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.

Zoledronic acid improves bone quality and muscle function in a high bone turnover state.

SUMMARY: Zoledronic acid (ZA) prevents muscle weakness in mice with bone metastases; however, its role in muscle weakness in non-tumor-associated metabolic bone diseases and as an effective treatment modality for the prevention of muscle weakness associated with bone disorders, is unknown. We demonstrate the role of ZA-treatment on bone and muscle using a mouse model of accelerated bone remodeling, which represents the clinical manifestation of non-tumor associated metabolic bone disease. ZA increased bone mass and strength and rescued osteocyte lacunocanalicular organization. Short-term ZA treatment increased muscle mass, whereas prolonged, preventive treatment improved muscle mass and function. In these mice, muscle fiber-type shifted from oxidative to glycolytic and ZA restored normal muscle fiber distribution. By blocking TGFß release from bone, ZA improved muscle function, promoted myoblast differentiation and stabilized Ryanodine Receptor-1 calcium channel. These data demonstrate the beneficial effects of ZA in maintaining bone health and preserving muscle mass and function in a model of metabolic bone disease. Context and significance: TGFß is a bone regulatory molecule which is stored in bone matrix, released during bone remodeling, and must be maintained at an optimal level for the good health of the bone. Excess TGFß causes several bone disorders and skeletal muscle weakness. Reducing excess TGFß release from bone using zoledronic acid in mice not only improved bone volume and strength but also increased muscle mass, and muscle function. Progressive muscle weakness coexists with bone disorders, decreasing quality of life and increasing morbidity and mortality. Currently, there is a critical need for treatments improving muscle mass and function in patients with debilitating weakness. Zoledronic acid's benefit extends beyond bone and could also be useful in treating muscle weakness associated with bone disorders.

Functional recovery and muscle atrophy in pre-clinical models of peripheral nerve transection and gap-grafting in mice: effects of 4-aminopyridine.

We recently demonstrated a repurposing beneficial effect of 4-aminopyridine (4-AP), a potassium channel blocker, on functional recovery and muscle atrophy after sciatic nerve crush injury in rodents. However, this effect of 4-AP is unknown in nerve transection, gap, and grafting models. To evaluate and compare the functional recovery, nerve morphology, and muscle atrophy, we used a novel stepwise nerve transection with gluing (STG), as well as 7-mm irreparable nerve gap (G-7/0) and 7-mm isografting in 5-mm gap (G-5/7) models in the absence and presence of 4-AP treatment. Following surgery, sciatic functional index was determined weekly to evaluate the direct in vivo global motor functional recovery. After 12 weeks, nerves were processed for whole-mount immunofluorescence imaging, and tibialis anterior muscles were harvested for wet weight and quantitative histomorphological analyses for muscle fiber cross-sectional area and minimal Feret's diameter. Average post-injury sciatic functional index values in STG and G-5/7 models were significantly greater than those in the G-7/0 model. 4-AP did not affect the sciatic functional index recovery in any model. Compared to STG, nerve imaging revealed more misdirected axons and distorted nerve architecture with isografting. While muscle weight, cross-sectional area, and minimal Feret's diameter were significantly smaller in G-7/0 model compared with STG and G-5/7, 4-AP treatment significantly increased right TA muscle mass, cross-sectional area, and minimal Feret's diameter in G-7/0 model. These findings demonstrate that functional recovery and muscle atrophy after peripheral nerve injury are directly related to the intervening nerve gap, and 4-AP exerts differential effects on functional recovery and muscle atrophy.

Bone-Muscle Crosstalk: Musculoskeletal Complications of Chemotherapy.

