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Autophagic Degradation Contributes to Muscle Wasting in Cancer Cachexia

Open ArchivePublished:February 11, 2013DOI:https://doi.org/10.1016/j.ajpath.2012.12.023
      Muscle protein wasting in cancer cachexia is a critical problem. The underlying mechanisms are still unclear, although the ubiquitin-proteasome system has been involved in the degradation of bulk myofibrillar proteins. The present work has been aimed to investigate whether autophagic degradation also plays a role in the onset of muscle depletion in cancer-bearing animals and in glucocorticoid-induced atrophy and sarcopenia of aging. The results show that autophagy is induced in muscle in three different models of cancer cachexia and in glucocorticoid-treated mice. In contrast, autophagic degradation in the muscle of sarcopenic animals is impaired but can be reactivated by calorie restriction. These results further demonstrate that different mechanisms are involved in pathologic muscle wasting and that autophagy, either excessive or defective, contributes to the complicated network that leads to muscle atrophy. In this regard, particularly intriguing is the observation that in cancer hosts and tumor necrosis factor α–treated C2C12 myotubes, insulin can only partially blunt autophagy induction. This finding suggests that autophagy is triggered through mechanisms that cannot be circumvented by using classic upstream modulators, prompting us to identify more effective approaches to target this proteolytic system.
      Cancer cachexia is a multifactorial syndrome characterized by anorexia, weight loss, and muscle wasting that significantly impairs patients’ quality of life and survival and reduces their tolerance to antineoplastic drug treatments. However, despite the relevance of cachexia to patient outcome, effective cachexia treatments are still lacking, and only recently has tumor-induced wasting been considered as a prognostic tool.
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      The views and practice of oncologists towards nutritional support in patients receiving chemotherapy.
      The mechanisms underlying muscle wasting in cancer cachexia are still unclear, although experimental and clinical studies have shown that hyperactivation of the ubiquitin-proteasome–dependent proteolytic system plays a critical role.
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      Muscle protein waste in tumor-bearing rats is effectively antagonized by a beta 2-adrenergic agonist (clenbuterol): role of the ATP-ubiquitin-dependent proteolytic pathway.
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      Increased muscle ubiquitin mRNA levels in gastric cancer patients.
      However, in the past few years, the involvement of autophagic-lysosomal proteolysis has also been proposed. The lysosomal degradative system contributes to different cellular processes, such as degradation of cytoplasmic proteins and organelles, limited cleavage or extensive degradation of various substrates, trafficking and recycling of molecules among internal organelles to and from the exterior of the cell, posttranslational maturation of secretory products, and storage of undigested material.
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      Delivery of proteins and organelles to the vacuole from the cytoplasm.
      Autophagy is the major process for degradation of cellular constituents, its rate being enhanced under stress conditions leading to organelle damage or under marked nutrient restriction (starvation), to recycle biomolecules for the synthesis of essential constituents.
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      Whole-body protein turnover in humans: past, present, and future.
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      and quite recently autophagy has been shown to contribute to starvation-induced muscle atrophy with a mechanism involving tumor necrosis factor α (TNF-α) receptor–associated factor (TRAF)6/Fn14 signaling.
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      Altered lysosomal function has also been reported in several myopathies,
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      Lysosomal proteolysis in skeletal muscle.
      and, more recently, autophagy-related genes have been shown to be induced in muscle by denervation or fasting through a FoxO3-dependent mechanism.
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      FoxO3 coordinately activates protein degradation by the autophagic/lysosomal and proteasomal pathways in atrophying muscle cells.
      FoxO3 has been proposed to regulate autophagy and proteasome-dependent proteolysis.
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      FoxO3 coordinately activates protein degradation by the autophagic/lysosomal and proteasomal pathways in atrophying muscle cells.
      However, a sort of hierarchy seems to exist between these two processes because a parallel study shows that autophagic degradation induced by starvation or FoxO3 overexpression is sufficient to determine muscle depletion, even if the ubiquitin-proteasome degradation is blocked using pharmacologic or genetic approaches.
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      FoxO3 controls autophagy in skeletal muscle in vivo.
      As for cancer cachexia, results obtained on muscles isolated from cachectic animals led us to rule out a substantial role for lysosomes in overall protein degradation.
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      Activation of the ATP-ubiquitin-proteasome pathway in skeletal muscle of cachectic rats bearing a hepatoma.
      On the other hand, an elevation of total lysosomal protease activity has been observed in the skeletal muscle and liver of tumor-bearing rats,
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      Cancer cachexia, malnutrition, and tissue protein turnover in experimental animals.
      and muscle depletion could be prevented by the treatment of tumor hosts with leupeptin, an inhibitor of cysteine proteases such as cathepsins B, H, and L.
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      • Baccino F.M.
      Pharmacological interference with tissue hypercatabolism in tumour-bearing rats.
      Finally, increased levels of cathepsin L mRNA have been reported in the skeletal muscle of septic or tumor-bearing rats, whereas cathepsin B gene expression has been shown to be enhanced in muscle biopsy samples obtained from patients with lung cancer.
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      Identification of cathepsin L as a differentially expressed message associated with skeletal muscle wasting.
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      • Goodship T.H.
      Skeletal muscle mRNA levels for cathepsin B, but not components of the ubiquitin-proteasome pathway, are increased in patients with lung cancer referred for thoracotomy.
      The present work has been aimed to investigate the relevance of autophagy to the pathogenesis of skeletal muscle wasting in cancer cachexia. Recently, few and scanty observations suggested that autophagy can be activated in the muscle of animals bearing Lewis lung carcinoma (LLC) or colon 26 (C26) tumor.
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      Targeted ablation of TRAF6 inhibits skeletal muscle wasting in mice.
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      Evidence for the contribution of insulin resistance to the development of cachexia in tumor-bearing mice.
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      Myostatin is a novel tumoral factor that induces cancer cachexia.
      • Cosper P.F.
      • Leinwand L.A.
      Cancer causes cardiac atrophy and autophagy in a sexually dimorphic manner.
      However, a comprehensive analysis of activation of the autophagic-lysosomal proteolytic pathway, including evaluation of autophagic flux, in cancer hosts is still lacking. The present study fills this gap. We compared cancer-induced muscle wasting in terms of activation of autophagy and that occurring in other experimental conditions, such as glucocorticoid-induced atrophy and aging. Previous data reported that autophagy significantly contributes to muscle wasting in different experimental conditions, such as denervation, starvation, and sepsis.
      • Zhao J.
      • Brault J.J.
      • Schild A.
      • Cao P.
      • Sandri M.
      • Schiaffino S.
      • Lecker S.H.
      • Goldberg A.L.
      FoxO3 coordinately activates protein degradation by the autophagic/lysosomal and proteasomal pathways in atrophying muscle cells.
      • Doyle A.
      • Zhang G.
      • Abdel Fattah E.A.
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      • Li Y.P.
      Toll-like receptor 4 mediates lipopolysaccharide-induced muscle catabolism via coordinate activation of ubiquitin-proteasome and autophagy-lysosome pathways.
      However, although some of the mechanisms underlying glucocorticoid-induced atrophy have already been described,
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      Glucocorticoids and muscle catabolism.
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      Insulin-like growth factor-I gene transfer by electroporation prevents skeletal muscle atrophy in glucocorticoid-treated rats.
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      Mechanisms of muscle atrophy induced by glucocorticoids.
      only two reports have provided little indirect evidence about the involvement of autophagy.
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      Branched-chain amino acids protect against dexamethasone-induced soleus muscle atrophy in rats.
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      Role of IGF-I and the TNFalpha/NF-kappaB pathway in the induction of muscle atrogenes by acute inflammation.
      The present study extensively investigates the effects of glucocorticoid administration on the activation of muscle autophagy, clarifying this point. As for aging, only a couple of reports
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      • Rasmussen B.B.
      Skeletal muscle autophagy and protein breakdown following resistance exercise are similar in younger and older adults.
      have analyzed the modulations of muscle autophagy. The present study extends such findings, providing a panel of autophagy markers in the aged muscle at protein levels, and compares their changes with those occurring in aged animals exposed to 40% calorie restriction (CR).
      The results show that autophagic-lysosomal degradation is induced in three different models of cancer-associated muscle atrophy and in glucocorticoid-treated animals, suggesting that this proteolytic system as a possible target for therapeutic strategies.

