Chemotherapy-related cachexia is associated with mitochondrial depletion and the activation of ERK1/2 and p38 MAPKs

Abstract

Cachexia affects the majority of cancer patients, with currently no effective treatments. Cachexia is defined by increased fatigue and loss of muscle function resulting from muscle and fat depletion. Previous studies suggest that chemotherapy may contribute to cachexia, although the causes responsible for this association are not clear. The purpose of this study was to investigate the mechanism(s) associated with chemotherapy-related effects on body composition and muscle function. Normal mice were administered chemotherapy regimens used for the treatment of colorectal cancer, such as Folfox (5-FU, leucovorin, oxaliplatin) or Folfiri (5-FU, leucovorin, irinotecan) for 5 weeks. The animals that received chemotherapy exhibited concurrent loss of muscle mass and muscle weakness. Consistently with previous findings, muscle wasting was associated with up-regulation of ERK1/2 and p38 MAPKs. No changes in ubiquitin-dependent proteolysis or in the expression of TGFβ-family members were detected. Further, marked decreases in mitochondrial content, associated with abnormalities at the sarcomeric level and with increase in the number of glycolytic fibers were observed in the muscle of mice receiving chemotherapy. Finally, ACVR2B/Fc or PD98059 prevented Folfiri-associated ERK1/2 activation and myofiber atrophy in C2C12 cultures. Our findings demonstrate that chemotherapy promotes MAPK-dependent muscle atrophy as well as mitochondrial depletion and alterations of the sarcomeric units. Therefore, these findings suggest that chemotherapy potentially plays a causative role in the occurrence of muscle loss and weakness. Moreover, the present observations provide a strong rationale for testing ACVR2B/Fc or MEK1 inhibitors in combination with anticancer drugs as novel strategies aimed at preventing chemotherapy-associated muscle atrophy.

Keywords: MAPKs; cachexia; chemotherapy; mitochondria; muscle wasting.

Figure 1. In vivo chemotherapy administration causes adipose tissue and skeletal muscle weight loss

Body weights (A), body composition assessment (fat and lean tissues) performed by means of EchoMRI (B–C), muscle (D) and organ (E) weights in mice treated with chemotherapy for up to 5 weeks (n = 4–6). Weights were normalized to the Initial Body Weight (IBW) and expressed as weight/100mg IBW. Overall food intake over the 5-week experimental period (F). Representative gut morphology in vehicle- and chemotherapy-treated animals (G). FBW: Final Body Weight; GSN: Gastrocnemius; Gem: Gemcitabine. Significance of the differences: *p < 0.05; **p < 0.01; ***p < 0.001 vs. Vehicle.

Figure 2. Folfiri-derived muscle atrophy associates with significant muscle weakness

Muscle cross sectional area (CSA) in the tibialis muscle of mice exposed to chemotherapy (A) and tibialis anterior morphology (H&E staining) (B). Whole body grip strength, reported as peak force (C) and specific (normalized) force (D), was measured by taking advantage of a grip strength meter and expressed as the average of the three top pulls from each animal (n = 4–6). Ex-vivo muscle contractility was performed on EDL muscle excised from animals administered chemotherapy for up to 5 weeks (E–F). Data expressed as means ± SEM. Significance of the differences: *p < 0.05; **p < 0.01; ***p < 0.001 vs. Vehicle.

Figure 3. The muscle of mice exposed to chemotherapy exhibits up-regulation of ERK1/2 and p38 MAPKs and down-regulation of mitochondrial proteins

Representative Western blotting (A) and quantification (B) for p-STAT3, STAT3, p-AKT, AKT, pMEK1/2, MEK1/2, p-ERK1/2, ERK1/2, p-p38, p38, PGC-1α, PGC-1β, Cytochrome C (Cyt-C), LC3B, Beclin-1 and p62 in muscle protein extracts from mice exposed to chemotherapy. Levels of phosphorylated proteins were normalized to the respective total protein expression. Tubulin was used as loading control. Data are expressed as Fold change vs. Vehicle and reported as means ± SEM. Significance of the differences: *p < 0.05; **p < 0.01; ***p < 0.001 vs. Vehicle.