PURPOSE OF REVIEW: Chemotherapy drugs combat tumor cells and reduce metastasis. However, a significant side effect of some chemotherapy strategies is loss of skeletal muscle and bone. In cancer patients, maintenance of lean tissue is a positive prognostic indicator of outcomes and helps to minimize the toxicity associated with chemotherapy. Bone-muscle crosstalk plays an important role in the function of the musculoskeletal system and this review will focus on recent findings in preclinical and clinical studies that shed light on chemotherapy-induced bone-muscle crosstalk. RECENT FINDINGS: Chemotherapy-induced loss of bone and skeletal muscle are important clinical problems. Bone antiresorptive drugs prevent skeletal muscle weakness in preclinical models. Chemotherapy-induced loss of bone can cause muscle weakness through both changes in endocrine signaling and mechanical loading between muscle and bone. Chemotherapy-induced changes to bone-muscle crosstalk have implications for treatment strategies and patient quality of life. Recent findings have begun to determine the role of chemotherapy in bone-muscle crosstalk and this review summarizes the most relevant clinical and preclinical studies.

Electroporation of Plasmid DNA into Mouse Skeletal Muscle.

Transient gene expression modulation in murine skeletal muscle by plasmid electroporation is a useful tool for assessing normal and pathological physiology. Overexpression or knockdown of target genes enables investigators to manipulate individual molecular events and, thus, better understand the mechanisms that impact muscle mass, muscle metabolism, and contractility. In addition, electroporation of DNA plasmids that encode fluorescent tags allows investigators to measure changes in subcellular localization of proteins in skeletal muscle in vivo. A key functional assessment of skeletal muscle includes the measurement of muscle contractility. In this protocol, we demonstrate that whole muscle contractility studies are still possible after plasmid DNA injection, electroporation, and gene expression modulation. The goal of this instructional procedure is to demonstrate the step-by-step method of DNA plasmid electroporation into mouse skeletal muscle to facilitate uptake and expression in the myofibers of the tibialis anterior and extensor digitorum longus muscles, as well as to demonstrate that skeletal muscle contractility is not compromised by injection and electroporation.

Loss of REDD1 prevents chemotherapy-induced muscle atrophy and weakness in mice.

BACKGROUND: Chemotherapy is an essential treatment to combat solid tumours and mitigate metastasis. Chemotherapy causes side effects including muscle wasting and weakness. Regulated in Development and DNA Damage Response 1 (REDD1) is a stress-response protein that represses the mechanistic target of rapamycin (mTOR) in complex 1 (mTORC1), and its expression is increased in models of muscle wasting. The aim of this study was to determine if deletion of REDD1 is sufficient to attenuate chemotherapy-induced muscle wasting and weakness in mice. METHODS: C2C12 myotubes were treated with carboplatin, and changes in myotube diameter were measured. Protein synthesis was measured by puromycin incorporation, and REDD1 mRNA and protein expression were analysed in myotubes treated with carboplatin. Markers of mTORC1 signalling were measured by western blot. REDD1 global knockout mice and wild-type mice were treated with a single dose of carboplatin and euthanized 7 days later. Body weight, hindlimb muscle weights, forelimb grip strength, and extensor digitorum longus whole muscle contractility were measured in all groups. Thirty minutes prior to euthanasia, mice were injected with puromycin to measure puromycin incorporation in skeletal muscle. RESULTS: C2C12 myotube diameter was decreased at 24 (P = 0.0002) and 48 h (P < 0.0001) after carboplatin treatment. Puromycin incorporation was decreased in myotubes treated with carboplatin for 24 (P = 0.0068) and 48 h (P = 0.0008). REDD1 mRNA and protein expression were increased with carboplatin treatment (P = 0.0267 and P = 0.0015, respectively), and this was accompanied by decreased phosphorylation of Akt T308 (P < 0.0001) and S473 (P = 0.0006), p70S6K T389 (P = 0.0002), and 4E-binding protein 1 S65 (P = 0.0341), all markers of mTORC1 activity. REDD1 mRNA expression was increased in muscles from mice treated with carboplatin (P = 0.0295). Loss of REDD1 reduced carboplatin-induced body weight loss (P = 0.0013) and prevented muscle atrophy in mice. REDD1 deletion prevented carboplatin-induced decrease of protein synthesis (P = 0.7626) and prevented muscle weakness. CONCLUSIONS: Carboplatin caused loss of body weight, muscle atrophy, muscle weakness, and inhibition of protein synthesis. Loss of REDD1 attenuates muscle atrophy and weakness in mice treated with carboplatin. Our study illustrates the importance of REDD1 in the regulation of muscle mass with chemotherapy treatment and may be an attractive therapeutic target to combat 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.