      Materials and Methods

      Reagents

      All the materials were supplied by Sigma-Aldrich (St. Louis, MO), unless differently specified.

      Animals and Experimental Design

      Experimental animals were cared for in compliance with the Italian Ministry of Health Guidelines and the Policy on Humane Care and Use of Laboratory Animals (NIH, 1996). The experimental protocol was approved by the Bioethical Committee of the University of Torino (Torino, Italy). Male Wistar rats weighing approximately 150 g and Balb-c and C57/BL6 mice weighing approximately 20 g were obtained from Charles River Laboratories (Wilmington, MA) and were maintained on a regular dark-light cycle (light from 8 AM to 8 PM), with free access to food and water during the whole experimental period.
      Tumor-bearing rats (n = 8) each received approximately 108 cells of i.p. inoculum of Yoshida AH-130 ascites hepatoma cells [a gift many years ago from Prof. Ugo Del Monte (University of Milano, Milano, Italy) and maintained in our laboratory by weekly i.p. transplantation], whereas tumor-bearing mice (n = 8) were inoculated s.c. dorsally with 5 × 105 C26 carcinoma cells [a gift from Prof. Mario P. Colombo (Istituto di Ricovero e Cura a Carattere Scientifico National Cancer Institute, Milano, Italy)] or i.m. with 5 × 105 LLC cells (ATCC, Manassas, VA). C26 and LLC cells were expanded to 50,000 cm2 in vitro [Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL of penicillin, 100 μg/mL of streptomycin, 100 μg/mL of sodium pyruvate, and 2 mmol/L l-glutamine maintained at 37°C in a humidified atmosphere of 5% CO2 in air], detached with trypsin, resuspended in sterile saline, and subsequently implanted in the animals at the concentrations indicated previously herein. Rats or mice inoculated with vehicle (saline) served as controls (n = 6). In another set of experiments, animals were randomized and divided into four groups: controls (n = 6) and C26 (n = 8), treated or not with insulin (INS). INS groups received daily 1.5 IU/kg body weight i.p. injections of INS during the whole experimental period. For autophagic flux experiments, control (n = 6) and C26 (n = 6) mice were administered either 0.4 mg/kg/d of colchicine or vehicle for 2 days before sacrifice (days 11 and 14 of tumor growth). Dexamethasone (Dexa) was administered i.p. daily for 7 days to 6-week-old Balb-c mice (n = 8) at a dose of 1 mg/kg. Vehicle (saline containing 5% ethanol)-treated mice served as controls (n = 6).
      Animal weight and food intake were recorded daily. Tumor-bearing rats and mice were sacrificed under anesthesia 7 and 14 days after tumor transplantation, respectively. Several muscles and organs were rapidly excised, weighed, frozen in isopentane cooled with liquid nitrogen, and stored at −80°C. As for CR, 6-month-old rats were fed ad libitum or were given 60% (w/w; 40% CR) of the food consumed by ad libitum animals until 24 months of age. For in vivo TNF administration, C57/BL6 mice weighing approximately 20 g received 40 μg/kg body weight i.m. injection of TNF as previously reported
      • Langen R.C.
      • Van Der Velden J.L.
      • Schols A.M.
      • Kelders M.C.
      • Wouters E.F.
      • Janssen-Heininger Y.M.
      Tumor necrosis factor-alpha inhibits myogenic differentiation through MyoD protein destabilization.
      • Langen R.C.
      • Schols A.M.
      • Kelders M.C.
      • van der Velden J.L.
      • Wouters E.F.
      • Janssen-Heininger Y.M.
      Muscle wasting and impaired muscle regeneration in a murine model of chronic pulmonary inflammation.
      in the right gastrocnemius and were sacrificed after 12 hours. Saline-treated mice, in place of using the contralateral muscle, served as controls to exclude TNF-dependent systemic effects.

      INS Tolerance Test

      Control, C26, and C26 INS-treated mice on day 11 of tumor growth were challenged with 1.5 IU/kg body weight i.p. INS. A blood drop was collected by saphenous vein puncture before and 15 and 45 minutes after INS administration, and glycemia was measured using the Glucocard G sensor strips and apparatus (A. Menarini Diagnostics, Firenze, Italy). Glycemia was also assessed before sacrifice by collecting the blood by cardiac puncture from anesthetized animals to assess the relationship between glycemia and body weight changes. Pearson’s correlation was calculated.

      Cell Cultures

      Murine C2C12 skeletal myoblasts (ATCC) were grown in high-glucose DMEM supplemented with 10% fetal bovine serum, 100 U/mL of penicillin, 100 μg/mL of streptomycin, 100 μg/mL of sodium pyruvate, and 2 mmol/L l-glutamine and maintained at 37°C in a humidified atmosphere of 5% CO2 in air. Differentiation was induced by shifting subconfluent cultures to DMEM supplemented with 2% horse serum (differentiation medium). At the same time, the cells were exposed to 100 ng/mL of TNF-α, 10 μg/mL of INS, or both and were collected after 48 hours; untreated cells served as controls. Such a differentiation scheme was chosen after preliminary screening of TNF-dependent autophagy induction in growing myoblasts, differentiating (2 days) or fully differentiated (6 days) myotubes. The differentiating cells showed the strongest autophagy alterations (data not shown) and were adopted for the following experiments. In a different set of experiments, TNF-α action was also tested at a lower dose (5 ng/mL) in fully differentiated myotubes. For autophagic flux measurements, 200 nmol/L bafilomycin A1 was added 6 hours before termination of the experiments, as previously reported.
      • Klionsky D.J.
      • Abeliovich H.
      • Agostinis P.
      • Agrawal D.K.
      • Aliev G.
      • Askew D.S.
      • et al.
      Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes.
      In other experiments, at the time of shifting subconfluent cultures to differentiation medium, the cells were exposed to 50 ng/mL of stable insulin-like growth factor 1 (IGF-1) synthetic peptide (LONG R
      • Bossola M.
      • Muscaritoli M.
      • Costelli P.
      • Bellantone R.
      • Pacelli F.
      • Busquets S.
      • Argiles J.
      • Lopez-Soriano F.J.
      • Civello I.M.
      • Baccino F.M.
      • Rossi Fanelli F.
      • Doglietto G.B.
      Increased muscle ubiquitin mRNA levels in gastric cancer patients.
      IGF-I, 85580C) or to neutralizing anti–IGF-1 antibody (I8773) in the presence or absence of 100 ng/mL of TNF-α and were collected after 48 hours; untreated cells served as controls.

      Primary Satellite Cells

      Single muscle fibers with associated satellite cells were isolated from the tibialis anterior muscles after digestion with collagenase. Single myofibers were plated on Matrigel-coated dishes in DMEM supplemented with 20% fetal bovine serum, 10% horse serum, 0.5% chick embryo extract, and penicillin-streptomycin. Three days later, the medium was replaced with proliferation medium (DMEM, 20% fetal bovine serum, 10% horse serum, 1% chick embryo extract) to promote proliferation of detached cells (delaminated satellite cells). After 5 days (only satellite cells remain on the plate), the medium was replaced with differentiation medium (2% horse serum and 0.5% chick embryo extract in DMEM).

