Regulation of skeletal-muscle–protein turnover in cancer-associated cachexia

CACHEXIA-ANOREXIA WORKSHOP

Regulation of Skeletal-Muscle–Protein Turnover in Cancer-Associated Cachexia Vickie E. Baracos, PhD From the Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada Cancer is frequently associated with anorexia, weight loss, negative nitrogen balance, and skeletal-muscle wasting. Depletion of skeletal-muscle mass is critical to overall survival of the patient, can prolong rehabilitation to normal function after recovery, and decreases quality of life in a palliative-care setting. The biochemical and physiologic bases of cancer-associated muscle wasting have been most fully investigated in animal models. These studies provide evidence for suppressed protein synthesis and activated proteolysis in cancer-associated muscle wasting and indicate a need for both anabolic and anticatabolic therapies. Several humoral factors of host or tumor origin are implicated in altered muscle-protein metabolism, including cytokines, metabolites of arachidonic acid, and a proteolysisinducing glycoprotein; their interrelationships are less well characterized. Several catabolic mediators may share common downstream mechanisms because they ultimately activate the ATP-, ubiquitin-, and proteasome-dependent intracellular proteolytic system. Although important gaps in our current understanding remain, data available from animal studies can be used as a basis to develop relevant studies in human subjects. Nutrition 2000;16:1015–1018. ©Elsevier Science Inc. 2000 Key words: protein metabolism, anabolism, catabolism, proteolysis cachexia

BIOCHEMICAL CHARACTERIZATION OF MUSCLE ATROPHY IN TUMOR-BEARING ANIMALS Roles of Protein Synthesis and Degradation Muscle wasting may be due to increased protein catabolism (hypercatabolism) or decreased protein synthesis (hypoanabolism); the simultaneous presence of both results in the most intense muscular atrophy. Hypoanabolism implies a failure of the normal stimuli for muscle-protein synthesis and/or in the supply of amino acids and energy for net protein deposition to occur. Hypercatabolism, in contrast, implies participation of a different subset of mediators (see below) and activation of specific intracellular proteases. Hypoanabolism would be treated with an anabolic agent plus appropriate nutritional support, whereas antagonists of catabolic mediators and/or intracellular proteolysis would be used to treat hypercatabolism. Because the mechanisms and control of protein synthesis and degradation are functionally distinct, it follows that hypoanabolism and hypercatabolism must be treated differently and, if simultaneously present, must be treated separately. An anabolic therapy alone would obviously have limited efficacy in the face of uncontrolled protein catabolism. The respective roles of protein synthesis and degradation in cancer-associated cachexia clearly differ from tumor to tumor in animal models. Simple hypoanabolism has been documented,1 as has hypoanabolism and hypercatabolism.1,2 The available information is presently limited to untreated tumors, and specific effects

This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada. Correspondence to: Vickie E. Baracos, PhD, Department of Agricultural, Food and Nutritional Science, 410 Agriculture/Forestry Centre, University of Alberta, Edmonton T6G 2P5, AB, Canada. E-mail: vickie.baracos@ ualbert.ca Date accepted: May 18, 2000. Nutrition 16:1015–1018, 2000 ©Elsevier Science Inc., 2000. Printed in the United States. All rights reserved.

of radiotherapy or chemotherapy on muscle-protein turnover remain uncharacterized. Although drugs commonly used in chemotherapy are known to cause poor or negative nitrogen balance,3 it is unknown whether and at what level these treatments might influence the regulation of muscle-protein turnover. This important gap in our understanding may account for failure of some therapeutic approaches. Little information is available on muscleprotein turnover in cancer patients,4 but at least 40 prospective, randomized clinical trials have been reported, evaluating the efficacy of supplemental nutritional support in cancer patients receiving chemotherapy or radiation.5 The disappointing results of supplemental nutritional support in treated cancer patients point to the potential presence of a block in protein anabolism. In the majority of these patients, it was not even known whether a state of hypoanabolism existed, and nutritional support would not neccessarily be expected to ameliorate a state of hypercatabolism. In any case, it was unknown whether protein synthesis retained the capacity to respond to nutrient supply, and anabolic therapy to promote utilization of nutritional support was not provided.

