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  • The present results highlight the marked impair ment of


    The present results highlight the marked impair-ment of mitochondrial function in the muscle of cachectic TB mice. Whether reduced mitochondrial respiration is a cause or a consequence of muscle protein hypercatabolism is still unclear. Both condi-tions potentially occur: modulation of mitochondrial dynamics triggers muscle wasting [12,18], while proteasome and autophagy activation might pro-mote mitophagy [31]. A recent study demonstrates that mitochondrial damage and dysfunction precede the onset of muscle atrophy in LLC-bearing mice [32], suggesting that mitochondrial damage might be a driver of the wasting process in cancer cachexia. In the muscle of C26-bearing mice, the induction of genes pertaining to proteolysis, including those involved in autophagy and mitophagy, is associated with reduced expression of mitochondrial fusion-related genes, the more so in mice overexpressing TP53INP2. Both MFN2 and OPA1 deficiency were previously reported in sarcopenic muscles and the ablation of each single gene impaired mitochondrial quality control and triggered muscle atrophy [33,34]. Whether TP53INP2 directly regulates mitophagy and mitochondrial dynamics is still unknown; however, a sustained autophagy flux likely facilitates the removal of substrates targeted for degradation, including mitochondria. Future experiments stimulat-ing mitochondrial fusion and preventing mitophagy will clarify the implications of such alterations in driving cancer-related muscle wasting.
    In conclusion, the excessive autophagy induced in the muscle of TB mice in parallel to increased UPS activation causes the degradation of both proteins and mitochondria. Considering that bulk autophagy inhibition is not a viable strategy, targeting mitophagy and promoting mitochondrial quality/efficiency could represent a future therapeutic strategy aimed at supporting muscle function and possibly muscle 
    mass, when coupled to anabolic/anticatabolic drugs such as formoterol.
    Materials and Methods
    Animals and experimental design
    All animal experiments were performed in compli-ance with guidelines established by the Barcelona Science Park's Committee on Animal Care, or with the Italian Ministry of Health Guidelines and the Policy on Humane Care and Use of Laboratory Animals (NRC 2011). Mice were maintained on a regular dark–light Necrosulfonamide (light from 8 AM to 8 PM), with free access to food and water during the whole experimental period.
    Mouse strains
    Cancer cachexia was induced in 6-week-old male and female Balb/c mice (for C26 tumor) or 6-week-old male C57BL/6 mice (for LLC tumor) obtained from Charles River Laboratories.
    The transgenic mouse line (Tg) overexpressing TP53INP2 in skeletal muscle under the control of the myosin-light chain 1 promoter/enhancer was gener-ated in a C57BL/6 genetic background as described before [10]. For modeling cancer-induce cachexia, Tg mice were backcrossed with Balb/c mice in order to obtain successful C26 cell engraftment and a reproducible cachectic phenotype. Based on previ-ously published data [35], the F2 generation of C57BL/6J mice backcrossed to Balb/c strain is permissive for C26 growth and is comparable for induction of body weight loss, as compared to pure BALB/c mice in both male and female animals.
    Cancer cachexia induction
    For C26 experiments, mice were randomized and divided into two groups, namely, controls and tumor bearers. Tumor bearing (C26) mice were inoculated subcutaneously in the back with 5 × 105 Colon26 (C26) cells. C26 cells were maintained in vitro in DMEM (Invitrogen) supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 100 μg/ ml sodium pyruvate, 2 mM L-glutamine, at 37 °C in a humidified atmosphere of 5% CO2 in air. The day of tumor implantation, cells were trypsinized, resus-pended in sterile saline and injected. For LLC experiments, TB mice were inoculated intramuscu-larly with 5 × 105 LLC cells obtained from exponen-tial growth. Animal weight and food intake were recorded every other day at the beginning of the experiment or daily once the mice started to lose weight. Control and TB mice were sacrificed under anesthesia 14 days after cell implantation. Several
    Autophagy and mitochondria in cancer cachexia 2683
    muscles and tissues were rapidly excised, weighed, frozen in melting isopentane cooled with liquid nitrogen and stored at − 80 °C.
    Genetic autophagy inhibition
    Four distinct short hairpin RNA sequences targeting mouse Becn1 gene were tested in C2C12 myoblasts for knockdown efficiency. The sequences were cloned in the pGFP-V-RS plasmid (Origene). The GI357932 sh-RNA showed the highest Beclin-1 knockdown efficiency (insert sequence: 5′-CAC-
    CATGCAGGTGAGCTTCGTGTGCCAGC-3′). A plasmid harboring a scrambled (SCR) sequence was used as control for the contralateral leg. As an alternative to Becn1 silencing, a BCL-2 triple mutant (Thr69Ala, Ser70Ala e Ser84Ala) was overexpressed in order to inhibit Beclin-1 activation (the plasmid was kindly gifted by Beth Levine, see Ref. [36]). In vivo transfection was performed by intramuscular injection of plasmids (20 μg) in the TA muscles followed by electroporation using an ECM830 apparatus (BTX) as previously described [37].