      Western Blot Analysis

      Approximately 50 mg of gastrocnemius muscle was homogenized in 80 mmol/L Tris-HCl, pH 6.8, containing 100 mmol/L dithiothreitol, 70 mmol/L SDS, and 1 mmol/L glycerol, with freshly added protease and phosphatase inhibitor cocktails; kept on ice for 30 minutes; centrifuged at 15,000 × g for 10 minutes at 4°C; and the supernatant collected. Protein concentration was assayed using bovine serum albumin as working standard. C2C12 cells were lysed on radioimmunoprecipitation assay buffer (50 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mmol/L phenylmethylsulfonyl fluoride) with freshly added protease and phosphatase inhibitor cocktails. Equal amounts of protein (30 μg) were heat denaturated in sample-loading buffer (50 mmol/L Tris-HCl, pH 6.8, 100 mmol/L dithiothreitol, 2% SDS, 0.1% bromophenol blue, 10% glycerol), resolved by SDS-PAGE, and transferred to nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA). The filters were blocked with Tris-buffered saline containing 0.05% Tween and 5% nonfat dry milk and then were incubated overnight with antibodies directed against beclin-1 (B6186), LC3B (L7583), p62 (610832; BD Biosciences, San Jose, CA), p-Akt (Ser473, 9271; Cell Signaling Technology Inc., Beverly, MA), atrogin-1 (AP2041; ECM Biosciences, Versailles, KY), and tubulin (T5168). Peroxidase-conjugated IgG (Bio-Rad Laboratories) was used as secondary antibody. Quantification of the bands was performed by densitometric analysis using specific software version 2006c (TotalLab; Nonlinear Dynamics, Newcastle on Tyne, UK).

      RT-PCR

      Total RNA was obtained using TriPure isolation reagent (Roche Applied Science, Indianapolis, IN) following the manufacturer’s instructions. RNA concentration was determined fluorometrically using RiboGreen reagent (Invitrogen, Carlsbad, CA). RNA integrity was checked by electrophoresis on 1.2% agarose gel containing 0.02 mol/L morpholinopropanesulfonic acid and 18% formaldehyde. Total mRNA was retrotranscribed using an iScript cDNA synthesis kit (Bio-Rad Laboratories). Transcript levels were determined by real-time PCR using the SsoFast EvaGreen supermix and the MiniOpticon thermal cycler (Bio-Rad Laboratories). Primer sequences (forward and reverse) were as follows: atrogin1, 5′-AGCGACCTCAGCAGTTACTGC-3′ and 5′-CTTCTGGAATCCAGGATGGC-3′; beclin-1, 5′-TGAATGAGGATGACAGTGAGCA-3′ and 5′-CACCTGGTTCTCCACACTCTTG-3′; cathepsin L, 5′-GTGGACTGTT-CTCACGCTCAAG-3′ and 5′-TCCGTCCTTCGCTTCATAGG-3′; LC3B, 5′-CACTGCTCTGTCTTGTGTAGGTTG-3′ and 5′-TCGTTGTGCCTTTATTAGTGCATC-3′; p62, 5′-CCCAGTGTCTTGGCATTCTT-3′ and 5′-AGGGAAAGCAGAGGAAGCTC-3′; and NBR1, 5′-CCCCAGATTGGTTTACAAGC-3′ and 5′-TCCACCGTTTCCTTAACCAC-3.

      Cathepsin Enzymatic Activity

      Cathepsin B+L activity was determined by evaluating the cleavage of a specific chromogenic substrate.
      • Tessitore L.
      • Costelli P.
      • Baccino F.M.
      Pharmacological interference with tissue hypercatabolism in tumour-bearing rats.
      Fifty milligrams of gastrocnemius muscle was homogenized in 0.25 mol/L sucrose, 25 mmol/L Hepes, pH 7.4, and 2 mmol/L EDTA and then was centrifuged at 2000 × g for 5 minutes at 4°C. The supernatant was collected and centrifuged at 70,000 × g for 30 minutes at 4°C. The pellet was suspended in 0.1 mol/L sodium acetate, pH 5.5, and 1 mmol/L EDTA. Aliquots of 10 μg of protein were incubated for 60 minutes at 37°C in the presence of 100 μmol/L substrate (Z-Phe-Arg-pNA; Enzo Life Sciences Inc., Farmingdale, NY). The incubation buffer contained 0.5 mol/L sodium acetate, pH 5.5, 5 mmol/L dithiothreitol, and 2.5 mmol/L EDTA. Absorbance was read using a spectrophotometer at 410 nm (PerkinElmer, Waltham, MA), with free p-nitroanilide as the working standard.

      Immunofluorescence

      C2C12 monolayers were washed with PBS and fixed in 1:1 acetone:methanol. Samples were then probed with the following primary antibodies: LC3B (L7543) and p62 (610832; BD Biosciences). Detection was performed using a fluorescein isothiocyanate–conjugated rabbit IgG secondary antibody. Nuclei were stained with Hoechst 33342 fluorochrome, and the images were captured using an epi-illuminated fluorescence microscope (Axiovert 35; Carl Zeiss MicroImaging GmbH, Jena, Germany).

      Statistical Analysis

      All the results are expressed as means ± SD, except for gene expression (means ± SEM). Representative Western blots show independent samples. The significance of the differences was evaluated by analysis of variance followed by Tukey’s test.

      Results

      Autophagy Is Induced in C26-Bearing Mice

      C26 carcinoma induces progressive loss of body and muscle weight (Supplemental Figure S1). Muscle expression of proteins accepted as markers of autophagy has been assessed in the gastrocnemius muscle on days 11 and 14 of tumor growth, representing the initial and advanced stages of muscle wasting, respectively. Beclin-1, a main upstream regulator of autophagic sequestration,
      • Funderburk S.F.
      • Wang Q.J.
      • Yue Z.
      The Beclin 1-VPS34 complex: at the crossroads of autophagy and beyond.
      is already markedly increased on day 11, suggesting that autophagy activation is an early event in tumor-induced muscle depletion (Figure 1A). Second, levels of microtubule-associated protein 1 light chain 3B isoform I (LC3B-I) do not change in tumor-bearing mice, whereas the lipidated form (LC3B-II), a reliable marker of autophagosome formation,
      • Klionsky D.J.
      • Abeliovich H.
      • Agostinis P.
      • Agrawal D.K.
      • Aliev G.
      • Askew D.S.
      • et al.
      Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes.
      is significantly elevated at both experimental time points (Figure 1A). Moreover, the levels of p62/SQSTM1 have been assayed as a measure of substrate sequestration into autophagosomes; p62 binds LC3 and substrates marked for degradation by ubiquitylation.
      • Klionsky D.J.
      • Abeliovich H.
      • Agostinis P.
      • Agrawal D.K.
      • Aliev G.
      • Askew D.S.
      • et al.
      Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes.
      Similar to Beclin-1 and LC3B-II, p62 accumulates at both experimental points (Figure 1A), which could reflect either induction of autophagic sequestration or reduced autophagosome clearance.
      • Bjorkoy G.
      • Lamark T.
      • Pankiv S.
      • Overvatn A.
      • Brech A.
      • Johansen T.
      Monitoring autophagic degradation of p62/SQSTM1.
      To clarify this point, a flux experiment was performed by treating controls and C26 hosts with colchicine, a microtubule-destabilizing agent that interacts with tubulin.
      • Ju J.S.
      • Varadhachary A.S.
      • Miller S.E.
      • Weihl C.C.
      Quantitation of “autophagic flux” in mature skeletal muscle.
      The results show that LC3B-II levels, already higher than in controls in the C26 hosts, further increase after colchicine administration (Figure 1B), demonstrating that autophagy is, indeed, activated. The reduction of tubulin expression in colchicine-treated animals (Figure 1B) versus untreated mice likely reflects specific drug-induced degradation,
      • Ren Y.
      • Zhao J.
      • Feng J.
      Parkin binds to alpha/beta tubulin and increases their ubiquitination and degradation.
      demonstrating that the treatment protocol adopted leads to bioactive colchicine levels in the skeletal muscle. Despite the fact that the flux experiment was designed to achieve data for days 11 and 14 of tumor growth, only the first time point could be evaluated; tumor-bearing animals administered colchicine on days 12 and 13 did not reach day 14 and died before sacrifice.
      Figure thumbnail gr1
      Figure 1A: Densitometric analysis and representative patterns of expression of autophagy-related proteins in the gastrocnemius muscle of control (C) and C26 bearers 11 and 14 days after tumor transplantation. B: Densitometric analysis and representative patterns of expression of LC3B protein in the gastrocnemius of C and C26 hosts on day 11 of tumor growth either untreated or treated with colchicine (Col). TUB, tubulin. Data are expressed as means ± SD percentages of C. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 versus C; P < 0.01 versus Col; P < 0.01 versus C26.
      However, the possibility that reduced autophagosome clearance might occur in the present experimental setting cannot be discarded. A gene study shows that p62 transcript is increased on day 14 of tumor growth (Figure 2A), whereas the enzymatic activity of lysosomal cathepsins is reduced on day 14 but not on day 11 (Figure 2B), suggesting that p62 accumulation may result from transcriptional induction and, possibly, reduced degradation. The gene study also gives evidence for increased levels of mRNAs coding for atrogin-1, cathepsin L, LC3B, and NBR1, a protein with functional similarity to p62 (Figure 2A), further supporting the occurrence of sustained induction of autophagy. Notably, autophagy induction in C26-bearing mice is not a feature specific to the skeletal muscle, being also detectable in the liver, wherein the pattern of LC3B and p62 expression is comparable with that observed in the gastrocnemius muscle (Supplemental Figure S2A).
      Figure thumbnail gr2
      Figure 2Expression of genes involved in atrophy (A) and cathepsin B+L activity (B) in the gastrocnemius of control (C) and C26 hosts on days 11 and 14 of tumor growth. Data are expressed as means ± SD percentages of C. Gene expression results are expressed as means ± SEM. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 versus C.

