Abstract

Objective

   We tested the hypothesis that exercise training would attenuate
   metabolic impairment in a model of severe cancer cachexia.

Methods

   We used multiple in vivo and in vitro methods to explore the mechanisms
   underlying the beneficial effects induced by exercise training in
   tumor-bearing rats.

Results

   Exercise training improved running capacity, prolonged lifespan,
   reduced oxidative stress, and normalized muscle mass and contractile
   function in tumor-bearing rats. An unbiased proteomic screening
   revealed COP9 signalosome complex subunit 2 (COPS2) as one of the most
   downregulated proteins in skeletal muscle at the early stage of cancer
   cachexia. Exercise training normalized muscle COPS2 protein expression
   in tumor-bearing rats and mice. Lung cancer patients with low endurance
   capacity had low muscle COPS2 protein expression as compared to
   age-matched control subjects. To test whether decrease in COPS2 protein
   levels could aggravate or be an intrinsic compensatory mechanism to
   protect myotubes from cancer effects, we performed experiments in vitro
   using primary myotubes. COPS2 knockdown in human myotubes affected
   multiple cellular pathways, including regulation of actin cytoskeleton.
   Incubation of cancer-conditioned media in mouse myotubes decreased
   F-actin expression, which was partially restored by COPS2 knockdown.
   Direct repeat 4 (DR4) response elements have been shown to positively
   regulate gene expression. COPS2 overexpression decreased the DR4
   activity in mouse myoblasts, and COPS2 knockdown inhibited the effects
   of cancer-conditioned media on DR4 activity.

Conclusions

   These studies demonstrated that exercise training may be an important
   adjuvant therapy to counteract cancer cachexia and uncovered novel
   mechanisms involving COPS2 to regulate myotube homeostasis in cancer
   cachexia.

   Keywords: Cancer cachexia, Muscle wasting, Atrophy, Endurance exercise,
   Response elements

Highlights

     * •
       Exercise training prolongs lifespan, reduces oxidative stress, and
       improves skeletal muscle function in tumor-bearing rats.
     * •
       Muscle COP9 signalosome complex subunit 2 (COPS2) protein is
       downregulated in cancer cachexia.
     * •
       Exercise training restores COPS2 protein expression to control
       levels.
     * •
       Cancer-conditioned media decreased F-actin expression in myotubes,
       which is partially restored by COPS2 knockdown.
     * •
       COPS2 overexpression decreases DR4 activity and COPS2 knockdown
       inhibits cancer-conditioned media effects on DR4 activity.

1. Introduction

   While there are multiple and complex mechanisms underlying skeletal
   muscle wasting, early onset of cachexia is clearly characterized by
   metabolic dysfunction [[67][1], [68][2], [69][3], [70][4], [71][5],
   [72][6]]. Several studies have reported a disruption of muscle
   oxidative metabolism in cancer animal models and cancer patients, which
   may be the trigger for cause of muscle atrophy [[73]1,[74][7], [75][8],
   [76][9], [77][10], [78][11], [79][12], [80][13], [81][14], [82][15]].
   Disrupted oxidative metabolism induces abnormal production of reactive
   oxygen species that ultimately impairs protein quality control and
   skeletal muscle contractile capacity [[83][16], [84][17], [85][18],
   [86][19]]. Therefore, oxidative stress seems a major regulator of
   muscle mass and function, and current studies are necessary to
   determine whether preventing disrupted oxidative metabolism at early
   stages would counteract muscle atrophy and weakness in cancer cachexia.

   Aerobic exercise training improves muscle oxidative metabolism and
   induces pleiotropic effects, including altered rates of blood flow,
   energy production, and substrate utilization [[87]20]. Exercise
   training has been extensively applied as a non-pharmacological
   treatment for endocrine, cardiovascular, and neurological diseases
   [[88][21], [89][22], [90][23], [91][24], [92][25], [93][26]], and
   recent studies have suggested that exercise training is also safe and
   tolerable for cancer patients during and following primary cancer
   therapy [[94][27], [95][28], [96][29]]. Although well-controlled
   studies investigating the specific effects of exercise training in
   advanced cancer patients are challenging because of severe fatigue and
   concurrent therapies, exercise training also seems to promote benefits
   in cancer cachexia animal models [[97]16,[98][30], [99][31], [100][32],
   [101][33], [102][34], [103][35]]. However, the molecular mechanisms
   underlying the beneficial effects of exercise training in cancer
   cachexia are poorly understood.

   In our study we tested the hypothesis that exercise training would
   counteract metabolic impairment in a model of cancer cachexia. We first
   established a cancer rat model with homogeneous tumor growth and
   mortality rate, and we determined the effects of different exercise
   training protocols in this model. To explore the mechanisms underlying
   the effects of exercise training, we applied an unbiased
   mass-spectrometry-based proteomic screening in the skeletal muscle of
   sedentary and trained tumor-bearing rats. Our main findings indicate
   that COP9 signalosome complex subunit 2 (COPS2), also known as thyroid
   receptor interacting protein 15 (TRIP15) or ALIEN, is one of the most
   downregulated proteins in skeletal muscle at the early stage of cancer
   cachexia, and its expression is normalized by exercise training. Thus,
   we performed a series of in vitro studies to investigate the potential
   role of COPS2 to maintain homeostasis in muscle cells.

2. Materials and methods

2.1. Ethics

   This study was approved by the Ethical Committee of the School of
   Physical Education and Sport, University of São Paulo. All animal
   procedures were performed in accordance with the Guidelines for the
   Care and Use of Laboratory Animals (National Institutes of Health,
   USA), and with ethical principles in animal research adopted by the
   Brazilian Council for the Control of Animal Experimentation. Human
   experiments were approved by the Ethical Committee of Instituto do
   Cancer do Estado de São Paulo, University of São Paulo (protocol
   #1.731.362) and written informed consent was obtained from all
   participants.

2.2. Animal models

   Ten-week-old male Wistar rats and C57BL/6 mice were used in this study.
   The sample size used for each experiment is indicated in the figure
   legends. Animals were housed in an animal facility under controlled
   temperature (21 °C) with 12:12 h light:dark cycle and had ad libitum
   access to standard laboratory food and water, except for the pair-fed
   experiment in which the amount of food provided to a healthy control
   group of rats was matched daily to that consumed by the tumor-bearing
   experimental group. To induce bone cancer in rats, Walker 256 tumor
   cells were injected into the femoral cavity as previously described
   [[104]36]. Suspensions of tumor cells in 5 μL of PBS were used for
   injection in the bone marrow. SHAM surgery was performed on the control
   rats. Dipyrone (Medley Farmacêutica Ltda., Brazil), an ampyrone
   sulfonate analgesic, was administered through the water during the
   entire protocol to minimize rat suffering. LLC or B16 tumor cells were
   injected subcutaneously in the right flank as previously described
   [[105]31]. One day following tumor cell injection, mice were randomly
   assigned into experimental groups. Rats were euthanized by decapitation
   under isoflurane anesthesia and mice were euthanized by cervical
   dislocation under isoflurane anesthesia. For ethical purposes, rats and
   mice were euthanized if they appeared moribund, indicating a low
   probability of surviving for greater than 24 h, and were removed from
   the analysis.