Bone metastases induce metabolic changes and mitophagy in mice.

NEW FINDINGS: What is the central question of this study? Cachexia causes severe changes in skeletal muscle metabolism and function and is a key predictor of negative outcomes in cancer patients: what are the changes in whole animal energy metabolism and mitochondria in skeletal muscle? What is the main finding and its importance? There is decreased whole animal energy expenditure in mice with cachexia. They displayed highly dysmorphic mitochondria and mitophagy in skeletal muscle. ABSTRACT: Cachexia causes changes in skeletal muscle metabolism. Mice with MDA-MB-231 breast cancer bone metastases and cachexia have decreased whole animal energy metabolism and increased skeletal muscle mitophagy. We examined whole animal energy metabolism by indirect calorimetry in mice with MDA-MB-231 breast cancer bone metastases, and showed decreased energy expenditure. We also examined skeletal muscle mitochondria and found that mitochondria in mice with MDA-MB-231 bone metastases are highly dysmorphic and have altered protein markers of mitochondrial biogenesis and dynamics. In addition, LC3B protein was increased in mitochondria of skeletal muscle from cachectic mice, and colocalized with the mitochondrial protein Tom20. Our data demonstrate the importance of mitophagy in cachexia. Understanding these changes will help contribute to defining treatments for cancer cachexia.

Gene-Metabolite Network Linked to Inhibited Bioenergetics in Association With Spaceflight-Induced Loss of Male Mouse Quadriceps Muscle.

Prolonged residence of mice in spaceflight is a scientifically robust and ethically ratified model of muscle atrophy caused by continued unloading. Under the Rodent Research Program of the National Aeronautics and Space Administration (NASA), we assayed the large-scale mRNA and metabolomic perturbations in the quadriceps of C57BL/6j male mice that lived in spaceflight (FLT) or on the ground (control or CTR) for approximately 4 weeks. The wet weights of the quadriceps were significantly reduced in FLT mice. Next-generation sequencing and untargeted mass spectroscopic assays interrogated the gene-metabolite landscape of the quadriceps. A majority of top-ranked differentially suppressed genes in FLT encoded proteins from the myosin or troponin families, suggesting sarcomere alterations in space. Significantly enriched gene-metabolite networks were found linked to sarcomeric integrity, immune fitness, and oxidative stress response; all inhibited in space as per in silico prediction. A significant loss of mitochondrial DNA copy numbers in FLT mice underlined the energy deprivation associated with spaceflight-induced stress. This hypothesis was reinforced by the transcriptomic sequencing-metabolomics integrative analysis that showed inhibited networks related to protein, lipid, and carbohydrate metabolism, and adenosine triphosphate (ATP) synthesis and hydrolysis. Finally, we discovered important upstream regulators, which could be targeted for next-generation therapeutic intervention for chronic disuse of the musculoskeletal system. © 2020 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research.

Zoledronic Acid Improves Muscle Function in Healthy Mice Treated with Chemotherapy.