      Autophagy Is Not a Specific Feature of the C26 Hosts

      To assess whether the enhanced autophagic degradation is peculiar to C26-bearing mice or also occurs in other experimental models of cancer cachexia, autophagic markers have been evaluated in the gastrocnemius muscle of rats bearing Yoshida AH-130 hepatoma or of mice transplanted with LLC. For the former, muscle depletion induced by 7 days of tumor growth (Figure 3A) is associated with increased LC3B lipidation (LC3B-II) and p62 accumulation, whereas no changes in Beclin-1 levels occur (Figure 3B). On the other side, muscle wasting in LLC hosts (Figure 3C) is characterized by up-regulation of Beclin-1 and p62 (Figure 3D).
      Figure thumbnail gr3
      Figure 3Gastrocnemius muscle mass and expression of autophagy-related proteins in the of AH-130 (A and B) and LLC (C and D) tumor-bearing animals. Data are expressed as means ± SD percentages of control (C). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 versus C. TUB, tubulin.
      These observations indicate that autophagy is activated in all the experimental models of cancer cachexia analyzed, although with different patterns. These results provide the first comprehensive approach to the contribution of autophagy to the pathogenesis of skeletal muscle wasting in cancer cachexia, confirming and significantly extending previous observations (see Introduction and Discussion). In addition, these results support the idea that a single marker is not sufficient to depict the state of activation of the autophagic-lysosomal degradative system.
      • Klionsky D.J.
      • Abeliovich H.
      • Agostinis P.
      • Agrawal D.K.
      • Aliev G.
      • Askew D.S.
      • et al.
      Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes.

      Activation of Autophagy in Cancer-Unrelated Muscle Wasting

      To investigate whether the modulations of autophagy in cancer-related muscle atrophy are comparable with those observed in other conditions associated with muscle wasting, autophagic markers were analyzed in Dexa-induced muscle atrophy and in aging-related sarcopenia.
      Dexa mice received daily i.p. injections of Dexa, a synthetic glucocorticoid whose catabolic action has been thoroughly investigated.
      • Schakman O.
      • Gilson H.
      • Kalista S.
      • Thissen J.P.
      Mechanisms of muscle atrophy induced by glucocorticoids.
      Body and muscle weight loss induced by 7-day Dexa administration (Figure 4A) are associated with increased expression of Beclin-1 and both LC3B isoforms, whereas p62 virtually disappeared (Figure 4B). The different patterns of p62 expression in Dexa-treated mice compared with that observed in tumor-bearing animals are associated with lack of p62 transcriptional induction (Figure 4C) and with normal cathepsin enzymatic activity (Figure 4D). The gene study also shows increased expression of atrogin-1, cathepsin L, and NBR1 (Figure 4C). These observations are consistent with previous data reporting that autophagy significantly contributes to muscle wasting in different experimental conditions, such as denervation, starvation, and sepsis.
      • Zhao J.
      • Brault J.J.
      • Schild A.
      • Cao P.
      • Sandri M.
      • Schiaffino S.
      • Lecker S.H.
      • Goldberg A.L.
      FoxO3 coordinately activates protein degradation by the autophagic/lysosomal and proteasomal pathways in atrophying muscle cells.
      • Doyle A.
      • Zhang G.
      • Abdel Fattah E.A.
      • Eissa N.T.
      • Li Y.P.
      Toll-like receptor 4 mediates lipopolysaccharide-induced muscle catabolism via coordinate activation of ubiquitin-proteasome and autophagy-lysosome pathways.
      The present results provide a panel of autophagy markers (at the gene and protein levels) and show that efficient autophagosome clearance is achieved in the muscle of glucocorticoid-treated animals, as inferred by the markedly reduced p62 levels.
      Figure thumbnail gr4
      Figure 4A: Loss of body weight and skeletal muscle mass in Dexa-treated mice. B: Densitometric analysis and representative patterns of expression of autophagy-related proteins in the gastrocnemius (GSN) muscle of control (C) and Dexa-treated mice. Expression of genes involved in atrophy (C) and cathepsin B+L activity (D) in the GSN of C and Dexa-treated mice. TIB, tibialis; TUB, tubulin. Data are means ± SD percentages of C. Gene expression results are expressed as means ± SEM. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 versus C. i.b.w., initial body weight.
      Cancer and glucocorticoids induce muscle depletion within a short period (7 to 14 days, depending on the model) and can be considered acute wasting conditions. In contrast, sarcopenia, associated with aging, is a chronic state, and the underlying mechanisms could be different. To test the relevance of autophagy to sarcopenia, the same markers reported previously herein were measured in aged mice and rats. In 22-month-old mice, Beclin-1 and LC3B-I protein levels fall, whereas p62 markedly accumulates compared with 3-month-old mice (Figure 5A), suggesting reduced autophagic degradation. As for the rats, the comparison between 6- and 24-month-old animals shows the accumulation of p62 (Figure 5B). In an attempt to stimulate autophagic flux, rats were submitted to 40% CR, an antiaging strategy previously shown to activate liver autophagy.
      • Del Roso A.
      • Vittorini S.
      • Cavallini G.
      • Donati A.
      • Gori Z.
      • Masini M.
      • Pollera M.
      • Bergamini E.
      Ageing-related changes in the in vivo function of rat liver macroautophagy and proteolysis.
      In the present conditions, CR reduces LC3B-II and p62 levels in the skeletal muscle, likely releasing the autophagy blockade (Figure 5B).
      Figure thumbnail gr5
      Figure 5Densitometric analysis and representative patterns of expression of autophagy-related proteins in the gastrocnemius (GSN) of 3- and 22-month-old mice (A) and 6- and 24-month-old rats (B) fed ad libitum (AL) or with 40% CR. TUB, tubulin. Data are expressed as means ± SD percentages of 3-month-old mice (A) or AL 6-month-old rats (B). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 versus 3-month-old mice (A) or AL 6-month-old rats (B); P < 0.05 versus AL 24 mo.

