghsr-/- ). METHODS: Five to 7-month-old male C57BL/6J Ghsr+/+ and Ghsr-/- mice were inoculated with 1 × 106 heat-killed (HK) or live LLC cells (tumour implantation, TI). When tumours were palpable (7 days after TI), tumour-bearing mice were injected with vehicle (T + V) or ghrelin twice/day for 14 days (T + G, 0.8 mg/kg), while HK-treated mice were given vehicle (HK + V). Body weight and grip strength were evaluated before TI and at termination (21 days after TI). Hindlimb muscles were collected for analysis. RESULTS: Less pronounced body weight (BW) loss (87.70 ± 0.98% vs. 83.92 ± 1.23%, percentage of baseline BW in tumour-bearing Ghsr+/+ vs. Ghsr-/- , P = 0.008), and lower upregulation of ubiquitin-proteasome system (UPS, MuRF1/Trim63, 5.71 ± 1.53-fold vs. 9.22 ± 1.94-fold-change from Ghsr+/+ HK + V in tumour-bearing Ghsr+/+ vs. Ghsr-/- , P = 0.036) and autophagy markers (Becn1, Atg5, Atg7, tumour-bearing Ghsr+/+  < Ghsr-/- , all P < 0.02) were found in T + V Ghsr+/+ vs. Ghsr-/- mice. Ghrelin attenuated LLC-induced UPS marker upregulation in both genotypes, [Trim63 was decreased from 5.71 ± 1.53-fold to 1.96 ± 0.47-fold in Ghsr+/+ (T + V vs. T + G: P = 0.032) and 9.22 ± 1.94-fold to 4.72 ± 1.06-fold in Ghsr-/- (T + V vs. T + G: P = 0.008)]. Only in Ghsr+/+ mice ghrelin ameliorated LLC-induced grip strength loss [improved from 89.24 ± 3.48% to 97.80 ± 2.31% of baseline (T + V vs. T + G: P = 0.042)], mitophagy markers [Bnip3 was decreased from 2.28 ± 0.56 to 1.38 ± 0.14-fold (T + V vs. T + G: P ≤ 0.05)], and impaired mitochondrial respiration [State 3u improved from 698.23 ± 73.96 to 934.37 ± 95.21 pmol/min (T + V vs. T + G: P ≤ 0.05)], whereas these markers were not improved by ghrelin Ghsr-/- . Compared with Ghsr+/+ , Ghsr-/- tumour-bearing mice also showed decreased response to ghrelin in BW [T + G-treated Ghsr+/+ vs. Ghsr -/- : 91.75 ± 1.05% vs. 86.18 ± 1.13% of baseline BW, P < 0.001)], gastrocnemius (T + G-treated Ghsr+/+ vs. Ghsr-/- : 96.9 ± 2.08% vs. 88.15 ± 1.78% of Ghsr+/+ HK + V, P < 0.001) and quadriceps muscle mass (T + G-treated Ghsr+/+ vs. Ghsr-/- : 96.12 ± 2.31% vs. 88.36 ± 1.94% of Ghsr+/+ HK + V, P = 0.01), and gastrocnemius type IIA (T + G-treated Ghsr+/+ vs. Ghsr-/- : 1250.49 ± 31.72 vs. 1017.62 ± 70.99 µm2 , P = 0.027) and IIB fibre cross-sectional area (T + G-treated Ghsr+/+ vs. Ghsr-/- : 2496.48 ± 116.88 vs. 2183.04 ± 103.43 µm2 , P = 0.024). CONCLUSIONS: Growth hormone secretagogue receptor-1a mediates ghrelin's effects on attenuating LLC-induced weakness but not muscle mass loss by modulating the autophagy-lysosome pathway, mitophagy, and mitochondrial respiration.