Identification of Mediators and Their Role in Abnormal Protein Turnover The mediators of the observed changes in protein turnover in tumor-bearing animals are not fully defined. Although anorexia is often associated with cancer, cachexia is distinct from simple starvation and more like the condition produced by major injury or sepsis.6 Through the use of pair-fed controls,1 it has been shown that anorexia does not fully explain wasting of muscle protein and elevated protein breakdown. In some animal models, cachexia develops in the complete absence of anorexia.2 It seems likely that cancer cachexia is provoked by humoral mediators originating from the host and/or the tumor.7 A list of factors implicated in the regulation of muscle-protein synthesis and degradation is shown in Table I. Although some features of cachexia may be induced by hormone infusion, no single combination of hormones can induce all of its manifestations.7 In contrast, many of the perturbations 0899-9007/00/$20.00 PII S0899-9007(00)00407-X

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Nutrition Volume 16, Number 10, 2000 TABLE I. FACTORS IMPLICATED IN REGULATION OF MUSCLE-PROTEIN TURNOVER

Factor Hormones Insulin Insulin-like growth factor-I Growth hormone Glucagon Glucocorticoids ␤-Adrenergic agonists Adrenaline Thyroid hormones (normal) Thyroid (excess) Substrates/metabolites Glucose Ketone bodies Glutamine Arginine Branched-chain amino acids Activity Contractile work Disuse (inactivity) Passive stretch Proinflammatory mediators Prostaglandin E2 Prostaglandin F2␣ Interleukin-1␤ Interleukin-6 Tumor necrosis factor-␣ Interferon-␥ Ciliary neurotrophic factor Proteolysis-inducing glycoprotein

Protein synthesis

Proteolysis

Overall promotes

Increase Increase Increase Decrease No effect Increase ? Increase Small increase

Decrease Decrease No effect No effect Increase Decrease Increase Small increase Large increase

Protein deposition Protein deposition Protein deposition Atrophy Atrophy Protein deposition Atrophy Protein deposition Atrophy

No effect No effect Increase Increase Increase

Decrease Decrease Decrease Decrease Decrease

Protein Protein Protein Protein Protein

Increase Decrease Increase

Decrease Increase No effect

Protein deposition Atrophy Protein deposition

No effect Increase Decrease Decrease Decrease ? ? Decrease

Increase No effect Increase Increase Increase Increase Increase Increase

Atrophy Protein deposition Atrophy Atrophy Atrophy Atrophy Atrophy Atrophy

associated with cancer cachexia can be reproduced by the administration of pro-inflammatory cytokines interleukin-1␤ (IL-1␤), interleukin-6 (IL-6), tumor necrosis factor-␣ (TNF-␣), and interferon-␥ (IFN-␥).8 Cytokines infused or injected into animals decrease skeletal-muscle–protein mass through increased rates of protein degradation and decreased protein synthesis and aminoacid uptake.8 The family of known “cachectins” is still growing. Ciliary neurotrophic factor (CNTF) is a 23-kDa protein expressed by cells of the central and peripheral nervous systems which shows structural and functional similarities to IL-6. In rats, implantation of CNTF-secreting cells resulted in a fatal syndrome of cachexia, with anorexia and loss of skeletal muscle.9 The role of CNTF in cancer-associated muscle wasting is unknown. A proteolysisinducing glycoprotein of tumor origin has been described.2,10,11 This factor is clearly not a cytokine, and it is the first factor of tumor origin thought to participate in regulation of muscle-protein turnover. Cytokines exert their biologic effects through binding to specific membrane receptors. Cytokine receptors and receptormediated mechanisms leading to intracellular protein catabolism have not been well studied. Skeletal muscle has recently been shown to express mRNA-encoding receptors for IL-1, IL-6, IFN-␥, and TNF-␣.12 The stimulation of muscle with cytokines induces expression of these receptors, providing a mechanism for amplification of cytokine responses at the muscle level. The role of modulation of muscle sensitivity to cytokines during the evolution of cancer cachexia is unknown. Systematic modulation of the production or action of factors known to regulate muscle-protein turnover is required to identify causal factors in muscle wasting. This experimental approach has been used by multiple investigators in a variety of animal models

deposition deposition deposition deposition deposition

(Table II) to obtain evidence for a causal role for cytokines. Tumor-bearing animals passively immunized with antibodies against individual cytokines and cytokine or cytokine receptorgene– knockout approaches13,14 have been used. Rats bearing the Yoshida ascites hepatoma showed enhanced rates of muscleprotein degradation, which were inhibited by administration of an anti-TNF immunoglobulin G15 but not by IL-1 receptor antagonist.16 A role for TNF is also supported by a study showing that pentoxifylline prevented muscle atrophy and increased proteolysis