      Autophagy in Cancer-Induced Muscle Atrophy Is Only Partially Modulated by INS

      Autophagy is mainly regulated by the PI3K/Akt/mTOR (mammalian Target of rapamycin) pathway, and previous data showed that INS administration effectively prevented muscle protein hypercatabolism in rats bearing AH-130 hepatoma.
      • Tessitore L.
      • Costelli P.
      • Baccino F.M.
      Pharmacological interference with tissue hypercatabolism in tumour-bearing rats.
      To test whether INS effectiveness in preventing muscle wasting could rely on down-regulation of the autophagic degradation, the hormone was administered to C26-bearing mice, the model system where autophagy seems more marked. In contrast to the results previously obtained in the AH-130 hosts,
      • Tessitore L.
      • Costelli P.
      • Baccino F.M.
      Pharmacological interference with tissue hypercatabolism in tumour-bearing rats.
      INS administration does not attenuate cachexia in the C26 hosts (Figure 6A). Beclin-1 and p62 protein levels are reduced in INS-treated tumor hosts are reduced in comparison with untreated animals but remain significantly higher than in controls, demonstrating that INS inhibition of autophagy is far from being complete (Figure 6B). Such a pattern is not peculiar to the skeletal muscle because comparable results can be observed in the liver (Supplemental Figure S2B). The gene study analysis shows that atrogin-1, Beclin-1, and p62 transcripts are reduced by INS, whereas cathepsin L and LC3B are even increased (Figure 6C). The partial restoration of physiologic autophagy exerted by INS could reflect a condition of mild resistance to this hormone. A study reported in the literature suggests that muscle wasting in C26 hosts is associated with INS resistance.
      • Asp M.L.
      • Tian M.
      • Wendel A.A.
      • Belury M.A.
      Evidence for the contribution of insulin resistance to the development of cachexia in tumor-bearing mice.
      To clarify this point, we performed an INS tolerance test. The results show that control and tumor-bearing animals either treated with INS or not respond comparably to INS challenge, thus excluding the occurrence of INS resistance (Supplemental Figure S3B). In addition, the direct correlation existing between basal glycemia and body weight changes over the experimental period (Supplemental Figure S3C) suggests that at least in this experimental setting, hypoglycemia rather than INS resistance contributes to the loss of body weight.
      Figure thumbnail gr6
      Figure 6A: Loss of body weight and skeletal muscle mass in 1.5 IU/kg body weight INS-treated animals. B: Densitometric analysis and representative patterns of expression of autophagy-related proteins in the gastrocnemius (GSN) muscle of control (C) and C26 hosts either treated or untreated with INS. C: Expression of genes involved in atrophy in the GSN muscle of C, C26, and INS-treated C26 hosts. TIB, tibialis; TUB, tubulin. Data are expressed as means ± SD percentages of C. Gene expression results are expressed as means ± SEM. ∗P < 0.05, ∗∗∗P < 0.001 versus C; P < 0.05, ††P < 0.01 versus C26. i.b.w., initial body weight.

      TNF-α Directly Stimulates Autophagic Degradation in Skeletal Muscle Cell Cultures