2.3. Human studies

   We recruited six male patients with histologically confirmed metastatic
   non-small-cell lung cancer (NSCLC). The patients were diagnosed with
   either squamous cell carcinoma (n = 3) or adenocarcinoma (n = 3) and
   were not previously treated with any cancer therapy. We also recruited
   4 age- and sex-matched control subjects. All patients with NSCLC and
   the control subjects were tobacco smokers. This is a sub-cohort of
   study [106]NCT03960034 registered on clinicaltrial.gov. Inclusion
   criteria included a) advanced stage IVa or IVb histologically-proven
   patients with NSCLC; b) Eastern Cooperative Oncology Group Performance
   status 0–2 treatment-naïve; c) current smokers or ex-smokers; d) normal
   renal, hepatic, and hematological functions; e) ability to perform the
   physical functional tests; and f) ability to to read and sign the
   consent form. Exclusion criteria included a) any previous systemic
   treatment for metastatic disease, and b) diagnosis of tumor driver
   mutation (e.g., epidermal growth factor receptor, anaplastic lymphoma
   kinase). Medications in use in this cohort included Metformin (1
   patient and 1 control subject), Enalapril (1 patient), Dimenhydrinate
   (1 patient), Losartan potassium (1 patient and 1 control), Tandrilax (1
   patient), and Dipyrone (1 patient). To provide proof-of-concept, we
   included only cancer patients in pre-cachexia stages because our
   proteomic screening from tumor-bearing rats was performed in an early
   stage of cachexia before an established muscle wasting condition. This
   allowed us to apply physical tests to determine the VO2 peak and muscle
   biopsies for further immunoblot assay. Patients with NSCLC had lower
   hemoglobin levels and higher albumin levels than control subjects, as
   shown in [107]Table S4. No cases with anorexia were included in this
   study and patients with NSCLC and control subjects had similar food
   intake. Cardiopulmonary exercise testing was conducted to determine
   VO[2]peak as previously described [[108]82]. Percutaneous muscle
   biopsies were obtained from the vastus lateralis muscle using a 5-mm
   modified Allendale-Bergstrom needle [[109]82]. Local anesthesia with
   1–2 mL of lidocaine 2% solution was performed. Muscle samples were
   immediately frozen in liquid nitrogen and subsequently stored
   at −80 °C. Exercise testing and muscle biopsy procedures were performed
   during the same week, with an interval of at least 3 days between each
   procedure. The experiments were conducted at the Instituto do Cancer do
   Estado de São Paulo and Instituto do Coração, HCFMUSP, São Paulo,
   Brazil.

2.4. Cell culture

   Human skeletal myoblasts (Thermo Fisher Scientific; A11440) were
   differentiated into myotubes in Dulbecco's modified Eagle's medium
   (DMEM; Gibco) supplemented with 2% horse serum and 1% pen/strep.
   Primary mouse myoblasts were isolated and differentiated into myotubes
   as previously described [[110]83,[111]84]. C2C12, HeLa, B16, and LLC
   cells were cultured in DMEM supplemented with 10% fetal bovine serum
   and 1% pen/strep. To collect conditioned media, HeLa, B16, and LLC
   cells were plated at 50% confluency and incubated in DMEM serum-free
   media for 24 h. For incubation in myotubes, cell-conditioned media were
   mixed with fresh media (ratio 1:1) and supplemented with 5% horse serum
   (or 2% for C2C12 experiments). Primary mouse myoblast-derived myotubes
   were incubated with conditioned media for 12 h, 24 h, 36 h, or 48 h
   depending on each specific readout. Myotubes were washed with saline
   before protein or RNA extractions. Oxygen consumption rates (OCR) and
   extracellular acidification rates (ECAR) were measured in myotubes
   using extracellular flux analysis (XF96, Agilent Seahorse, MA USA) in
   sodium bicarbonate-free DMEM supplemented with 31.7 mM NaCl, 10 mM
   glucose, and 2 mM glutamax (pH 7.4 adjusted using NaOH) as previously
   described [[112]85]. MitoTracker Red (Thermo Fisher Scientific; M7512)
   was used to stain mitochondria in live myotubes according to the
   manufacturer's instructions. To overexpress COPS2, mouse myotubes were
   transduced with adenoviruses (MOI 100) expressing GFP or mouse COPS2
   (ADV-255916; Vector Biolabs, Malvern, PA, USA). Twelve hours after
   transduction, the medium was replaced with fresh differentiation
   medium. To knockdown COPS2, specific siRNA sequences (Thermo Fisher
   Scientific) were used for human (AM16708; Assay ID 140004) or mouse
   (AM16708; Assay ID 161305) cells using Lipofectamine RNAiMAX
   Transfection Reagent (Thermo Fisher Scientific; 13778-075) in Opti-MEM
   Media (Thermo Fisher Scientific; 31985062). Transfections with scramble
   siRNA (4390843) were used for the control groups. For experiments
   involving adenovirus transduction or siRNA transfection in C2C12
   myoblasts, media was replaced with DMEM supplemented with 2% horse
   serum and 1% Pen/Strep to avoid additional cell growth. All cells used
   in this study were maintained at 37 °C in 5% CO2.

2.5. Running capacity and exercise training protocols for mice and rats

   Rats and mice were subjected to a maximum incremental running test as
   previously described [[113]63]. The maximum speed achieved by each
   animal was recorded. Tumor cells or control solution were injected two
   days after the maximum incremental running test. Exercise training
   protocols started one day following injection and were comprised of
   daily treadmill running sessions for 10 consecutive days. Each aerobic
   interval training session consisted of 3 bouts at 85% of maximum speed
   for 4 min with intervals at 60% of maximum speed for 3 min. Animals in
   the aerobic continuous training protocol ran at 60% of maximum speed,
   and the duration was adjusted to match the total volume of the aerobic
   interval training sessions, resulting in the same total running
   distance between aerobic interval and continuous training
   [[114]31,[115]54]. Running speed was adjusted as cachexia progressed to
   maintain the relative exercise intensity (% of max running speed)
   throughout the study. Running speed was reduced by 20% when the animal
   failed to exercise at the target intensity. The same reduced intensity
   was then applied from the onset of the next session. Most of the rats
   injected with Walker 256 tumor cells had exercise intensities reduced
   between the 8th and 10th day post-injection (dpi).

2.6. Ex vivo skeletal muscle contractility

   Contractile capacity of the extensor digitorum longus (EDL) muscle was
   measured. The EDL was carefully harvested and immediately used for
   ex vivo experiments. The muscles were mounted between force transducers
   in a tissue bath containing aerated Krebs Ringer's buffer and
   platinum-based electrodes for electrical stimulation. Force output was
   recorded after successive electrical stimulation as previously
   described [[116]31,[117]63].

2.7. Circulating lactate, cytokine concentrations and insulin tolerance test

   Serum lactate and plasma glucose were determined using
   spectrophotometric techniques [[118]86]. IL-1β, IL-6, IL-10, and TNF-α
   concentrations were determined from the serum and protein extracted
   from plantaris muscle using the Milliplex Map, rat cytokine/chemokine
   magnetic bead panel (Millipore, Sigma, Switzerland) according to the
   manufacturer's instructions. To test insulin tolerance, rats were
   fasted for 6 h and administered insulin (0.75 units/kg body weight) by
   intraperitoneal injection. Blood samples were collected to measure
   plasma glucose concentration at specified time points after injection.

2.8. Glutathione redox status, citrate synthase and catalase activities

   Glutathione levels were measured with the Glutathione Fluorescent
   Detection Kit (K006-F5; Arbor Assay, USA). To block free GSH,
   2-Vinylpyridine (2VP) was used according to the manufacturer's
   instructions. Citrate synthase activity was determined using the
   Citrate Synthase Assay Kit (CS 0720, Sigma–Aldrich, USA). Catalase
   activity was determined by the hydrogen peroxide (H[2]O[2])
   decomposition, which was assessed by following the decay in sample
   absorbance at 240 nm in the presence of 10 mM H[2]O[2].

2.9. Protein carbonyls, dihydroethidium fluorescence staining and lipid
hydroperoxides

   Protein carbonyls were assessed with the OxyBlot Protein Detection Kit
   (S7150; Millipore, Switzerland). Soluble proteins (20 μg) were
   denatured by SDS and derivatized by Dinitrophenylhydrazine (DNPH).
   Proteins were subjected to electrophoresis and immunoblotting according
   to the manufacturer's instructions. For dihydroethidium (DHE)
   fluorescence staining, muscle cross-sections (10 μm) were incubated
   with DHE (5 μM) in a light-protected incubator at 37 °C for 30 min. The
   sections were washed with phosphate buffered saline and fluorescence
   was assessed by confocal microscopy. Quantitative analysis of
   fluorescent images was performed with ImageJ (NIH, USA). Lipid
   hydroperoxides were evaluated using the modified ferrous
   oxidationxylenol (FOX) orange technique [[119]65], and malondialdehyde
   (MDA) and 4-hydroxyalkenals (HAE) were assessed using an ALDetect Lipid
   Peroxidation Assay Kit (Enzo Life Sciences International, NY, USA).

2.10. Proteasome activity

   26 S proteasome activity was measured by using a substrate for
   proteasome chymotrypsin-like catalytic site (Suc LLVY-AMC, Enzo Life
   Sciences, USA) as previously described [[120]87]. Fluorescent product
   formation was followed in the presence or absence of epoxomicin
   (20 μM), a highly specific inhibitor of chymotrypsin-like proteasome
   activity, and the difference between the two rates was considered 26 S
   proteasomal activity.