Figure 4. Folfiri-mediated muscle wasting is not associated with ubiquitin-dependent proteolysis or with increased expression of TGFβ-associated ligands or markers of myogenesis

Chymotrypsin-like proteasome activity (U/ml) was performed on muscle from mice exposed to either Folfiri or Folfox and expressed as means ± SEM. Significance of the differences: *p < 0.05 vs. Vehicle (A). Gene expression levels for Atrogin-1, MuRF-1, Fbxo21 (SMART), Fbxo30 (MUSA1), Fbxo31 (B), TGF-β1, TGF-β2, myostatin, Activin A (C), MyoD, Myogenin and Pax-7 (D) was performed by qRT-PCR (Light Cycler 96, Roche, Indianapolis, IN). Gene expression was normalized to TBP levels. Data (fold change vs. vehicle) expressed as means ± SEM.

Figure 5. Chemotherapy causes marked reduction in mitochondrial activity and increase in the number of glycolytic muscle fibers

Succinate dehydrogenase (SDH) staining was performed on 8 μm-thick sections from tibialis muscle frozen in liquid nitrogen-cooled isopentane (A). Quantification of signal intensity (expressed in pixels) (B), fiber-specific CSA (expressed as % of vehicle) (C) and number of oxidative (dark blue) and glycolytic (light blue) fibers (expressed as % of vehicle) (D) was assessed. Scale bar: 100 μm. Significance of the differences: *p < 0.05, **p < 0.01, ***p < 0.001 vs. Vehicle.

Figure 6. Chemotherapy causes depletion of mitochondria and aberrant muscle morphological features

Electron microscopy micrographs (magnification: 30,000x) of EDL muscles from mice exposed to chemotherapy are reported in (A). White arrows indicate mitochondria. Black arrows indicate the Z-line. Brackets identify the I-bands. Scale bar: 500nm. Quantification of mitochondrial amount (number per field) (B) and size (minimum diameter, nm) (C) was performed. Significance of the differences: *p < 0.05, **p < 0.01 vs. Vehicle.

Figure 7. RNA-Seq analysis reveals down-regulation of mitochondrial metabolism and up-regulation of acute phase response proteins, lipid transportation and energy metabolism

Next-Generation RNA-sequencing was performed on whole RNA extracted from skeletal muscle of Vehicle- and Folfiri-treated animals (n = 4). RNA-Seq reads were mapped to the mouse genome (mm 9). Only statistically significant differentially expressed genes (False Discovery Rate < 5%) between Vehicle- and Folfiri-exposed muscles are reported in the figure.

Figure 8. ACVR2B/Fc and PD98059 MEK1 inhibitor prevent Folfiri-associated muscle atrophy

C2C12 murine myotubes were exposed to Folfiri in combination with either ACVR2B/Fc (10 μg/ml) or PD98059 (20 μM) for 48 h and later stained for Myosin Heavy Chain (MHC), visualized as green (A) or red (E) staining. Quantification of fiber size (n = 500–600) is reported in (B–F). Representative Western blotting (C–G) and quantification (D–H) for pMEK1/2, MEK1/2, pERK1/2, ERK1/2, pAKT and AKT in total protein extracts from C2C12 cultures. Tubulin was used as loading control. Scale bar: 100 μm. Data expressed as means ± SEM. Significance of the differences: *p < 0.05; **p < 0.01.

Figure 9. Representative model of chemotherapy-dependent cachexia

Based on our observations, chemotherapy causes mitochondrial depletion and (directly or indirectly) activation of ERK1/2 and p38 MAPKs-dependent pathways. Altogether, these alterations might lead to cachexia, characterized by loss of muscle mass and increased muscle weakness. Promoting muscle growth by taking advantage of ACVR2B/Fc or blocking ERK1/2 activation by means of the MEK1 pharmacologic inhibitor PD98059 can prevent muscle wasting.