      Muscle wasting in cancer cachexia is largely mediated by proinflammatory cytokines. Indeed, protein hypercatabolism can be partially reversed by treating rats bearing the AH-130 hepatoma with anti–TNF-α antibodies or with pentoxifylline, an inhibitor of TNF-α synthesis.
      • Costelli P.
      • Bossola M.
      • Muscaritoli M.
      • Grieco G.
      • Bonelli G.
      • Bellantone R.
      • Doglietto G.B.
      • Baccino F.M.
      • Rossi Fanelli F.
      Anticytokine treatment prevents the increase in the activity of ATP-ubiquitin- and Ca(2+)-dependent proteolytic systems in the muscle of tumour-bearing rats.
      • Llovera M.
      • Carbo N.
      • Garcia-Martinez C.
      • Costelli P.
      • Tessitore L.
      • Baccino F.M.
      • Agell N.
      • Bagby G.J.
      • Lopez-Soriano F.J.
      • Argiles J.M.
      Anti-TNF treatment reverts increased muscle ubiquitin gene expression in tumour-bearing rats.
      We examined whether TNF-α has the ability to directly activate the autophagic process in differentiating C2C12 myocytes cultured in the presence of the cytokine for 48 hours. Consistent with a previous study obtained with the cytokine TWEAK (TNF-like weak inducer of apoptosis),
      • Dogra C.
      • Changotra H.
      • Wedhas N.
      • Qin X.
      • Wergedal J.E.
      • Kumar A.
      TNF-related weak inducer of apoptosis (TWEAK) is a potent skeletal muscle-wasting cytokine.
      TNF-α caused a reduction of Akt phosphorylation (Figure 7A). This observation seems to contrast with previous data showing increased levels of phosphorylated Akt in TNF-α–treated C2C12 myotubes
      • Moylan J.S.
      • Smith J.D.
      • Chambers M.A.
      • McLoughlin T.J.
      • Reid M.B.
      TNF induction of atrogin-1/MAFbx mRNA depends on Foxo4 expression but not AKT-Foxo1/3 signaling.
      ; the discrepancy may well result from the fact that the experiments shown herein were performed on differentiating C2C12 myocytes (see Materials and Methods). The reduced levels of active Akt are associated with Beclin-1 induction and LC3B-II accumulation (Figure 7A) in the absence of significant means ± SD changes in atrogin-1 expression [control = 0.55 ± 0.15 arbitrary units (AU), TNF = 0.39 ± 0.15 AU, n = 3]. Activation of autophagy is also suggested by increased p62 (Figure 7B) and dotted LC3B staining (control = 4 ± 2 cells with >10 puncta per 60 nuclei, TNF = 41 ± 9 cells with >10 puncta per 60 nuclei, n = 3, P = 0.0024) (Figure 8B). A comparable pattern can also be observed in primary satellite cells exposed to TNF-α (Supplemental Figure S4A) and in C2C12 myotubes exposed to low TNF-α concentrations (5 ng/mL) (Supplemental Figure S4B). Finally, autophagic flux was evaluated by incubating C2C12 cultures with bafilomycin A1, a drug able to inhibit the vacuolar H+-ATPase, resulting in reduced acidification and, therefore, lysosomal-dependent degradation of the autophagosome cargo.
      • Ju J.S.
      • Varadhachary A.S.
      • Miller S.E.
      • Weihl C.C.
      Quantitation of “autophagic flux” in mature skeletal muscle.
      These results show that LC3B-II levels in bafilomycin A1–treated cells markedly increase with respect to those in controls. When cultures are exposed to TNF-α and bafilomycin A1, LC3B-II levels are significantly higher than those measured in cells treated with bafilomycin A1 alone (Figure 8A and Supplemental Figure S4B). This observation suggests that the rate of LC3B-II formation is increased by the cytokine.
      • Rubinsztein D.C.
      • Cuervo A.M.
      • Ravikumar B.
      • Sarkar S.
      • Korolchuk V.
      • Kaushik S.
      • Klionsky D.J.
      In search of an “autophagomometer”.
      Conversely, LC3B-II levels in C2C12 cultures treated with 100 ng/mL of TNF-α alone (Figure 8A) are not different from those detected in cells exposed to TNF-α and bafilomycin A1. Such an observation suggests that TNF-α inhibits autophagosome degradation rather than enhancing their formation. However, lysosomal proteolysis should be completely blocked by bafilomycin; an LC3B-II increase over bafilomycin-induced levels should result from enhanced autophagosome formation.
      • Rubinsztein D.C.
      • Cuervo A.M.
      • Ravikumar B.
      • Sarkar S.
      • Korolchuk V.
      • Kaushik S.
      • Klionsky D.J.
      In search of an “autophagomometer”.
      This conclusion is strengthened by the results obtained on C2C12 myotubes exposed to TNF-α 5 (Supplemental Figure S4B). The results obtained on C2C12 cells are consistent with those reported for the muscle of the C26 hosts, where inhibition of autophagosome degradation and increased sequestration are likely to occur. To ascertain whether autophagy can be activated by TNF-α in vivo, healthy mice received 40 μg/kg body weight i.m. injection of this cytokine
      • Langen R.C.
      • Van Der Velden J.L.
      • Schols A.M.
      • Kelders M.C.
      • Wouters E.F.
      • Janssen-Heininger Y.M.
      Tumor necrosis factor-alpha inhibits myogenic differentiation through MyoD protein destabilization.
      • Langen R.C.
      • Schols A.M.
      • Kelders M.C.
      • van der Velden J.L.
      • Wouters E.F.
      • Janssen-Heininger Y.M.
      Muscle wasting and impaired muscle regeneration in a murine model of chronic pulmonary inflammation.
      and were sacrificed 12 hours later. Supplemental Figure S5 shows that LC3B-II levels are increased in TNF-α–treated muscles compared with muscles of control mice, more so when the animals also received colchicine, indicating the enhancement of autophagic flux.
      Figure thumbnail gr7
      Figure 7C2C12 myotubes (2-day differentiation) treated for 48 hours with TNF-α in the presence or absence of INS. A: Densitometric analysis and representative patterns of expression of autophagy-related proteins and of phospho-Akt. TUB, tubulin. Data (n = 3) are expressed as means ± SD percentages of controls (C). ∗P < 0.05, ∗∗P < 0.01 versus C; P < 0.05 versus TNF. B: p62 and LC3B immunostaining (green: p62 or LC3B-II; blue: Hoechst).
      Figure thumbnail gr8
      Figure 8Densitometric analysis of expression of autophagy-related proteins in C2C12 cells cultured in the presence or absence of bafilomycin A1 (BAF; A) and/or INS (B). TUB, tubulin. Data (n = 3) are expressed as means ± SD percentages of controls (C). ∗P < 0.05, ∗∗P < 0.01 versus C; P < 0.05 versus BAF; P < 0.05 versus BAF INS.
      The data reported previously herein indicate that exposure of muscle cell cultures to TNF-α results in up-regulation of autophagy. To clarify whether this effect is responsive to anabolic regulation, C2C12 cells were cotreated with TNF-α and INS. As expected, the results show that INS alone increases means ± SD Akt phosphorylation (control = 0.21 ± 0.05 AU, INS = 0.31 ± 0.03 AU, n = 3, P < 0.05) and reduces atrogin-1 expression (control = 0.55 ± 0.15 AU, INS = 0.28 ± 0.05 AU, n = 3, P < 0.05) and LC3B-II levels (Figure 7A). These observations, together with the enhanced immunostaining for p62 (Figure 7B), suggest that basal autophagy is inhibited. When INS is coupled with TNF-α, no significant changes could be observed in means ± SD atrogin-1 expression (INS = 0.28 ± 0.05 AU, INS+TNF = 0.24 ± 0.06 AU, n = 3) and Akt phosphorylation (INS = 0.31 ± 0.03 AU, INS+TNF = 0.18 ± 0.10 AU, n = 3) (Figure 7A), although a tendency toward reduction is apparent. Despite INS exposure, the TNF-α–induced Beclin-1 and LC3B-II increases remained detectable (Figure 7A). In the presence of bafilomycin A1, INS resulted in LC3B-II accumulation lower than in cells exposed to the lysosomal inhibitor alone (Figure 8B). However, in INS- and bafilomicyn A1–treated cultures, LC3B-II levels in the presence of TNF-α were higher than those observed in the absence of the cytokine, although they did not reach those obtained in cells exposed to bafilomycin A1 alone (Figure 8B). Such observations suggest that INS reduces basal autophagic flux but is unable to completely prevent the activation induced by TNF-α exposure. This hypothesis was confirmed by LC3B immunostaining (INS = 5 ± 1 cells with >10 puncta per 60 nuclei, TNF+INS = 60 ± 9 cells with >10 puncta per 60 nuclei, n = 3, P = 0.00025) (Figure 7B). Similar results have been obtained by treating the cells with recombinant stable IGF-1; treatment with an IGF-1 neutralizing antibody was sufficient to activate autophagy, although to levels lower than those attained by TNF-α (Supplemental Figure S6).