2.11. Gel free proteomics and mass spectrometry analysis

   Plantaris muscle extracts were homogenized in degassed 2D buffer (7 M
   Urea, 2 M Thiourea, 2.5% CHAPS) without DTT (dithiothreitol) at pH 5.0.
   Protein (600 μg) was precipitated in methanol (4 V), chloroform (1 V),
   and H2O (3 V) and frozen for further labeling to proceed with gel-free
   proteomics. Each sample was digested with trypsin and purified and
   analyzed by mass spectrometry. For gel-free proteomics in the Walker
   rat model muscle samples, technical duplicates were performed for each
   of the 5 biological independent biological samples. For gel-free
   proteomics in B16 mouse model samples, technical duplicates were
   performed in a pool of 8 samples in each experiment. Peptides were
   analyzed on an LC-MS/MS platform consisting of an Easy-nLC 1000 UHPLC
   system (Thermo Fisher Scientific Inc, USA) interfaced with an
   LTQ-Orbitrap Elite hybrid mass spectrometer (Thermo Fisher Scientific
   Inc, USA) via a nanospray ESI ion source (Proxeon, Odense, Denmark) as
   previously described [[121]38].

   Raw data files were analyzed in Proteome Discoverer 1.4 (Thermo Fisher
   Scientific Inc., US) using the SEQUEST HT search engine with the
   December 2013 version of the rat protein sequence database from UniProt
   (UniProt Consortium). The enzyme specified as trypsin with a maximum of
   two allowed missed cleavages was searched. Precursor mass tolerance was
   10 ppm and fragment mass tolerance was 0.6 Da. The Percolator tool was
   used for peptide validation and a cutoff value of 0.01 was used for
   false discovery rate. Only peptides with high confidence were used for
   final protein identification. To further understand the biological
   relevance of the identified proteins, we performed functional
   enrichment analysis in the context of the Kyoto Encyclopedia of Genes
   and Genomes (KEGG) databases using the Enrichr43 and QIAGEN’S Ingenuity
   Pathway Analysis (IPA).

   The mass spectrometry (MS) proteomics data have been deposited into the
   ProteomeXchange Consortium via the PRIDE partner repository with the
   dataset identifier PXD013226 [[122]88]. Proteins were quantified by
   processing MS data using Max Quant [[123]89]. Preview 2.3.5 [Protein
   Metrics Inc.] was used to inspect the raw data to determine the optimal
   search criteria [[124]90]. Specifically, the following search
   parameters were used: enzyme specified as Trypsin with maximum two
   missed cleavages allowed; protein N-terminal acetylation,
   carbamidomethylation of cysteine and oxidation of methionine as dynamic
   post-translational modification. These were imported into MaxQuant
   which uses m/z and RT values to align each run against every other
   sample with a 2-minute window match-between-run function and a
   20-minute overall sliding window using a clustering-based technique.
   These are further queried against the rat proteome downloaded from
   Uniprot ([125]https://www.uniprot.org/proteomes/UP000002494) and
   MaxQuant's internal contaminants database using Andromeda built into
   MaxQuant. Both protein and peptide identifications FDR were set to 1%
   thus only peptides with high confidence and a minimum length of 7 amino
   acids were used for final protein group identification. Peak abundances
   were extracted by integrating the area under the peak curve. Each
   protein group abundance was normalized by the total abundance of all
   identified peptides for each run and protein by calculated median
   summing all unique and razor peptide ion abundances for each protein
   using a label free quantification (LFQ) algorithm [[126]89] with
   minimum peptides ≥ 1. These LFQ values were log transformed with base
   2.

2.12. Immunoblotting and immunofluorescence

   Protein isolation and immunoblotting were performed as previously
   described [[127]65,[128]91]. Primary antibodies were incubated
   overnight and used to probe for COPS2/TRIP15/ALIEN using Abcam
   Anti-CSN2 (ab83225; not commercially available anymore) or Bethyl CSN2
   antibody (Cat# A300-027 A). The c-terminal Abcam Anti-CSN2 (Cat#
   ab155774) was used for the specific immunofluorescence experiments in
   C2C12 myoblasts. Ponceau or GAPDH (Cell Signaling Technology, USA,
   2118 S and Santa Cruz, USA, #sc-20358) were used as loading controls.
   Membranes were imaged using the ChemiDoc Touch System (Bio-Rad, USA) or
   the LI-COR Odyssey Infrared Imaging System (LI-COR, Inc., USA). To
   understand antibody binding in COPS2 structure, PYMOL was used where a
   COPS PBD file was downloaded from Unitprot and the sequence was
   uploaded for structural analysis. COPS2 (CSN2 subunit) was colored
   blue, the N-terminal was colored green and the C-terminal 62 residue
   sequence targeted by the Abcam antibody (Cat# ab155774) was colored red
   as shown in [129]Figure S8A. Alexa Fluor™ 488 Phalloidin (Thermo Fisher
   Scientific; A12379) was used to stain F-actin in myotubes.

2.13. Microarray analyses

   Human myotubes transcriptome was determined using Clariom S Assay,
   human (Thermo Fisher Scientific) using the Thermo Fisher Scientific
   facility services (Santa Clara, CA, USA). All data analysis was done in
   R [[130]92], using the packages “Limma” [[131]93] and “Oligo” [[132]94]
   through Bioconductor. Affymetrix data were first normalized by using
   robust multi-array averages. These normalized data were then fit
   through a linear model. The empirical Bayes statistics for differential
   expression was used to calculate all statistical values. Graphs were
   generated using ggplot2 [[133]95]. Microarray data were submitted to
   NCBI Gene Expression Omnibus (GEO) Access code: [134]GSE151591.

2.14. Quantitative RT-qPCR

   RNA was isolated from tissue using an RNA extraction kit (Direct-zol™
   RNA MiniPrep, Zymo Research, Irvine, CA, USA) or RNeasy Plus Universal
   Kits (Qiagen, Hilden, Germany). RNA was reverse-transcribed using
   standard reagents (High Capacity Reverse Transcription Kits, Applied
   Biosystems) and cDNA was amplified with Power SYBR Green PCR master mix
   (Applied Biosystems), using the ABI 7900HT real-time PCR system. For
   each gene, mRNA expression was calculated relative to Hprt for mouse
   cell lines and rats, and relative to TBP for human cells. Primer
   sequences are provided in [135]Table S5.

2.15. Preparation of plasmid vectors and luciferase reporter assay

   Vector pGL4.10 [luc2] (Promega) was linearized by digesting with
   HindIII and XhoI (New England Biolabs), and was then purified using the
   QIA quick PCR purification kit (Qiagen 28104) Two ssDNA oligos (DR4
   sequence with overhangs) were annealed to form a dsDNA oligo with
   specific HindIII and XhoI overhangs. The linearized vector and dsDNA
   oligo were ligated together using T4 DNA ligase (NEB) according to
   manufacturer protocol. The product was then transformed into competent
   cells and plated onto ampicillin agar plates. Single colonies were
   picked the following day and grown in LB overnight. The cells were then
   harvested and mini prepped with Qiaprep spin Miniprep Kit (Qiagen
   27104). Purified plasmid DNA was sent for sequencing to confirm
   successful and correct ligation. A Dual-Luciferase Reporter Assay
   System (Promega) was used to determine DR4 luciferase activity
   (firefly) and each individual well was normalized via Renilla
   luciferase internal control.

2.16. Statistical analysis

   Values are presented as mean ± standard error (SE). Individual values
   are also presented for in vivo data. Analyses were conducted using
   Graph Pad Prism 8 (Graph Pad Software Inc., USA). Unpaired Student's
   t-test was used to test the differences between the two experimental
   groups. One- or two-way analysis of variance (ANOVA) was used to
   compare more than two experimental groups. Whenever significant effects
   were found in ANOVA, Fisher's least significance difference test was
   used for multiple comparison purposes. A log-rank test was performed to
   compare survival rate. Statistical significance was set at p < 0.05.