Carboplatin is a chemotherapy drug used to treat solid tumors but also causes bone loss and muscle atrophy and weakness. Bone loss contributes to muscle weakness through bone-muscle crosstalk, which is prevented with the bisphosphonate zoledronic acid (ZA). We treated mice with carboplatin in the presence or absence of ZA to assess the impact of bone resorption on muscle. Carboplatin caused loss of body weight, muscle mass, and bone mass, and also led to muscle weakness as early as 7 days after treatment. Mice treated with carboplatin and ZA lost body weight and muscle mass but did not lose bone mass. In addition, muscle function in mice treated with ZA was similar to control animals. We also used the anti-TGFß antibody (1D11) to prevent carboplatin-induced bone loss and showed similar results to ZA-treated mice. We found that atrogin-1 mRNA expression was increased in muscle from mice treated with carboplatin, which explained muscle atrophy. In mice treated with carboplatin for 1 or 3 days, we did not observe any bone or muscle loss, or muscle weakness. In addition, reduced caloric intake in the carboplatin treated mice did not cause loss of bone or muscle mass, or muscle weakness. Our results show that blocking carboplatin-induced bone resorption is sufficient to prevent skeletal muscle weakness and suggests another benefit to bone therapy beyond bone in patients receiving chemotherapy. © 2019 American Society for Bone and Mineral Research.

Chemotherapy-induced loss of bone and muscle mass in a mouse model of breast cancer bone metastases and cachexia.

BACKGROUND: Chemotherapy used to treat malignancy can lead to loss of skeletal muscle mass and reduced force production, and can reduce bone volume in mice. We have shown that bone-muscle crosstalk is a key nexus in skeletal muscle function and bone homeostasis in osteolytic breast cancer bone metastases. Because chemotherapy has significant negative side effects on bone mass, and because bone loss can drive skeletal muscle weakness, we have examined the effects of chemotherapy on the musculoskeletal system in mice with breast cancer bone metastases. METHODS AND RESULTS: Six-week-old Female athymic nude mice were inoculated with 105 MDA-MB231 human breast cancer cells into the left ventricle and bone metastases were confirmed by X-ray. Mice were injected with carboplatin at a dose of 60mg/kg once per week starting 4 days after tumor inoculation. Skeletal muscle was collected for biochemical analysis and extensor digitorum longus (EDL) whole muscle contractility was measured. The femur and tibia bone parameters were assessed by microCT and tumor burden in bone was determined by histology. Healthy mice treated with carboplatin lose whole body weight and have reduced individual muscle weights (gastrocnemius, tibialis anterior (TA), and EDL), reduced trabecular bone volume (BV/TV), and reduced EDL function. Mice with MDA-MB-231 bone metastases treated with carboplatin lose body weight, and have reduced EDL function as healthy mice treated with carboplatin. Mice with MDA-MB-231 bone metastases plus carboplatin do have reduced proximal tibia BV/TV compared to carboplatin alone, but carboplatin does reduce tumor burden in bone. CONCLUSIONS: Our data shows that carboplatin treatment, aimed at reducing tumor burden, contributes to cachexia and trabecular bone loss. The muscle atrophy and weakness may occur through bone-muscle crosstalk and would lead to a feed-forward cycle of musculoskeletal degradation. Despite anti-tumor effects of chemotherapy, musculoskeletal impairment is still significant in mice with bone metastases.

Chronic Treatment with Multi-Kinase Inhibitors Causes Differential Toxicities on Skeletal and Cardiac Muscles.

Despite recent progress, chemotherapy remains the preferred treatment for cancer. We have shown a link between anticancer drugs and the development of cachexia, i.e., body wasting accompanied by muscle loss. The multi-kinase inhibitors (MKIs) regorafenib and sorafenib, used as second-line treatment for solid tumors, are frequently accompanied by several side effects, including loss of muscle mass and strength. In the present study we aimed to investigate the molecular mechanisms associated with the occurrence of muscle toxicities in in vivo conditions. Hence, we treated 8-week old healthy CD2F1 male mice with MKIs for up to six weeks and observed decreased skeletal and cardiac muscle mass, consistent with muscle weakness. Modulation of ERK1/2 and GSK3ß, as well as increased expression of markers of autophagy, previously associated with muscle atrophy conditions, were shown in skeletal muscle upon treatment with either drug. MKIs also promoted cardiac abnormalities consistent with reduced left ventricular mass, internal diameter, posterior wall thickness and stroke volume, despite unchanged overall function. Notably, different signaling pathways were affected in the heart, including reduced expression of mitochondrial proteins, and elevated AKT, GSK3ß, mTOR, MEK1/2 and ERK1/2 phosphorylation. Combined, our data demonstrate detrimental effects on skeletal and cardiac muscle in association with chronic administration of MKIs, although different mechanisms would seem to contribute to the cachectic phenotype in the two tissues.