      Discussion

      The present study demonstrates that muscle wasting in three different experimental models of cancer cachexia is associated with increased autophagy, as evaluated by analyzing three of the most representative markers of this degradative pathway: Beclin-1 as an indicator of autophagy induction, LC3B conversion to the lipidated form to measure autophagosome abundance, and p62/SQSTM1 as a marker of substrate sequestration and eventual degradation. Analysis of these proteins gives an approximate estimate of autophagic flux, ie, the extent of protein sequestration and subsequent degradation into lysosomes. To further strengthen the data, autophagic flux was measured by treating C26 hosts with colchicine.
      • Ju J.S.
      • Varadhachary A.S.
      • Miller S.E.
      • Weihl C.C.
      Quantitation of “autophagic flux” in mature skeletal muscle.
      Although the markers of autophagy mentioned previously herein do not strictly share the same pattern of expression in the cachexia models used in the present study, the net result is that in all of them an increase of autophagic degradation is likely to occur. This observation represents an important new finding. Indeed, although muscle wasting in cancer cachexia is considered to depend mainly on hyperactivation of proteasomal degradation,
      • Costelli P.
      • Garcia-Martinez C.
      • Llovera M.
      • Carbo N.
      • Lopez-Soriano F.J.
      • Agell N.
      • Tessitore L.
      • Baccino F.M.
      • Argiles J.M.
      Muscle protein waste in tumor-bearing rats is effectively antagonized by a beta 2-adrenergic agonist (clenbuterol): role of the ATP-ubiquitin-dependent proteolytic pathway.
      • Attaix D.
      • Combaret L.
      • Bechet D.
      • Taillandier D.
      Role of the ubiquitin-proteasome pathway in muscle atrophy in cachexia.
      • Acharyya S.
      • Guttridge D.C.
      Cancer cachexia signaling pathways continue to emerge yet much still points to the proteasome.
      • Khal J.
      • Wyke S.M.
      • Russell S.T.
      • Hine A.V.
      • Tisdale M.J.
      Expression of the ubiquitin-proteasome pathway and muscle loss in experimental cancer cachexia.
      • Zhang G.
      • Jin B.
      • Li Y.P.
      C/EBPbeta mediates tumour-induced ubiquitin ligase atrogin1/MAFbx upregulation and muscle wasting.
      only recently, Beclin-1 and LC3B induction was shown in LLC-bearing mice
      • Paul P.K.
      • Gupta S.K.
      • Bhatnagar S.
      • Panguluri S.K.
      • Darnay B.G.
      • Choi Y.
      • Kumar A.
      Targeted ablation of TRAF6 inhibits skeletal muscle wasting in mice.
      ; however, Bnip3 overexpression and increased LC3B-II levels in the C26 hosts were previously reported.
      • Asp M.L.
      • Tian M.
      • Wendel A.A.
      • Belury M.A.
      Evidence for the contribution of insulin resistance to the development of cachexia in tumor-bearing mice.
      • Lokireddy S.
      • Wijesoma I.W.
      • Bonala S.
      • Wei M.
      • Sze S.K.
      • McFarlane C.
      • Kambadur R.
      • Sharma M.
      Myostatin is a novel tumoral factor that induces cancer cachexia.
      Finally, autophagy was proposed to contribute to C26-induced cardiac atrophy.
      • Cosper P.F.
      • Leinwand L.A.
      Cancer causes cardiac atrophy and autophagy in a sexually dimorphic manner.
      To the best of our knowledge, the data shown in the present study represent the most comprehensive analysis available that demonstrates how the autophagic system is poised toward activation in the skeletal muscle of C26-bearing mice. Similar conclusions can be drawn from results obtained in rats bearing Yoshida AH-130 hepatoma, where hyperactivation of muscle lysosomal proteases was already shown by Tessitore et al,
      • Tessitore L.
      • Costelli P.
      • Bonetti G.
      • Baccino F.M.
      Cancer cachexia, malnutrition, and tissue protein turnover in experimental animals.
      and in mice implanted with the LLC tumor, these latter confirming but extending the findings by Paul et al.
      • Paul P.K.
      • Gupta S.K.
      • Bhatnagar S.
      • Panguluri S.K.
      • Darnay B.G.
      • Choi Y.
      • Kumar A.
      Targeted ablation of TRAF6 inhibits skeletal muscle wasting in mice.
      These results highlight the relevance of autophagy to cancer-induced muscle wasting, more so when considering the fact that not only the upstream signals that activate protein breakdown are able to coordinate autophagy and proteasome induction
      • Paul P.K.
      • Kumar A.
      TRAF6 coordinates the activation of autophagy and ubiquitin-proteasome systems in atrophying skeletal muscle.
      • Zhao J.
      • Brault J.J.
      • Schild A.
      • Goldberg A.L.
      Coordinate activation of autophagy and the proteasome pathway by FoxO transcription factor.
      but also that typical proteasome substrates (eg, ubiquitylated proteins) can be alternatively degraded through the acidic vacuolar proteolysis.
      • Johansen T.
      • Lamark T.
      Selective autophagy mediated by autophagic adapter proteins.
      A careful analysis of these results suggests that despite the fact that autophagy is activated in cancer cachexia and Dexa-induced muscle atrophy but not in sarcopenic muscles, different mechanisms are operating in the specific experimental conditions. As for cancer cachexia, all the markers of autophagy are up-regulated in the C26 hosts. The marked accumulation of p62 that contrasts with the observation that autophagic flux is clearly enhanced, at least in day 11 C26 hosts, is particularly interesting (Figure 1B). Such increased p62 levels could reflect transcriptional induction, as reported in desmin-related cardiomyopathy.
      • Zheng H.
      • Tang M.
      • Zheng Q.
      • Kumarapeli A.R.
      • Horak K.M.
      • Tian Z.
      • Wang X.
      Doxycycline attenuates protein aggregation in cardiomyocytes and improves survival of a mouse model of cardiac proteinopathy.
      Consistently, p62 gene expression is markedly increased in the muscle of the C26 hosts. Autophagic flux measurement was not possible in day 14 C26 hosts because colchicine-treated animals died before sacrifice, suggesting that autophagy inhibition negatively impinges on survival when metabolic homeostasis is markedly compromised.
      Despite the induction of autophagy, muscle lysosomal cathepsin activities are reduced in day 14 C26 bearers, a finding that may reflect selective disturbances in lysosomal function, such as lysosomal enzyme mistargeting.
      • Eskelinen E.L.
      • Illert A.L.
      • Tanaka Y.
      • Schwarzmann G.
      • Blanz J.
      • Von Figura K.
      • Saftig P.
      Role of LAMP-2 in lysosome biogenesis and autophagy.
      The possibility that during tumor growth muscle protein degradation relies initially on the lysosomal system and only subsequently on other proteolytic pathways cannot be excluded. Consistently, lysosomal protease activity is reduced only in day 14 C26 hosts (advanced cachexia), in parallel with increased atrogin-1 mRNA levels and calpastatin degradation, the latter being a marker of calpain hyperactivation (unpublished data).
      As for Dexa-induced atrophy, the results show that Beclin-1 and LC3B-II increase, whereas p62 virtually disappears, indicating that substrates sequestered into autophagosomes are readily degraded by lysosomes. The lack of p62 accumulation in the skeletal muscle of Dexa-treated mice is associated with unchanged levels of p62 transcript. In addition, this observation suggests that even if rapid muscle atrophy observed in tumor bearers and glucocorticoid-treated mice is characterized by autophagic degradation, the molecular signals involved are different and may require distinct therapeutic approaches.
      Finally, autophagic degradation is reduced in the skeletal muscle of aged animals, whereas it is reactivated by CR, consistent with recent data.
      • Wohlgemuth S.E.
      • Seo A.Y.
      • Marzetti E.
      • Lees H.A.
      • Leeuwenburgh C.
      Skeletal muscle autophagy and apoptosis during aging: effects of calorie restriction and life-long exercise.
      Reduced muscle mass occurs in aging and CR,
      • Hepple R.T.
      • Qin M.
      • Nakamoto H.
      • Goto S.
      Caloric restriction optimizes the proteasome pathway with aging in rat plantaris muscle: implications for sarcopenia.
      ; however, the pattern of the autophagic markers is quite different. Indeed, although Beclin-1 and LC3B-II are comparable in aged animals, or even reduced compared with young individuals, p62 levels are markedly increased, likely because autophagy is not activated and waste substrates accumulate. Although impairment of autophagic degradation during aging is a well-known event in the liver, the present observations represent a new finding as little data are available for the skeletal muscle.
      • Wohlgemuth S.E.
      • Seo A.Y.
      • Marzetti E.
      • Lees H.A.
      • Leeuwenburgh C.
      Skeletal muscle autophagy and apoptosis during aging: effects of calorie restriction and life-long exercise.
      It is likely that the abnormal mitochondria and sarcomere disorganization in aged skeletal muscle result from impaired autophagy. Consistently, muscle-specific deletion of Atg7, a crucial autophagy gene, leads to phenotypic alterations resembling sarcopenia.
      • Masiero E.
      • Agatea L.
      • Mammucari C.
      • Blaauw B.
      • Loro E.
      • Komatsu M.
      • Metzger D.
      • Reggiani C.
      • Schiaffino S.
      • Sandri M.
      Autophagy is required to maintain muscle mass.
      CR is associated with p62 levels that are still increased with respect to young animals but markedly reduced compared with aged rats, suggesting partial restoration of autophagic flux. These observations are in agreement with a previous report by Wohlgemuth et al
      • Wohlgemuth S.E.
      • Seo A.Y.
      • Marzetti E.
      • Lees H.A.
      • Leeuwenburgh C.
      Skeletal muscle autophagy and apoptosis during aging: effects of calorie restriction and life-long exercise.
      that showed partial restoration of reduced expression of autophagic markers in aged rats by 8% CR.
      In the C26 host, energy deficit due to potential hypoglycemia and body weight loss and by mitochondrial damage (unpublished data) is a possible trigger of autophagy not only in the skeletal muscle but also in the liver.
      • Czaja M.J.
      Functions of autophagy in hepatic and pancreatic physiology and disease.
      Such a speculation would fit with the recently proposed model of battery-operated tumor growth, where autophagic degradation is suggested to start in the tumor stroma and then to spread systemically to provide a continuous energy transfer from the host to the tumor.
      • Martinez-Outschoorn U.E.
      • Whitaker-Menezes D.
      • Pavlides S.
      • Chiavarina B.
      • Bonuccelli G.
      • Casey T.
      • Tsirigos A.
      • Migneco G.
      • Witkiewicz A.
      • Balliet R.
      • Mercier I.
      • Wang C.
      • Flomenberg N.
      • Howell A.
      • Lin Z.
      • Caro J.
      • Pestell R.G.
      • Sotgia F.
      • Lisanti M.P.
      The autophagic tumor stroma model of cancer or “battery-operated tumor growth”: a simple solution to the autophagy paradox.
      The onset of cancer cachexia is also associated with a proinflammatory environment.
      • Penna F.
      • Minero V.G.
      • Costamagna D.
      • Bonelli G.
      • Baccino F.M.
      • Costelli P.
      Anti-cytokine strategies for the treatment of cancer-related anorexia and cachexia.
      • Argiles J.M.
      • Busquets S.
      • Toledo M.
      • Lopez-Soriano F.J.
      The role of cytokines in cancer cachexia.
      Anti–TNF-α strategies have been shown to effectively prevent the hyperactivation of proteasomal degradation in the AH-130 hosts.
      • Costelli P.
      • Bossola M.
      • Muscaritoli M.
      • Grieco G.
      • Bonelli G.
      • Bellantone R.
      • Doglietto G.B.
      • Baccino F.M.
      • Rossi Fanelli F.
      Anticytokine treatment prevents the increase in the activity of ATP-ubiquitin- and Ca(2+)-dependent proteolytic systems in the muscle of tumour-bearing rats.
      • Llovera M.
      • Carbo N.
      • Garcia-Martinez C.
      • Costelli P.
      • Tessitore L.
      • Baccino F.M.
      • Agell N.
      • Bagby G.J.
      • Lopez-Soriano F.J.
      • Argiles J.M.
      Anti-TNF treatment reverts increased muscle ubiquitin gene expression in tumour-bearing rats.
      Recent data show that TRAF6 induces the expression of muscle-specific E3 ubiquitin ligases and autophagy-related molecules in the skeletal muscle on denervation and in LLC-bearing mice.
      • Paul P.K.
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      Targeted ablation of TRAF6 inhibits skeletal muscle wasting in mice.
      Moreover, TWEAK has been reported to induce the activation of autophagy and proteasome in C2C12 myotubes.
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      • Kumar A.
      TWEAK causes myotube atrophy through coordinated activation of ubiquitin-proteasome system, autophagy, and caspases.
      The results obtained in the present study show that TNF-α directly stimulates autophagic degradation in C2C12 and isolated murine satellite cells, suggesting another possible trigger of autophagy in cancer cachexia.
      A relevant issue raised in the present work concerns the observation that TNF-α exposure in vitro and tumor growth in vivo partially overcome the well known INS/IGF-1 ability to down-regulate autophagy. These results could explain the virtual lack of effect of IGF-1/INS–based strategies in preventing cancer-induced muscle wasting.
      • Penna F.
      • Bonetto A.
      • Muscaritoli M.
      • Costamagna D.
      • Minero V.G.
      • Bonelli G.
      • Rossi Fanelli F.
      • Baccino F.M.
      • Costelli P.
      Muscle atrophy in experimental cancer cachexia: is the IGF-1 signaling pathway involved?.
      • Schmidt K.
      • von Haehling S.
      • Doehner W.
      • Palus S.
      • Anker S.D.
      • Springer J.
      IGF-1 treatment reduces weight loss and improves outcome in a rat model of cancer cachexia.
      Recently, a study reported that increased IGF-1 receptor engagement in the skeletal muscle results in rapid IRS-1 phosphorylation and proteasome-dependent degradation.
      • Shi J.
      • Luo L.
      • Eash J.
      • Ibebunjo C.
      • Glass D.J.
      The SCF-Fbxo40 complex induces IRS1 ubiquitination in skeletal muscle, limiting IGF1 signaling.
      On the contrary, IGF-1 overexpression effectively prevents glucocorticoid-induced muscle atrophy and the sarcopenia of aging,
      • Schakman O.
      • Gilson H.
      • de Coninck V.
      • Lause P.
      • Verniers J.
      • Havaux X.
      • Ketelslegers J.M.
      • Thissen J.P.
      Insulin-like growth factor-I gene transfer by electroporation prevents skeletal muscle atrophy in glucocorticoid-treated rats.
      • Penna F.
      • Bonetto A.
      • Muscaritoli M.
      • Costamagna D.
      • Minero V.G.
      • Bonelli G.
      • Rossi Fanelli F.
      • Baccino F.M.
      • Costelli P.
      Muscle atrophy in experimental cancer cachexia: is the IGF-1 signaling pathway involved?.
      although no information regarding the effect on autophagy in these model systems is available.

