Skeletal muscle atrophy commonly occurs in acute and chronic disease. The expression of the muscle-specific E3 ligases atrogin-1 (MAFbx) and muscle RING finger 1 (MuRF1) is induced by atrophy stimuli such as glucocorticoids or absence of IGF-I/insulin and subsequent Akt signaling. We investigated whether glycogen synthase kinase-3β (GSK-3β), a downstream molecule in IGF-I/Akt signaling, is required for basal and atrophy stimulus-induced expression of atrogin-1 and MuRF1, and myofibrillar protein loss in C(2)C(12) skeletal myotubes. Abrogation of basal IGF-I signaling, using LY294002, resulted in a prominent induction of atrogin-1 and MuRF1 mRNA and was accompanied by a loss of myosin heavy chain fast (MyHC-f) and myosin light chains 1 (MyLC-1) and -3 (MyLC-3). The synthetic glucocorticoid dexamethasone (Dex) also induced the expression of both atrogenes and likewise resulted in the loss of myosin protein abundance. Genetic ablation of GSK-3β using small interfering RNA resulted in specific sparing of MyHC-f, MyLC-1, and MyLC-3 protein levels after Dex treatment or impaired IGF-I/Akt signaling. Interestingly, loss of endogenous GSK-3β suppressed both basal and atrophy stimulus-induced atrogin-1 and MuRF1 expression, whereas pharmacological GSK-3β inhibition, using CHIR99021 or LiCl, only reduced atrogin-1 mRNA levels in response to LY294002 or Dex. In conclusion, our data reveal that myotube atrophy and myofibrillar protein loss are GSK-3β dependent, and demonstrate for the first time that basal and atrophy stimulus-induced atrogin-1 mRNA expression requires GSK-3β enzymatic activity, whereas MuRF1 expression depends solely on the physical presence of GSK-3β.

Cachexia is a prevalent phenomenon of non-small cell lung cancer (NSCLC) which is responsible for increased mortality and deterioration of physical performance. Preclinical research indicates that systemic inflammation induces cachexia-related muscle wasting through muscular Nuclear Factor-kappa B (NF-κB) signaling and subsequent ubiquitin proteasome system (UPS)-mediated proteolysis. As these pathways could be a target for early intervention strategies, it needs to be elucidated whether increased activation of these pathways is already present in early stage NSCLC cachexia. The aim of the present study was therefore to assess muscular NF-κB and UPS activation in patients with NSCLC pre-cachexia. Sixteen patients with newly diagnosed stages I-III NSCLC having <10% weight loss and ten healthy controls were studied. Body composition, systemic inflammation and exercise capacity were assessed in all subjects and NF-κB and UPS activity in vastus lateralis muscle biopsies in a subset. Patients showed increased plasma levels of C-reactive protein (CRP) (P<0.001), soluble Tumor Necrosis Factor receptor 1 (sTNF-R1) (P<0.05), fibrinogen (P<0.001) and decreased levels of albumin (P<0.001). No changes in fat free body mass or skeletal muscle NF-κB and UPS activity were observed, while peak oxygen consumption ( [Formula: see text] ) was significantly decreased in patients compared with healthy controls. In conclusion, this exploratory study demonstrates significantly reduced exercise capacity in NSCLC pre-cachexia despite maintenance of muscle mass and unaltered indices of UPS activation. The absence of muscular NF-κB-dependent inflammatory signaling supports the notion that transition of systemic to local inflammation is required to initiate UPS-dependent muscle wasting characteristic for (experimental) cachexia.

Background Cachexia, highly prevalent in patients with non-small cell lung cancer (NSCLC), impairs quality of life and is associated with reduced tolerance and responsiveness to cancer therapy and decreased survival. MicroRNAs (miRNAs) are small non-coding RNAs that play a central role in post-transcriptional gene regulation. Changes in intramuscular levels of miRNAs have been implicated in muscle wasting conditions. Here, we aimed to identify miRNAs that are differentially expressed in skeletal muscle of cachectic lung cancer patients to increase our understanding of cachexia and to allow us to probe their potential as therapeutic targets. Methods A total of 754 unique miRNAs were profiled and analysed in vastus lateralis muscle biopsies of newly diagnosed treatment-naïve NSCLC patients with cachexia (n = 8) and age-matched and sex-matched healthy controls (n = 8). miRNA expression analysis was performed using a TaqMan MicroRNA Array. In silico network analysis was performed on all significant differentially expressed miRNAs. Differential expression of the top-ranked miRNAs was confirmed using reverse transcriptionquantitative real-time PCR in an extended group (n = 48) consisting of NSCLC patients with (n = 15) and without cachexia (n = 11) and healthy controls (n = 22). Finally, these miRNAs were subjected to univariate and multivariate Cox proportional hazard analysis using overall survival and treatment-induced toxicity data obtained during the follow-up of this group of patients. Results We identified 28 significant differentially expressed miRNAs, of which five miRNAs were up-regulated and 23 were down-regulated. In silico miRNA-target prediction analysis showed 158 functional gene targets, and pathway analysis identified 22 pathways related to the degenerative or regenerative processes of muscle tissue. Subsequently, the expression of six topranked miRNAs was measured in muscle biopsies of the entire patient group. Five miRNAs were detectable with reverse transcription-quantitative real-time PCR analysis, and their altered expression (expressed as fold change, FC) was confirmed in muscle of cachectic NSCLC patients compared with healthy control subjects: miR-424-5p (FC = 4.5), miR-424-3p (FC = 12), miR-450a-5p (FC = 8.6), miR-144-5p (FC = 0.59), and miR-451a (FC = 0.57). In non-cachectic NSCLC patients, only miR-424-3p was significantly increased (FC = 5.6) compared with control. Although the statistical support was not sufficient to imply these miRNAs as individual predictors of overall survival or treatment-induced toxicity, when combined in multivariate analysis, miR-450-5p and miR-451a resulted in a significant stratification between short-term and long-term survival. Conclusions We identified differentially expressed miRNAs putatively involved in lung cancer cachexia. These findings call for further studies to investigate the causality of these miRNAs in muscle atrophy and the mechanisms underlying their differential expression in lung cancer cachexia.

Background and purposeRadiation-esophagitis and weight loss are frequently observed toxicities in patients treated with concurrent chemo-radiotherapy (CT-RT) for non-small cell lung cancer (NSCLC) and might be related. The purpose was to investigate whether weight loss already starts early after initiation of CT-RT and precedes radiation-esophagitis.Materials and methodsIn a retrospective cohort, weight and esophagitis grade ≥2 were assessed during the first weeks of (CT-)RT in patients treated with concurrent (n = 102) or sequential (n = 92) therapy. In a prospective validation study, data on body weight, esophagitis grade ≥2, nutritional intake and muscle strength were obtained before, during and following CT-RT.ResultsIn the retrospective cohort, early weight loss was observed in concurrently treated patients (p = 0.002), independent of esophagitis ≥ grade 2. Early weight loss was also observed in the prospective cohort (p = 0.003) and was not accompanied by decreases in nutritional intake. In addition lower limb muscle strength rapidly declined (p = 0.042). In the later weeks of treatment, further body weight loss occurred (p < 0.001) despite increased nutritional supplementation and body weight was only partly recovered after 4 weeks post CT-RT (p = 0.003).ConclusionsWeight loss during concurrent CT-RT for NSCLC starts early and prior to onset of esophagitis, requiring timely and intense nutritional rehabilitation.Electronic supplementary materialThe online version of this article (doi:10.1007/s13539-013-0127-5) contains supplementary material.

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