Cancer- and Chemotherapy-Induced Musculoskeletal Degradation.

Mobility in advanced cancer patients is a major health care concern and is often lost in advanced metastatic cancers. Erosion of mobility is a major component in determining quality of life but also starts a process of loss of muscle and bone mass that further devastates patients. In addition, treatment options become limited in these advanced cancer patients. Loss of bone and muscle occurs concomitantly. Advanced cancers that are metastatic to bone often lead to bone loss (osteolytic lesions) but may also lead to abnormal deposition of new bone (osteoblastic lesions). However, in both cases there is a disruption to normal bone remodeling and radiologic evidence of bone loss. Many antitumor therapies can also lead to loss of bone in cancer survivors. Bone loss releases cytokines (TGFß) stored in the mineralized matrix that can act on skeletal muscle and lead to weakness. Likewise, loss of skeletal muscle mass leads to reduced bone mass and quality via mechanical and endocrine signals. Collectively these interactions are termed bone-muscle cross-talk, which has garnered much attention recently as a prime target for musculoskeletal health. Pharmacological approaches as well as nutrition and exercise can improve muscle and bone but have fallen short in the context of advanced cancers and cachexia. This review highlights our current knowledge of these interventions and discusses the difficulties in treating severe musculoskeletal deficits with the emphasis on improving not only bone mass and muscle size but also functional outcomes. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

A "Connexin" Responsible for the Fatal Attraction of Cancer to Bone.

Tumor cell interactions with the bone microenvironment are vital for the establishment and progression of bone metastases. Recently in Cancer Cell, Wang et al. (2018) showed that cells of the osteoblast lineage are critical for the delivery of calcium to tumor cells through gap junctions, pointing toward potential therapies for bone metastases.

Bone-Induced Expression of Integrin ß3 Enables Targeted Nanotherapy of Breast Cancer Metastases.

Bone metastases occur in approximately 70% of metastatic breast cancer patients, often leading to skeletal injuries. Current treatments are mainly palliative and underscore the unmet clinical need for improved therapies. In this study, we provide preclinical evidence for an antimetastatic therapy based on targeting integrin ß3 (ß3), which is selectively induced on breast cancer cells in bone by the local bone microenvironment. In a preclinical model of breast cancer, ß3 was strongly expressed on bone metastatic cancer cells, but not primary mammary tumors or visceral metastases. In tumor tissue from breast cancer patients, ß3 was significantly elevated on bone metastases relative to primary tumors from the same patient (n = 42). Mechanistic investigations revealed that TGFß signaling through SMAD2/SMAD3 was necessary for breast cancer induction of ß3 within the bone. Using a micelle-based nanoparticle therapy that recognizes integrin αvß3 (αvß3-MPs of ∼12.5 nm), we demonstrated specific localization to breast cancer bone metastases in mice. Using this system for targeted delivery of the chemotherapeutic docetaxel, we showed that bone tumor burden could be reduced significantly with less bone destruction and less hepatotoxicity compared with equimolar doses of free docetaxel. Furthermore, mice treated with αvß3-MP-docetaxel exhibited a significant decrease in bone-residing tumor cell proliferation compared with free docetaxel. Taken together, our results offer preclinical proof of concept for a method to enhance delivery of chemotherapeutics to breast cancer cells within the bone by exploiting their selective expression of integrin αvß3 at that metastatic site. Cancer Res; 77(22); 6299-312. ©2017 AACR.

The Role of TGFß in Bone-Muscle Crosstalk.