      Conclusions

      The results reported in the present study show that autophagy contributes to generate muscle atrophy in cancer cachexia and in Dexa-treated animals, although the underlying mechanisms in the two conditions are likely different. In contrast, sarcopenia of aging, a slow but progressive physiologic loss of muscle mass, does not seem to depend on autophagy. These results further support the idea that the final event, eg, muscle wasting, likely derives from the combination of different mechanisms; autophagy participates in the complicated network that leads to the loss of muscle mass to a variable extent. Finally, catabolic stimuli, such as the presence of a tumor or, more specifically, proinflammatory cytokines, cannot be fully antagonized, at least in terms of autophagic degradation by INS, one of the most powerful inhibitors of autophagy. These results are particularly intriguing and could suggest that activation of autophagy in these model systems is so enforced that it cannot be circumvented by simple inhibitor-based strategies.

      Supplemental Data

      • Supplemental Figure S1

        Loss of body (A) and skeletal muscle (B) weight in controls (C) (n = 6) and C26 hosts (n = 8) on days 11 (d11) and 14 (d14) of tumor growth. Data are expressed as means ± SD as percentages of control (C). *P < 0.05, **P < 0.01, and ***P < 0.001 versus C. GSN, gastrocnemius; i.b.w., initial body weight; TIB, tibialis.

      • Supplemental Figure S2

        Densitometric analysis and representative patterns of expression of autophagy-related proteins in the liver of controls (C) and C26 hosts (A) or C, C26 bearers, and INS-treated C26 hosts (B) on day 14 of tumor growth. Data are expressed as means ± SD percentages of C. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 versus C; §§P < 0.01 versus C26. TUB, tubulin.

      • Supplemental Figure S3

        Daily food intake (A) and INS tolerance test (B) in controls (C), C26 hosts, and INS-treated C26 hosts. C: Correlation between glycemia and Δ i.b.w. in C26 hosts either untreated (r = 0.693) or INS treated (r = 0.939). i.b.w., initial body weight.

      • Supplemental Figure S4

        A: LC3B immunostaining in primary cultures of tibialis-derived satellite cells untreated or treated for 48 hours with 100 ng/mL of TNF-α (green, LC3B; blue, Hoechst). B: Densitometric analysis and representative patterns of expression of LC3B-I and LC3B-II in C2C12 myotubes exposed to 5 ng/mL of TNF-α. ∗P < 0.05, ∗∗P < 0.01 versus C; P < 0.05 versus TNF. BAF, bafilomycin A1; C, control; TUB, tubulin.

      • Supplemental Figure S5

        Densitometric analysis and representative patterns of expression of LC3B-I and LC3B-II in the muscle of control (C) and TNF-α–treated mice in the presence or absence of colchicine (Col) administration (see Materials and Methods). Data are expressed as means ± SD as percentages of control (C). ∗P < 0.05, ∗∗P < 0.01 versus C. TUB, tubulin.

      • Supplemental Figure S6

        A: LC3B immunostaining in C2C12 myotubes (2-day differentiation) untreated or treated for 48 hours with 100 ng/mL of TNF-α in the presence or absence of 50 ng/mL of IGF-1 or 2 μg/mL of anti–IGF-1 antibody (a-IGF-1). B: Densitometric analysis and representative patterns of expression of LC3B and phospho-Akt. Data (n = 3) are expressed as means ± SD percentages of control (C). ∗P < 0.05, ∗∗P < 0.01 versus C. TUB, tubulin.

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