3. Results

3.1. Injection of Walker 256 tumor cells into the bone marrow induces severe
cancer cachexia in rats

   To establish a model of cancer cachexia with homogeneous tumor growth
   and mortality rate, we injected Wistar rats with 10^6 Walker 256 tumor
   cells subcutaneously in the right flank or in the bone marrow via an
   osteotomy in the femur [[136]36]. We observed a more homogeneous
   mortality rate ([137]Figure S1A), greater decline in the ambulation
   test performance ([138]Figure S1B), and greater mechanical hyperalgesia
   ([139]Figure S1C) in rats injected with tumor cells in the bone marrow
   than in rats injected subcutaneously in the flank. Furthermore, we
   observed solid tumors in all rats injected in the bone marrow, while 4
   of 24 of rats (16%) injected subcutaneously did not present visual
   tumors until 20 days post injection (dpi) and were removed from the
   experiment. We next injected different concentrations of tumor cells in
   the bone marrow and found that higher concentrations resulted in a more
   homogeneous mortality rate ([140]Figure S1D). Thus, all subsequent
   experiments were done with rats injected in the bone marrow with 10^6
   Walker 256 tumor cells, and the term “tumor-bearing rats” will be used
   from this point onward to refer to this model.

   To further determine whether tumor-bearing rats develop cancer
   cachexia, we assessed changes in total and lean mass as well as in
   ambulation performance. Tumor-bearing rats had consistent reduction in
   total and lean mass ([141]Figure 1A–D) in conjunction with a decline in
   the ambulation test performance ([142]Figure 1E). Tumor-bearing rats
   also had higher circulating lactate concentrations ([143]Figure 1F) and
   lower basal glucose levels compared with healthy control rats
   ([144]Figure 1G). Tumor-bearing rats did not show changes in food
   intake until 10 dpi, but demonstrated a significant reduced intake
   starting at 12 dpi ([145]Figure 1H,I). To investigate whether changes
   in the muscle mass were secondary to reduced food intake, we performed
   a pair-fed experiment in which the daily food intake of healthy control
   rats was paired to that of tumor-bearing rats. Tumor-bearing rats had
   decreased plantaris and gastrocnemius muscle mass, while pair-fed
   healthy control rats showed preserved muscle mass compared with the ad
   libitum-fed control group ([146]Figure 1J). Soleus muscle mass was not
   affected by neither cancer nor reduced food intake ([147]Figure 1J).
   Collectively, these initial findings demonstrate that Walker 256
   tumor-bearing rats is a model of severe cancer cachexia with
   homogeneous tumor growth and mortality rate, and skeletal muscle
   atrophy is not only a consequence of reduced food intake.

Figure 1.

   [148]Figure 1
   [149]Open in a new tab

   Bone marrow injection of Walker 256 tumor cells in rats. (A)
   Representative DXA images of Walker 256 tumor-bearing rats at 16 days
   post injection (dpi). (B) Body mass delta changes in Walker 256
   tumor-bearing rats. n = 15–21. (C) Lean mass. n = 5–6. (D)
   Representative images of left hindlimb. (E) Ambulation test. n = 10–12.
   (F) Circulating lactate levels. n = 6–11. (G) Insulin tolerance test.
   n = 6–8. (H) Food intake assessed in group-housed rats. n = 4–5. (I)
   Food intake assessed in single-housed rats in metabolic cages. 7–8. (J)
   Plantaris, Gastrocnemius, and Soleus mass relative to controls.
   n = 5–6. (K) Running capacity. n = 10. (L) Correlation between reduced
   running capacity (pre–post delta change) and survival after tumor cell
   injection. n = 10. (M) Study design to test exercise training effects.
   Walker 256 tumor-bearing rats were subjected to aerobic interval (AIT)
   or continuous (ACT) training. Data are presented as mean ± s.e.m.
   ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001. n.s. not significant.

3.2. Exercise training improves running capacity and prolongs survival in
Walker 256 tumor-bearing rats

   Tumor-bearing rats had impaired running capacity ([150]Figure 1K),
   which was directly correlated with time of death after tumor cell
   injections ([151]Figure 1L). Therefore, the data suggest that running
   capacity predicts longevity in this cancer model. To test whether
   exercise training would improve running capacity, we subjected
   tumor-bearing rats to two different protocols of aerobic exercise
   training (interval and continuous), and we evaluated their running
   capacity at 12 dpi ([152]Figure 1M). Because reduced running capacity
   was associated with a reduced lifespan, we also evaluated the effects
   of these two protocols on the mortality rate. Both aerobic interval and
   continuous training improved running capacity ([153]Figure 2A) and
   prolonged the lifespan ([154]Figure 2B), with no significant changes in
   the tumor size ([155]Figure S2A) or circulating serum TNFα levels
   ([156]Figure S2B). Additionally, tumor-bearing rats had increased
   wet/dry lung mass difference, a marker of lung edema [[157]37], which
   was normalized by both aerobic interval and continuous training
   protocols ([158]Table S1). These data demonstrate that aerobic interval
   training improves running capacity and prolongs survival in
   tumor-bearing rats.

Figure 2.

   [159]Figure 2
   [160]Open in a new tab

   Effects of exercise training on running capacity, survival rate and
   skeletal muscle in Walker 256 tumor-bearing rats. Tumor-bearing rats
   were submitted to aerobic interval (AIT) or continuous (ACT) training.
   (A) Running capacity. n = 22–23 rats. (B) Survival rate. n = 26–27
   rats. Different letters indicate significant difference between groups.
   (C) Citrate synthase activity. n = 6–11 rats. (D) Total glutathione and
   (E) ratio between reduced (GSH) and oxidized (GSSG) glutathione.
   n = 6–10 rats. (F) Quantification of carbonyl protein levels. n = 6–8
   rats. (G) 26 S proteasome activity. n = 6–11 rats. (H) Skeletal muscle
   mass normalized to tibia length. n = 4–6 rats. (I) Muscle contraction
   capacity at 18 dpi. n = 3 rats. Data are presented as mean ± s.e.m.
   ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.001. n.s. not
   significant. dpi: days post injection.

3.3. Exercise training restores redox homeostasis and improves muscle
function in tumor-bearing rats

   To determine the effects of exercise training on the skeletal muscle of
   tumor-bearing rats, we designed new experiments involving only aerobic
   interval training because our data indicated similar effects on running
   capacity between aerobic interval and continuous training protocols. We
   observed reduced activity of citrate synthase in the plantaris muscle
   from tumor-bearing rats, while exercise training partially
   reestablished citrate synthase activity toward control levels
   ([161]Figure 2C). Furthermore, plantaris muscle from tumor-bearing rats
   had changes in several makers of oxidative stress, including reduced
   levels of total glutathione ([162]Figure 2D) and GSH:GSSG ratio
   ([163]Figure 2E), as well as increased levels of 4-hydroxyalkenals
   (HAE) ([164]Figure S2C), malondialdehyde (MDA) ([165]Figure S2D), and
   protein carbonyl levels ([166]Figure 2F). MDA and HAE are indicators of
   lipid peroxidation and protein carbonylation is an irreversible
   oxidation reaction and a biomarker of oxidative damage
   [[167]38,[168]39]. Exercise training did not change MDA and HAE levels
   ([169]Figures S2C–D), however, it did remarkably increase glutathione
   levels ([170]Figure 2D–E) and decrease protein carbonylation
   ([171]Figure 2F). There were no significant changes for catalase
   activity ([172]Figure S2E) or fluorescent DHE oxidation products
   ([173]Figure S2F) among the experimental groups. Finally, we evaluated
   the proteasome activity in the plantaris muscle because the
   ubiquitin-proteasome proteolytic system is responsible for the removal
   of damaged proteins and plays a major role in protein quality control
   [[174]16,[175]40]. Interestingly, aerobic interval training increased
   proteasomal activity by ∼2-fold ([176]Figure 2G).

   We analyzed skeletal muscle mass from tumor-bearing rats at early (11
   dpi) and late (18 dpi) stages of cancer cachexia. Analyses at 18 dpi
   were restricted to about half of the initial cohort that survived until
   this stage. We did not find significant differences among all
   experimental groups in the skeletal muscle mass at 11 dpi
   ([177]Figure 2H and [178]Figure S2G). Tumor-bearing rats had muscle
   atrophy in glycolytic muscles at 18dpi ([179]Figure 2H), with no
   significant differences in soleus muscle ([180]Figure S2G). Remarkably,
   exercise training attenuated muscle mass atrophy ([181]Figure 2H) and
   fully restored the muscle contractile function to the control levels
   ([182]Figure 2I and [183]Fig. S2H). Collectively, these data indicate
   that exercise training reduces protein carbonyl levels, normalizes
   GSH:GSSG ratio, and increases proteasomal activity in the plantaris
   muscle of tumor-bearing rats, likely contributing to restoration of
   redox homeostasis and protein quality control. Additionally, exercise
   training attenuates muscle atrophy and improves skeletal muscle
   contractile function in tumor-bearing rats.