TABLE II. MODULATION OF FACTORS REGULATING MUSCLE-PROTEIN CATABOLISM IN TUMOR-BEARING ANIMALS

Tumor model Yoshida ascites hepatoma, AH130 Lewis lung carcinoma Yoshida sarcoma Colon 26 adenocarcinoma Methylcholanthrene-induced sarcoma MAC 16 adenocarcinoma

Factors implicated in muscle catabolism/wasting TNF-␣, PGE2, Not IL-1 TNF-␣ TNF-␣ IL-6, Not TNF-␣ IFN-␥, Not TNF-␣ Proteolysis-inducing glycoprotein, Not TNF-␣

IL-1, interleukin-1; IL-6, interleukin-6; IFN-␥, interferon-␥; PGE2, Prostaglandin E2; TNF-␣, tumor necrosis factor-␣.

Nutrition Volume 16, Number 10, 2000 in muscles of Yoshida-sarcoma– bearing rats.17 Llovera et al. implanted Lewis lung carcinoma into both wild-type and genedeficient mice for the TNF-␣ receptor type I and showed that gene-deficient mice had less muscle wasting and activation of protein catabolism in response to the tumor.14 In contrast to the foregoing studies, an anti-TNF antibody did not modify cachexia in cachectic mice implanted with a cell line derived from murine colon-26 adenocarcinoma and several studies have provided evidence for involvement of IL-6 in this model.18 Langstein et al.19 implanted rats with a transplantable methylcholanthrene-induced sarcoma. In these animals, anorexia and body-weight loss were reduced in animals injected with anti-IFN-␥ but not with anti-TNF. Finally, muscle catabolism in mice bearing the MAC 16 adenocarcinoma was independent of TNF and was caused by a proteolysis-inducing glycoprotein.11 Available studies have generated a complex picture, suggesting that cytokines contributing to muscle wasting in tumor-bearing animals may differ with tumor type and that in some tumors cytokines may not be involved. Because one cytokine may act by provoking the synthesis of another, further work is needed to clarify the primary role of cytokines implicated in the work done to date. The relationship between cytokines and mediators such as the proteolysis-inducing glycoprotein is unknown. It may be that tumor-associated catabolism in humans falls into distinct subtypes, as seen in animal models. The two most highly characterized experimental models apparently differ entirely in the causal mediators. In the Yoshida ascites hepatoma AH130, muscle-protein catabolism is attributable to a cytokine, TNF-␣, and a metabolite of arachidonic acid,1,15 the proteolysis-inducing glycoprotein, is absent from this model (M.J. Tisdale, personal communication). In the MAC 16 adenocarcinoma, increased muscle-protein catabolism is caused by the proteolysis-inducing glycoprotein and does not involve TNF-␣.2,11 Although there may be common elements to all forms of cancer-associated muscle wasting, if several different catabolic mediators can independently induce muscle atrophy, then successful treatment aimed at these mediators will depend on the ability to identify the different catabolic phenotypes in patients. In addition to antagonism of the production or action of cytokines, diverse treatments have been applied to the problem of muscle wasting in experimental animal models. Anabolic treatments have included growth hormone and insulin,20 glutamine or ornithine ␣-ketoglutarate,21 ␩-acetyl cysteine,22 anabolic steroids,23 ␤-adrenergic agonists,24,25 progestational agents,26 and exercise.27 Non-steroidal antiinflammatory drugs and nutritional treatment with dietary ␻-3 fatty acids1,2,11,26,28 have also been used to suppress activated proteolysis through modulation of the production of metabolites of arachidonic acid. The application of these types of treatment to different patients will depend on an ability to determine in which categories of patients they are likely to be effective.

