Nuclear magnetic resonance in conjunction with functional genomics suggests mitochondrial dysfunction in a murine model of cancer cachexia

Abstract

Cancer patients commonly suffer from cachexia, a syndrome in which tumors induce metabolic changes in the host that lead to massive loss in skeletal muscle mass. Using a preclinical mouse model of cancer cachexia, we tested the hypothesis that tumor inoculation causes a reduction in ATP synthesis and genome-wide aberrant expression in skeletal muscle. Mice implanted with Lewis lung carcinomas were examined by in vivo 31P nuclear magnetic resonance (NMR). We examined ATP synthesis rate and the expression of genes that play key-regulatory roles in skeletal muscle metabolism. Our in vivo NMR results showed reduced ATP synthesis rate in tumor-bearing (TB) mice relative to control (C) mice, and were cross-validated with whole genome transcriptome data showing atypical expression levels of skeletal muscle regulatory genes such as peroxisomal proliferator activator receptor γ coactivator 1 ß (PGC-1ß), a major regulator of mitochondrial biogenesis and, mitochondrial uncoupling protein 3 (UCP3). Aberrant pattern of gene expression was also associated with genes involved in inflammation and immune response, protein and lipid catabolism, mitochondrial biogenesis and uncoupling, and inadequate oxidative stress defenses, and these effects led to cachexia. Our findings suggest that reduced ATP synthesis is linked to mitochondrial dysfunction, ultimately leading to skeletal muscle wasting and thus advance our understanding of skeletal muscle dysfunction suffered by cancer patients. This study represents a new line of research that can support the development of novel therapeutics in the molecular medicine of skeletal muscle wasting. Such therapeutics would have wide-spread applications not only for cancer patients, but also for many individuals suffering from other chronic or endstage diseases that exhibit muscle wasting, a condition for which only marginally effective treatments are currently available.

Figure 1

NMR spectra of in vivo ³¹P NMR saturation-transfer performed on the hind limb skeletal muscle of awake mice. Representative summed ³¹P-NMR spectra acquired from C and TB mice before (A) and after (B) saturation of the γ-ATP resonance. The arrow on γ-ATP indicates the position of saturation (sat) by rf irradiation (−2.4 ppm). ppm, chemical shift in parts per million.

Figure 2

Distribution of genes differentially expressed in gastrocnemius muscle of TB animals relative to C animals among 14 functional categories showed in the X axis, as identified by using Gene Ontology and KEGG metabolic pathways at P≤0.05. Gray bars indicate the number of upregulated genes while black bars correspond to down-regulated genes in the gastrocnemious muscle of TB animals versus C animals (left Y axis). The negative log10 of P-values represented by diamonds are indicated in the right Y axis.

Figure 3

Summary of the magnitudes of change in mRNA expression of mitochondrial function-related and other cancer-induced cachexia-related genes 14 days after cancer transplantation. IRS-1, insulin receptor substrate 1; IGF-1, insulin-like growth factor 1; IGFBP-5, insulin-like growth factor binding protein 5; PDK4, pyruvate dehydrogenase kinase 4; PGC-1ß, peroxisome proliferator-activated receptor-γ coactivator-1ß; UCP3, uncoupling protein 3; FoxO3α, Forkhead box O3α; ATROGIN-1, F-box only protein 32; UBE3a, ubiquitin protein ligase E3A; SOD-2, superoxide dismutase 2; GPX, glutathione peroxidase 3.

Figure 4

Putative PGC-1-mediated mechanism of cancer-induced skeletal muscle wasting. In this model, significant down-regulation of PGC-1ß expression causes dysregulation of mitochondria, resulting in reduced ATP synthesis and increased ROS production, most likely due to PGC-1-dependent upregulation of UCPs. Meanwhile, reduced PGC-1ß gene expression levels result in a failure to down-regulate expression of FoXO3α, which normally suppresses atrogen expression and proteolysis. Insulin-stimulated IGF-1/AKT pathway signaling is also significantly attenuated. Thus, AKT does not phosphorylate FoXOs and, consequently, favors further upregulation of atrogenes, thus leading to skeletal muscle wasting. Moreover, reduced IGF-1/AKT signaling further facilitates protein translation inhibition due to mTOR inactivation which also results in cachexia. Meanwhile, PGC-1ß may affect IGF-1 and IRS-1 expression thereby producing insulin resistance in skeletal muscle. IRS-1, insulin receptor substrate 1; IGF-1, insulin-like growth factor 1; PGC-1s, peroxisome proliferator-activated receptor-γ coactivators-1; UCPs, uncoupling proteins; FoXOs, forkhead box O proteins; ROS, reactive oxygen species; TNFα, tumor necrosis factor α; NF-κB, nuclear factor-κB.