3.4. Aerobic interval training reverses changes in the skeletal muscle
proteomic profile in tumor-bearing rats

   To explore the mechanisms underlying the benefits induced by exercise
   training during cachexia progression, we performed an unbiased gel-free
   mass-spectrometry-based proteomic analysis in the serum and plantaris
   muscle from 1) healthy control rats, 2) tumor-bearing rats, and 3)
   tumor-bearing rats submitted to the aerobic interval training protocol.
   Because our aim was to identify changes that occurred prior to onset of
   muscle atrophy, we used samples collected at 11 dpi.

   Exercise training did not change the expression of proteins affected in
   the serum of tumor-bearing rats ([184]Figures S3A–C) which led us to
   speculate that the main effects of exercise training would occur
   directly in the skeletal muscle tissue. In this content, plantaris
   proteomics identified a total of 1370 proteins across all experimental
   groups, with 59 proteins significantly downregulated and 14 proteins
   significantly upregulated in the tumor-bearing rats ([185]Figure 3A–B
   and [186]Table S3). Ingenuity Pathway Analysis using differently
   expressed proteins revealed Eukaryotic Initiation Factor 2 (EIF2)
   signaling, liver X receptor/retinoid X receptor (LXR/RXR) activation,
   farnesoid X receptor/retinoid X receptor (FXR/RXR) activation, and
   acute phase response signaling as the most significantly affected
   pathways in tumor-bearing rats ([187]Figure 3B). We next verified the
   effects of exercise training on the 73 proteins regulated in the
   plantaris muscle from tumor-bearing rats ([188]Figure 3C). Exercise
   training significantly normalized the expression of 9 proteins and did
   not significantly increase or decrease any protein to the same
   direction of the cancer effects ([189]Figure 3C). As a particularly
   interesting protein for further investigation, COPS2 was one of the
   most downregulated proteins in tumor-bearing rats and its expression
   was fully restored by exercise training as demonstrated by mass
   spectrometry ([190]Figure 3D) and immunoblot ([191]Figure S4A)
   analysis. Basically, COPS2 mRNA levels were not changed in
   tumor-bearing rats ([192]Figure S4B), suggesting that reduced COPS2
   protein content is a consequence of posttranslational modifications.

Figure 3.

   [193]Figure 3
   [194]Open in a new tab

   Effects of aerobic interval training (AIT) on muscle proteome in
   tumor-bearing rats and mice. (A) Volcano plot comparing the profile of
   the plantaris proteomes of control and tumor-bearing rats. n = 5 rats.
   (B) Number of proteins differentially expressed in the plantaris muscle
   from tumor-bearing rats. Overlap indicates proteins were not changed
   between groups Pathway enrichment analysis using upregulated and
   downregulated proteins. n = 5 rats. (C) Volcano plot showing the
   effects of AIT specifically in proteins changed in tumor-bearing rats.
   n = 5 rats. (D) COPS2 protein content in the plantaris muscle. n = 5
   rats. (E) Running capacity and (F) survival rate in B16 tumor-bearing
   mice submitted or not to AIT. n = 11–14. (G) COPS2 protein content in
   the plantaris muscle in B16 tumor-bearing mice submitted or not to AIT.
   n = pool of 8 mice for each group. (H) COPS2 protein content in the
   plantaris muscle in LLC tumor-bearing mice. n = 9–10 mice. (I) VO[2]
   peak from patients with NSCLC prior to first line therapy. n = 4–6
   subjects. (J) Quantification and representative immunoblot for COPS2
   protein content in the vastus lateralis muscle from patients with
   NSCLC. n = 4–6 subjects. Data are presented as mean ± s.e.m. ∗P < 0.05;
   ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.001; n.s. not significant. dpi:
   days post injection.

   We simultaneously we performed an experiment to test whether exercise
   training would affect COPS2 protein content in healthy control rats.
   Exercise training increased glutathione levels in plantaris muscle of
   healthy rats ([195]Figure S5) without additional effects in carbonyl
   levels, catalase activity, 26 S proteasome activity, and muscle mass
   ([196]Figures S5A–E), suggesting that the beneficial effects of
   exercise training are greater in rats under catabolism conditions than
   in healthy rats. Likewise, exercise training did not change COPS2
   protein content in healthy rats ([197]Figure S5F).

   To investigate whether the effects of cancer cachexia on COPS2
   expression were also recapitulated in another cancer model with
   exercise intolerance, we investigated sedentary and trained B16F10
   tumor-bearing mice. Consistent with data from the Walker 256 rat model,
   exercise training improved running capacity ([198]Figure 3E) and tended
   to prolong survival in B16F10 tumor-bearing mice ([199]Figure 3F)
   without affecting tumor progression ([200]Figures S6A–B). B16F10
   tumor-bearing mice had reduced running capacity but did not show
   significant body and muscle wasting by 16 dpi ([201]Figures S6C–D). At
   this same timepoint, mass spectrometry and immunoblot analysis
   demonstrated decreased COPS2 protein content in the plantaris muscle
   from B16F10 tumor-bearing mice, which was fully normalized by exercise
   training ([202]Figure 3G and [203]Figure S5E). These findings
   corroborate data from the Walker rat model, showing that exercise
   training improves running capacity and reverses cancer-induced decrease
   in muscle COPS2 protein expression.

3.5. Lewis lung carcinoma (LLC) tumor-bearing mice and lung cancer patients
have reduced muscle COPS2 protein content

   We previously demonstrated that exercise training attenuates tumor
   growth in Lewis lung carcinoma (LLC) tumor-bearing mice, resulting in
   obvious benefits as a result of reduced tumor growth, including
   improved muscle function and prolonged lifespan (Alves et al., 2018).
   We discovered that LLC tumor-bearing mice also present reduced levels
   of COPS2 protein content ([204]Figure 3H). Moreover, to determine
   whether muscle COPS2 protein content is associated with exercise
   tolerance in humans, we evaluated six patients with non-small-cell lung
   cancer (NSCLC), and four age- and gender-matched control subjects
   ([205]Table S4). The patients with NSCLC patients displayed lower
   VO[2]peak ([206]Figure 3I) than the control subjects. A vastus
   lateralis muscle biopsy was performed in the patients with NSCLC and
   control subjects under resting conditions. Immunoblot analysis revealed
   lower muscle COPS2 protein content in patients with NSCLC compared with
   age-matched controls ([207]Figure 3J). Thus, these findings demonstrate
   that patients with NSCLC who have impaired aerobic capacity have
   decreased muscle COPS2 protein content as observed in the rodent
   models.

3.6. Effects of COPS2 knockdown in primary human myotubes

   To investigate the molecular pathways regulated by COPS2 in myotubes,
   we used a specific siRNA to knockdown COPS2 in primary human myotubes
   ([208]Figure 4A,B). COPS2 knockdown did not change total protein
   content ([209]Figure 4C), suggesting that COPS2 knockdown does not
   induce substantial cell death or atrophy. Microarray analysis using 3
   independent experiments, and a cutoff at p < 0.05, exhibited 872 genes
   differently expressed after COPS2 knockdown ([210]Figure 4D).
   Functional enrichment analysis in the context of the Kyoto Encyclopedia
   of Genes and Genomes (KEGG) databases demonstrated cellular pathways
   affected by COPS2 knockdown, including multiple pathways related to the
   regulation of actin cytoskeleton ([211]Figure 4E). As demonstrated in a
   volcano plot ([212]Figure 4F) and validated using RT-qPCR
   ([213]Figure 4G), the two most downregulated genes were Alkylglycerone
   Phosphate Synthase (AGPS) and Cytoskeleton Associated Protein 4 (CKAP),
   while the two most upregulated genes were Chemerin Chemokine-Like
   Receptor 1 (CMKLR1) and Solute Carrier Family 7 Member 5 (SLC7A5).
   Among other important functions for cellular homeostasis of these
   genes, an interesting observation includes the upregulation of SLC7A5
   mRNA levels, which codes a membrane transporter to uptake large amino
   acids [[214]41] that can also mediate the transport of thyroid hormones
   translocation across the cell membrane [[215]42,[216]43]. Because COPS2
   was previously shown to act as a corepressor of thyroid hormones
   activity, we speculate that this increase in SLC7A5 mRNA levels could
   be an intrinsic compensatory cellular mechanism leading to increased
   thyroid hormone activity after COPS2 knockdown. Finally, we assessed
   whether these pathways could be associated with metabolic changes in
   myotubes with COPS2 knockdown using extracellular flux analysis.
   Interestingly, COPS2 knockdown decreased basal oxygen consumption rate
   (OCR) and increased basal extracellular acidification rate (ECAR)
   ([217]Figure 4H), indicating a basal metabolic switch in human
   myotubes. Thus, these experiments indicate that COPS2 knockdown can
   affect muscle cell homeostasis, including changes in pathways involved
   in the regulation of actin cytoskeleton.