PURPOSE OF REVIEW: The role of bone-derived factors in regulation of skeletal muscle function is an important emerging aspect of research into bone-muscle crosstalk. Implications for this area of research are far reaching and include understanding skeletal muscle weakness in cancer, osteoporosis, cachexia, rare diseases of bone, and aging. RECENT FINDINGS: Recent research shows that bone-derived factors can lead to changes in the skeletal muscle. These changes can either be anabolic or catabolic, and we focus this review on the role of TGFß in driving oxidative stress and skeletal muscle weakness in the setting of osteolytic cancer in the bone. The bone is a preferred site for breast cancer metastasis and leads to pathological bone loss. Osteolytic cancer in the bone leads to release of TGFß from the bone via osteoclast-mediated bone destruction. Our appreciation of crosstalk between the muscle and bone has recently expanded beyond mechanical force-driven events to encompass a variety of signaling factors originating in one tissue and communicating to the other. This review summarizes some previously known mediators of bone-to-muscle signaling and also recent work identifying a new role for bone-derived TGFß as a cause of skeletal muscle weakness in the setting of osteolytic cancer in the bone. Multiple points of potential therapeutic intervention are discussed.

Aromatase inhibitor-induced bone loss increases the progression of estrogen receptor-negative breast cancer in bone and exacerbates muscle weakness in vivo.

Aromatase inhibitors (AIs) cause muscle weakness, bone loss, and joint pain in up to half of cancer patients. Preclinical studies have demonstrated that increased osteoclastic bone resorption can impair muscle contractility and prime the bone microenvironment to accelerate metastatic growth. We hypothesized that AI-induced bone loss could increase breast cancer progression in bone and exacerbate muscle weakness associated with bone metastases. Female athymic nude mice underwent ovariectomy (OVX) or sham surgery and were treated with vehicle or AI (letrozole; Let). An OVX-Let group was then further treated with bisphosphonate (zoledronic acid; Zol). At week three, trabecular bone volume was measured and mice were inoculated with MDA-MB-231 cells into the cardiac ventricle and followed for progression of bone metastases. Five weeks after tumor cell inoculation, tumor-induced osteolytic lesion area was increased in OVX-Let mice and reduced in OVX-Let-Zol mice compared to sham-vehicle. Tumor burden in bone was increased in OVX-Let mice relative to sham-vehicle and OVX-Let-Zol mice. At the termination of the study, muscle-specific force of the extensor digitorum longus muscle was reduced in OVX-Let mice compared to sham-vehicle mice, however, the addition of Zol improved muscle function. In summary, AI treatment induced bone loss and skeletal muscle weakness, recapitulating effects observed in cancer patients. Prevention of AI-induced osteoclastic bone resorption using a bisphosphonate attenuated the development of breast cancer bone metastases and improved muscle function in mice. These findings highlight the bone microenvironment as a modulator of tumor growth locally and muscle function systemically.

Osteolytic Breast Cancer Causes Skeletal Muscle Weakness in an Immunocompetent Syngeneic Mouse Model.

Muscle weakness and cachexia are significant paraneoplastic syndromes of many advanced cancers. Osteolytic bone metastases are common in advanced breast cancer and are a major contributor to decreased survival, performance, and quality of life for patients. Pathologic fracture caused by osteolytic cancer in bone (OCIB) leads to a significant (32%) increased risk of death compared to patients without fracture. Since muscle weakness is linked to risk of falls which are a major cause of fracture, we have investigated skeletal muscle response to OCIB. Here, we show that a syngeneic mouse model of OCIB (4T1 mammary tumor cells) leads to cachexia and skeletal muscle weakness associated with oxidation of the ryanodine receptor and calcium (Ca2+) release channel (RyR1). Muscle atrophy follows known pathways via both myostatin signaling and expression of muscle-specific ubiquitin ligases, atrogin-1 and MuRF1. We have identified a mechanism for skeletal muscle weakness due to increased oxidative stress on RyR1 via NAPDH oxidases [NADPH oxidase 2 (Nox2) and NADPH oxidase 4 (Nox4)]. In addition, SMAD3 phosphorylation is higher in muscle from tumor-bearing mice, a critical step in the intracellular signaling pathway that transmits TGFß signaling to the nucleus. This is the first time that skeletal muscle weakness has been described in a syngeneic model of OCIB and represents a unique model system in which to study cachexia and changes in skeletal muscle.