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TABLE III. PROTEASES IMPLICATED IN CANCER-ASSOCIATED MUSCLE ATROPHY

Tumor model Yoshida ascites hepatoma AH130 Colon 26 adenocarcinoma MAC 16 adenocarcinoma Yoshida sarcoma Lewis lung carcinoma Leukemia L1210

Factors implicated in muscle catabolism/wasting ATP and ubiquitin dependent Lysosomal ATP and ubiquitin dependent Lysosomal ATP and ubiquitin dependent Lysosomal ATP and ubiquitin dependent ATP and ubiquitin dependent Lysosomal (ATP and ubiquitin dependence not determined)

ATP. A major pathway for protein breakdown in most cells is the soluble, ATP-dependent system, which involves the cofactor ubiquitin and the proteasome complex.30 Recent data indicate that this process may be the primary mechanism by which breakdown of muscle proteins, including myofibrillar components, increases in various catabolic processes, including denervation atrophy and fasting, glucocorticoid treatment, metabolic acidosis, trauma, and infection. The role of proteolytic systems in muscle has been explored in animal models of cachexia (reviewed by Attaix et al.30) (Table III). Experimental approaches have mainly included suppression of proteolysis with specific inhibitors in incubated muscles and expression of mRNA encoding different proteases and other elements of proteolytic systems such as ubiquitin.14,18,29,30 Data from animals bearing the Yoshida ascites hepatoma suggest that accelerated muscle proteolysis and muscle wasting in tumor-bearing rats result primarily from activation of the ATP-dependent pathway involving ubiquitin and the proteasome29; a relatively small but consistent lysosomal component was also observed. Although to date similar observations have not yet been made in muscles of cancer patients, related work in other animal models also suggest a key role for the ATP- and ubiquitin-dependent and lysosomal pathways.14,17,18,30,31 Although the activation of proteolysis in these models is clearly attributable to different catabolic stimuli, it appears that these converge at some level to induce the same proteolytic systems. This convergence may provide a key to intervention at the level of muscle-protein degradation.32

Intracellular Proteolysis in Cancer-Associated Cachexia

VERIFICATION IN HUMAN STUDIES AND APPLICATION OF KNOWLEDGE TO TREATMENT DEVELOPMENT

Like other cells, skeletal muscle contains several distinct proteolytic systems, and approaches for estimating their relative activities in incubated rat muscles have been described.29,30 Muscle cells contain a variety of lysosomal proteases, and this organelle is responsible for degradation of membrane protein and certain soluble proteins in normal muscle. Experiments with inhibitors of lysosomal acidification or of lysosomal proteases have shown that a lack of insulin and amino acids leads to an activation of this process. Skeletal muscle contains three Ca2⫹-activated proteases, calpains I and II, and p94, whose in vivo function in normal cells remains unclear, but they appear to promote overall degradation in various pathologic conditions or with in vitro treatments that raise cytosolic Ca2⫹ levels. The bulk of proteolysis in normal muscle and degradation of the contractile proteins appear to occur through a non-lysosomal process that is independent of Ca2⫹ and requires

More research on animal models is needed to develop a comprehensive understanding of the biochemical mechanisms of cancerassociated muscle wasting, but human studies are also warranted at this time. Little information is available regarding the roles of abnormal muscle protein synthesis and degradation and activities of muscle proteases in cancer patients experiencing negative nitrogen balance. Methods for estimation of these variables in human subjects are available, although these methods are somewhat more challenging to carry out than in animals.4,33 These determinations may be made on different cancer patients to determine the main features of abnormal protein metabolism over the course of the disease and therapy. Although the possibilities for mechanistic studies in human subjects are limited, it would be possible to evaluate the profile of putative anabolic and catabolic mediators in cancer patients to

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determine whether there is any parallel with the well-characterized animal models. It would then be possible to test the responsiveness of protein synthesis and degradation to specific interventions such as 1) contractile activity and stretch as anabolic stimuli, 2) pharmacologic stimulation of protein synthesis with factors such as insulin-like growth factor-1, progestational agents and anabolic steroids, 3) nutritional stimulation of protein synthesis by total nutrient mixtures and by specific anabolic amino acids such as glutamine, 4) nutritional (dietary fish oil or eicosapentaenoic acid) and pharmacologic (non-steroidal antiinflammatory agents) treatments to suppress protein catabolism through modulation of arachidonic acid metabolism, and 5) anti-cytokine therapies.

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