Figure 4.

   [218]Figure 4
   [219]Open in a new tab

   Effects of COPS2 knockdown in primary human myotubes. (A) COPS2 mRNA
   expression 48 h after transfection with a specific siRNA for COPS2.
   n = 3 independent experiments. (B) Immunoblot demonstrating efficiency
   of COPS2 knockdown. (C) Total protein content 48 h after COPS2
   knockdown. n = 3 independent experiments. (D) Heatmap including genes
   differentially regulated by COPS2 knockdown. n = 3 independent
   experiments. (E) Most significant pathways affected by COPS2 knockdown.
   (F) Volcano plot with highlights for the genes most differentially
   regulated by COPS2 knockdown. n = 3 independent experiments. (G)
   RT-qPCR validation for the 4 genes most regulated by COPS2 knockdown.
   n = 3 independent experiments. (H) Energy map showing changes in oxygen
   consumption rate (OCR) and extracellular acidification rate (ECAR)
   after COPS2 knockdown. n = 11 technical replications. Data are
   presented as mean ± s.e.m. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗∗P < 0.001; n.s.
   not significant.

3.7. LLC-conditioned media decreases COPS2 protein content in primary mouse
myoblast-derived myotubes

   To test whether factors produced by tumor cells could directly affect
   muscle COPS2 protein content, we established an in vitro model by
   challenging primary mouse myoblast-derived myotubes with tumor
   cell-conditioned media ([220]Figure 5A). Myotubes incubated with B16 or
   LLC-conditioned media had progressive loss of protein content
   ([221]Figure 5B,C). HeLa cell-conditioned medium was used as an
   additional control group and did not affect the myotube total protein
   content. We decided to use the LLC cell line for the following
   experiments because the effects of LLC-conditioned media were more
   pronounced than B16-conditioned media ([222]Figure 5B–D). Notably,
   incubation with LLC-conditioned media significantly decreased COPS2
   protein content in the myotubes ([223]Figure 4E). Thus, these findings
   demonstrate that LLC cells produce factors that directly affect
   myotubes reducing COPS2 protein expression.

Figure 5.

   [224]Figure 5
   [225]Open in a new tab

   Effects of cancer-conditioned media in primary mouse myoblast-derived
   myotubes. (A) Experimental design to challenge myotubes with
   cancer-conditioned media. (B and C) Total protein content in myotubes
   treated for (B) 24 or (C) 48 h with media conditioned in HeLa, B16F10
   or LLC tumor cells. n = 3 independent experiments. (D) Representative
   images of myotubes treated for 48 h with LLC-conditioned media. (E)
   COPS2 protein expression in myotubes treated with LLC conditioned
   media. n = 3 independent experiments. (F) Cops2 mRNA expression 48 h
   after transduction with an adenovirus vector-COPS2. n = 3 technical
   replications. (G) Immunoblot demonstrating efficiency of COPS2
   overexpression. (H and I) Total protein content and representative
   images of myotubes with COPS2 overexpression with or without treatment
   with LLC-conditioned media for 48 h n = 3–4 technical replications. (J)
   Total protein content of myotubes with COPS2 knockdown with or without
   treatment with LLC-conditioned media for 48 h n = 12 technical
   replications. (K) Total glutathione. n = 5 technical replications. (L
   and M) F-actin expression. n = 3–5 technical replications. (N) 26 S
   proteasome activity. n = 3 technical replications. (O) Slc7a5 mRNA
   expression. n = 6 technical replications. Data are presented as
   mean ± s.e.m. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. n.s.
   not significant.

   To test whether COPS2 overexpression or COPS2 knockdown could protect
   myotubes from LLC-induced catabolism, we first used an adenovirus
   vector to overexpress full length COPS2 protein in differentiated mouse
   myotubes ([226]Figure 5F,G). COPS2 overexpression did not attenuate
   loss of protein levels ([227]Figure 5H,I) or enhance metabolic demand
   and glutathione levels ([228]Figs. S7A and B) induced by
   LLC-conditioned media in myotubes. Conversely, COPS2 knockdown
   ([229]Figure S7C) significantly reduced the loss of protein levels
   ([230]Figure 5J) and exacerbated glutathione levels induced by
   LLC-conditioned media ([231]Figure 5K), suggesting that a decrease in
   muscle COPS2 protein expression could be a compensatory mechanism to
   attenuate cancer effects. This occurred with no significant changes in
   OCR or ECAR in mouse myotubes ([232]Figure S7D). Neither COPS2
   knockdown nor LLC-conditioned media affected MitoTracker signal in
   mouse myotubes ([233]Figures S7E and F).

   Because our microarray analysis has demonstrated that COPS2 knockdown
   affects multiple pathways that could regulate actin cytoskeleton, we
   next studied whether COPS2 knockdown and/or LLC-conditioned media would
   affect polymeric actin filament (F-actin) expression in mouse myotubes.
   Interestingly, incubation of mouse myotubes with LLC-conditioned media
   decreased F-actin expression, while COPS2 knockdown increased F-actin
   in both control myotubes and myotubes incubated with LLC-conditioned
   media ([234]Figures 5L and M). Moreover, LLC-conditioned media
   increased 26 proteasome activity and decreased the Slc7a5 mRNA
   expression ([235]Figure 5O), and these effects were attenuated by COPS2
   knockdown ([236]Figure 5N and O). Therefore, these data demonstrated
   that incubation with LLC-conditioned media decreases COPS2 protein
   content in myotubes, while COPS2 knockdown seems to compensate most of
   the LLC-conditioned media-induced effects in myotubes, suggesting that
   decreased COPS2 protein levels could be an intrinsic compensatory
   mechanism to protect myotubes from the effects of cancer.

3.8. COPS2 regulates direct repeat 4 (DR4) response element activity in
myotubes incubated with LLC-conditioned media

   COPS2 is a key component of the COP9 signalosome complex, which
   regulates the Cullin-RING E3 ubiquitin ligases [[237][44], [238][45],
   [239][46]]. However, COPS2 also acts as a transcriptional corepressor
   by interacting with nuclear hormone receptors and this cellular
   function is independent of the COP9 signalosome complex [[240][47],
   [241][48], [242][49]]. Based on this cellular function, we hypothesized
   that LLC-conditioned media would affect the activity of nuclear hormone
   receptors and COPS2 would regulate this process through transcriptional
   corepressor activity. We therefore performed a series of experiments
   using C2C12 immortalized mouse myoblast cell line to test this
   hypothesis.

   We first evaluated whether incubation with LLC-conditioned media
   affects nuclear COPS2 protein expression in these myoblasts. The
   C-terminal of COPS2 protein interacts with the other subunits of the
   assembled COP9 signalosome complex, while the N-terminal does not
   present protein-protein interactions [[243]45]. Primary antibodies
   previously used in this study target the COPS2 N-terminal and,
   therefore, stains overall cellular COPS2 protein expression
   ([244]Figure S8A and B). On the other hand, a specific antibody that
   targets only the COPS2 C-terminal (Abcam; Cat# ab155774) specifically
   stains the nuclear COPS2 expression in immunofluorescence experiments
   ([245]Figure 6A). Using this specific C-terminal antibody, we found
   that incubation of myoblasts with LLC-conditioned media for 16 h
   decreased nuclear COPS2 protein expression by ∼50% ([246]Figure 6B).