Combination therapy in a xenograft model of glioblastoma: enhancement of the antitumor activity of temozolomide by an MDM2 antagonist.

OBJECTIVE Improvement in treatment outcome for patients with glioblastoma multiforme (GBM) requires a multifaceted approach due to dysregulation of numerous signaling pathways. The murine double minute 2 (MDM2) protein may fulfill this requirement because it is involved in the regulation of growth, survival, and invasion. The objective of this study was to investigate the impact of modulating MDM2 function in combination with front-line temozolomide (TMZ) therapy in GBM. METHODS The combination of TMZ with the MDM2 protein-protein interaction inhibitor nutlin3a was evaluated for effects on cell growth, p53 pathway activation, expression of DNA repair proteins, and invasive properties. In vivo efficacy was assessed in xenograft models of human GBM. RESULTS In combination, TMZ/nutlin3a was additive to synergistic in decreasing growth of wild-type p53 GBM cells. Pharmacodynamic studies demonstrated that inhibition of cell growth following exposure to TMZ/nutlin3a correlated with: 1) activation of the p53 pathway, 2) downregulation of DNA repair proteins, 3) persistence of DNA damage, and 4) decreased invasion. Pharmacokinetic studies indicated that nutlin3a was detected in human intracranial tumor xenografts. To assess therapeutic potential, efficacy studies were conducted in a xenograft model of intracranial GBM by using GBM cells derived from a recurrent wild-type p53 GBM that is highly TMZ resistant (GBM10). Three 5-day cycles of TMZ/nutlin3a resulted in a significant increase in the survival of mice with GBM10 intracranial tumors compared with single-agent therapy. CONCLUSIONS Modulation of MDM2/p53-associated signaling pathways is a novel approach for decreasing TMZ resistance in GBM. To the authors' knowledge, this is the first study in a humanized intracranial patient-derived xenograft model to demonstrate the efficacy of combining front-line TMZ therapy and an inhibitor of MDM2 protein-protein interactions.

Chemotherapy-related cachexia is associated with mitochondrial depletion and the activation of ERK1/2 and p38 MAPKs.

Cachexia affects the majority of cancer patients, with currently no effective treatments. Cachexia is defined by increased fatigue and loss of muscle function resulting from muscle and fat depletion. Previous studies suggest that chemotherapy may contribute to cachexia, although the causes responsible for this association are not clear. The purpose of this study was to investigate the mechanism(s) associated with chemotherapy-related effects on body composition and muscle function. Normal mice were administered chemotherapy regimens used for the treatment of colorectal cancer, such as Folfox (5-FU, leucovorin, oxaliplatin) or Folfiri (5-FU, leucovorin, irinotecan) for 5 weeks. The animals that received chemotherapy exhibited concurrent loss of muscle mass and muscle weakness. Consistently with previous findings, muscle wasting was associated with up-regulation of ERK1/2 and p38 MAPKs. No changes in ubiquitin-dependent proteolysis or in the expression of TGFß-family members were detected. Further, marked decreases in mitochondrial content, associated with abnormalities at the sarcomeric level and with increase in the number of glycolytic fibers were observed in the muscle of mice receiving chemotherapy. Finally, ACVR2B/Fc or PD98059 prevented Folfiri-associated ERK1/2 activation and myofiber atrophy in C2C12 cultures. Our findings demonstrate that chemotherapy promotes MAPK-dependent muscle atrophy as well as mitochondrial depletion and alterations of the sarcomeric units. Therefore, these findings suggest that chemotherapy potentially plays a causative role in the occurrence of muscle loss and weakness. Moreover, the present observations provide a strong rationale for testing ACVR2B/Fc or MEK1 inhibitors in combination with anticancer drugs as novel strategies aimed at preventing chemotherapy-associated muscle atrophy.