Figure 6.

   [247]Figure 6
   [248]Open in a new tab

   Effects of LLC-conditioned media and COPS2 regulation on a specific
   direct repeat 4 (DR4) response element activity. (A) Immunofluorescence
   demonstrating specific nuclear staining for COPS2 using a specific
   C-terminal antibody in C2C12 myoblasts. (B) Quantification of nuclear
   COPS2 expression with or without treatment with LLC-conditioned media
   in C2C12 myoblasts for 16 h n = 6 independent experiments. (C)
   Experimental design to analyze the effects of LLC-conditioned media and
   COPS2 regulation on DR4 activity using a luciferase reporter assay. (D)
   DR4 activity after LLC-conditioned media treatment. n = 3 independent
   experiments. (E and F) DR4 activity after COPS2 (E) overexpression or
   (F) knockdown with or without cancer-conditioned media treatment. n = 3
   independent experiments. Data are presented as mean ± s.e.m. ∗P < 0.05,
   ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. n.s. not significant.

   Nuclear hormone receptors bind to hormone response elements in the DNA
   and regulate the gene expression in response to sensing molecules,
   including thyroid hormones [[249]50]. One of the most characterized
   thyroid hormone response elements is the direct repeat 4 (DR4), which
   is composed of two half-sites, such as the AGGTCA hexamer, interposed
   with 4 base pairs (e.g. AGGTCACTTCAGGTCA) [[250]50,[251]51]. We
   inserted a sequence with 2 of this DR4 response element in a
   luciferase-containing plasmid ([252]Figure S8C) and transfected into
   C2C12 myoblasts to further test the effects COPS2 overexpression or
   COPS2 knockdown with or without LLC-conditioned media incubation on DR4
   luciferase activity ([253]Figure 6C). LLC-conditioned media
   consistently decreased DR4 luciferase activity ([254]Figure 6D). COPS2
   overexpression also decreased DR4 luciferase activity without
   additional effects of LLC-conditioned media incubation
   ([255]Figure 6E). Notably, COPS2 knockdown not only increased DR4
   luciferase activity, but also inhibited the effects of LLC-conditioned
   media incubation ([256]Figure 6F). These experiments emphasize the role
   of COPS2 as a transcriptional corepressor and, most importantly,
   demonstrate that COPS2 is required to decrease DR4 activity in
   myoblasts incubated with cancer-conditioned media.

4. Discussion

   It is expected that more than 60% of patients with advanced stages of
   cancer will develop cachexia [[257]52]. Given the fast and devastating
   progression of cancer cachexia, identifying therapies to target
   skeletal muscle and improve oxidative metabolism at early stages of
   cachexia is a major research challenge. In the current study, we
   establish that exercise training counteracts cancer-induced oxidative
   stress and muscle wasting. While the mechanisms underlying cachexia are
   still not fully understood, for the first time our findings demonstrate
   that muscle COPS2 is one of the most downregulated proteins at the
   early stage of cachexia progression and exercise training can normalize
   muscle COPS2 to the control levels.

   Previous studies have demonstrated that high-intensity AIT promotes
   superior outcomes for cardiac patients [[258]53] and in a rat model of
   infarction [[259]54]. For example, an AIT protocol consisting of four
   bouts at 90% of maximal heart rate for 4 min, with intervals of 3 min
   at 70% of maximal heart rate, was superior to ACT protocol (70% of
   maximal heart rate) in reducing the blood glucose level, improving
   endothelial function and enhancing muscle biogenesis in metabolic
   syndrome patients [[260]55]. Therefore, in addition to traditional
   aerobic continuous training, we tested the effects of an aerobic
   interval training in this study. Our findings showed similar benefits
   on running capacity and lifespan after aerobic continuous training and
   aerobic interval training protocols in tumor-bearing rats. Thus, to
   simplify our study design in the next steps, we performed experiments
   with only aerobic interval training. With regard to the cancer cachexia
   models, we first tested the effects of exercise training in a severe
   cancer rat model with homogeneous tumor growth and mortality rate. To
   investigate whether the effects of exercise training would be
   recapitulated in another cancer model with exercise intolerance, we
   investigated sedentary and trained B16F10 tumor-bearing mice. Finally,
   we also provide data from the LLC tumor-bearing mice, in which we
   previously demonstrated that exercise training promotes important
   benefits [[261]31]. By using various models of cancer cachexia, this
   study extended conclusions from previous contributions and provides
   novel pre-clinical evidence that exercise training may be a beneficial
   co-therapy for cancer cachexia. One limitation of this study is that at
   this point we cannot extend any of our findings to models or patients
   undergoing chemotherapy or other treatments.

   A major finding of the current study is that exercise training
   prolonged the lifespan of tumor-bearing rats. We found that neither
   aerobic interval training nor aerobic continuous training could
   attenuate tumor progression in the Walker 256 (bone marrow) model,
   suggesting that improved survival was not due to a slower tumor growth
   rate. Respiratory failure is the main cause of death in cachectic
   cancer patients, and the most likely cause of death in the Walker 256
   model [[262]56,[263]57]. Both aerobic interval and continuous training
   protocols reduced lung edema in tumor-bearing rats. We thus speculate
   that reduced lung edema and secondary improvements of respiratory
   function are the major causes in the underlying prolonged lifespan in
   the tumor-bearing rats subjected to aerobic interval or continuous
   training protocols. While we focused on the effects of exercise
   training in the plantaris muscle in this study, future studies will be
   important to explore the diaphragm muscle function because it may
   explain the therapeutic effects of exercise on lung function.
   Additionally, skeletal muscle is a tissue profoundly affected by cancer
   but rarely inhabited by metastatic cancer cells [[264]58], and future
   studies are necessary to investigate the systemic effects of exercise
   training, including site-specific metastasis formation. Overall, the
   current findings suggest that exercise training is a potential adjuvant
   therapy for cancer patients. We advocate for the application of
   exercise protocols at early-stage development of cachexia and reinforce
   the importance of exercise protocols in the multimodal treatment for
   cancer cachexia [[265]59].

   Exercise training can increase the mobilization of immune cells to
   effectively target tumor cells and several studies have demonstrated
   beneficial effects of exercise training in de-escalating tumor growth
   in various animal models. Although the precise molecular mechanism of
   the underlying exercise training effects is not fully characterized, a
   previous study demonstrated that natural killer cell infiltration was
   significantly increased in B16 tumors from trained mice, and this
   effect was mediated by IL-6 signaling [[266]30]. Another
   exercise-induced effect is increased local vessel perfusion, which
   could facilitate the action of immune cells to suppress tumor growth
   [[267]60]. Among many other factors that could explain why not all
   exercise training protocols attenuate tumor progression in cancer
   models, we speculate that the main reason in our study was the rapid
   and severe progression of cancer in our model. The time-course of
   cancer progression in this model is most likely quicker than the
   adaptation induced by exercise training. In this context, most of the
   previous studies demonstrated benefits with exercise training beginning
   prior to tumor cell injection, and we consider that exercise training
   has limited effects when the protocol begins after tumor cell injection
   because it requires time to induce the appropriate adaptation. Other
   factors that could explain the various findings include the different
   cancer types and the location of cancer cell injection, as well as the
   high variability observed in some models such as in our B16
   tumor-bearing mice. Finally, identifying the best exercise training
   protocol to activate immune cells is a necessary step in future
   studies.

   Another striking finding of the current study is that exercise training
   improved the redox homeostasis in the skeletal muscle of tumor-bearing
   rats. Over the past years, our group has been studying the therapeutic
   effects of exercise training for cardiac cachexia. These studies
   demonstrated that cardiomyopathy is associated with systemic
   inflammation and exacerbated oxidative stress, leading to skeletal
   muscle atrophy and weakness, and that exercise training re-established
   the redox homeostasis and protein quality control toward control levels
   [[268][16], [269][17], [270][18],[271]54,[272]61]. For the first time
   we report that aerobic interval training improves redox homeostasis and
   attenuates muscle contractile dysfunction in tumor-bearing rats that
   survived more than two weeks. Of note, exercise training increased
   proteasome activity, which we understand to be an adaptation to remove
   oxidized proteins and maintain quality control, allowing novel protein
   synthesis. Furthermore, soleus muscle was the only assessed muscle that
   did not undergo atrophy in tumor-bearing rats. Soleus has a
   predominance of type I fibers and previous studies demonstrated that
   type I fibers display higher oxidative capacity and lower response to
   cachexia [[273]62]. This indicates that highly oxidative tissues have
   mechanisms to protect from the cachectic factors and this protection of
   oxidative muscle fibers has been attributed, at least in part, to the
   antioxidant capacity [[274][63], [275][64], [276][65]].

   Animal models have been useful in determining numerous mechanisms
   behind muscle wasting. However, animal models have important
   limitations and caution is necessary to translate those findings to
   patients. For example, a recent study reported that two of the most
   common xenograft mouse cachexia models do not recapitulate changes in
   gene expression of cachectic pancreatic ductal adenocarcinoma patients,
   a cancer type with a high prevalence of cachexia [[277]66]. For
   instance, Atg5 and Bnip3 mRNA expression, two genes related to
   autophagy process, were not changed in pancreatic ductal adenocarcinoma
   patients, but increased in skeletal muscle from both C26 and LLC mice
   [[278]66]. Importantly, other models such as orthotopic models or
   patient-derived xenograft exhibit improvements over classic xenograft
   models, but also present challenges in replicating human cancer
   cachexia. Finally, while tumor size usually represents less than 1% of
   body mass in humans, the tumor grows too fast and reaches more than 10%
   of the host body mass in most animal models. We have validated some of
   our findings in patients with lung cancer, showing that these patients
   have impaired aerobic capacity and decreased muscle COPS2 protein
   content as observed in the rodent models. In future studies, we will
   need to characterize other large cohorts, including other types of
   cancer and patients under treatment.

   We sought to determine the molecular mechanisms associated with muscle
   wasting in our model. To understand the implications of the proteins
   altered in the mass spectrometry analysis, we used pathway analysis to
   define the canonical cellular pathways affected at the early stage of
   cancer cachexia. We found that acute phase response signaling was one
   of the most upregulated pathways in the serum of tumor-bearing rats.
   This was not surprising, because the acute phase response is the innate
   systemic early-defense activated by neoplasia and inflammation
   [[279]67]. Two other affected pathways in the tumor-bearing rats were
   LXR/RXR activation and FXR/RXR activation. LXR/RXR and FXR/RXR
   heterodimers are members of the nuclear receptor superfamily and key
   players in the control of metabolic pathways, including lipid and
   glucose metabolism [[280][68], [281][69], [282][70], [283][71]]. RXR
   heterodimers act as ligand-dependent transcriptional regulators and
   increase the DNA-binding efficiency of their partners, including not
   only LXR or FXR, but also the peroxisome proliferator-activated
   receptor α (PPARα), retinoic acid receptor (RAR), vitamin D receptor
   (VDR), thyroid hormone receptor (TR), and others [[284][68], [285][69],
   [286][70], [287][71]]. Thus, future investigations should focus on the
   precise upstream mechanisms that regulate the activation or inhibition
   of RXR heterodimers during cancer cachexia progression.

   Muscle COPS2 was one of the most downregulated proteins in
   tumor-bearing rats and mice and aerobic interval training normalized
   the muscle COPS2 protein content. COPS2 is expressed in most tissues
   (gtexportal.org/home), acts as a transcriptional corepressor by
   interacting with nuclear hormone receptors [[288][47], [289][48],
   [290][49]], and it is also a component of the COP9 signalosome complex
   that is responsible for deneddylation of Cullin-RING E3 ubiquitin
   ligases [[291][44], [292][45], [293][46]]. A recent study reported that
   only COPS2, but not other members of the COPS signalosome complex, is
   implicated in pluripotency maintenance of human embryonic stem cells
   [[294]72]. The role of COPS2 for skeletal muscle homeostasis has not
   been previously determined. Because we did not observe changes in other
   subunits of the COP9 signalosome complex in tumor-bearing rats using
   mass spectrometry analysis, we suggest that decreased COPS2 protein
   content is more likely a result of changes in its corepressor role
   during the development of cancer cachexia. Interestingly, in our
   experiments COPS2 knockdown not only increased the DR4 response element
   activity, but also inhibited the effects of LLC-conditioned media in
   this DR4 activity, suggesting that COPS2 acts as a transcriptional
   corepressor and is required to mediate the effects of
   cancer-conditioned media incubation. Additionally, it is well
   established that binding of agonist ligands to RXR would result in
   dissociation of corepressors [[295]48,[296][68], [297][69], [298][70],
   [299][71]]. Based on this collective evidence, we speculate that COPS2
   is dissociated and is degraded as a compensatory mechanism to protect
   myotubes from the cachexia-induced factors under muscle-wasting
   conditions. However, because exercise training attenuates the oxidative
   stress and other potential stressors, this compensatory mechanism would
   not be activated. Finally, COPS2 knockdown affected multiple pathways
   that could regulate actin cytoskeleton and COPS2 knockdown increased
   F-actin expression in myotubes. The actin cytoskeleton is a collection
   of actin filaments and regulatory proteins that regulate cell
   contractility [[300]73,[301]74]. Indeed, the actin cytoskeleton is the
   primary force-generating machinery in the cell and while the mechanisms
   linking COPS2 cellular function to the regulation of actin cytoskeleton
   are unknown, we speculate that decreases in COPS2 could also be a
   compensatory process to attenuate the loss of contractile capacity in
   skeletal muscle. Future research will be necessary to understand these
   protein-protein interactions in the context of oxidative stress.

   Our findings demonstrate that LLC cells produce factors that directly
   affect myotubes reducing COPS2 protein expression. Identifying factors
   produced by tumors or other tissues in response to tumor progression
   that could induce muscle atrophy is of major scientific interest.
   Recent discoveries have uncovered some of these catabolic mediators,
   including fatty acids [[302]75], microvesicles [[303]76], TGF-β
   [[304]77], parathyroid hormone-related protein [[305]78], Ataxin-10
   [[306]79] and multiple pro-inflammatory cytokines [[307]14]. Upcoming
   studies investigating these factors in the context of DR4 response
   element activity in myoblasts/myotubes will be highly relevant.

   The current study has important clinical implications. Despite the
   variability commonly observed in human studies, we found reduced muscle
   COPS2 protein content in lung cancer patients compared with healthy
   controls. Cachexia is a negative prognostic in patients with NSCLC
   [[308]80], and identifying simple biomarkers that could detect skeletal
   muscle impairment prior to observable changes in the body composition
   is critical in clinical settings. While a muscle biopsy is invasive and
   difficult to incorporate into clinical practice, potential circulating
   factors associated with LXR/RXR or FXR/RXR activation, including
   COPS2/TRIP15/ALIEN, are potential candidates for testing. This study
   focused on the role of COPS2 in the skeletal muscle. However, by mining
   other studies, we found that Yang et al. (2016) applied a proteomics
   approach in the serum of gastric cancer patients and healthy controls
   and COPS2 was the most significant differently expressed protein
   between cancer patients and controls, leading the authors to conclude
   that COPS2 constitutes a novel biomarker for gastric cancer diagnosis
   [[309]81]. Based on these findings, we have initiated a study to
   determine circulating COPS2 levels in a large cohort of patient with
   lung cancer (clinicaltrial.gov [310]NCT03960034).

   In summary, the current study raises the concept of exercise training
   as a co-therapy for cancer cachexia and uncovers muscle COPS2 as one of
   the most downregulated proteins in cancer cachexia conditions which is
   normalized by exercise training.

Authors' contributions

   CRRA and PCB conceived and designed the study. CRRA, WN, NRA, VAV, GCT,
   LRGB, CGC and DF carried out animal procedures. CRRA and PRJ conducted
   in vitro procedures and assays. LH, AS, and GS carried out MS analysis.
   CRRA, WN, EJE, PRJ, VAV, GCT, LRGB, CGC, DF, and PCB acquired data. WN,
   MJNNA, CEN, and GCJ performed human studies. CEN, GS, UW, MFH, GCJR,
   RC, KJS, JLR, LJG, and PCB provided laboratory support and supervised
   the experiments. CRRA and EJE performed data analysis. CRRA drafted the
   manuscript. All authors interpreted the data, participated in
   manuscript review, and approved the final manuscript.

Acknowledgment