Preserved muscle oxidative metabolic phenotype in newly diagnosed non-small cell lung cancer cachexia

Abstract

Background: Cachexia augments cancer-related mortality and has devastating effects on quality of life. Pre-clinical studies indicate that systemic inflammation-induced loss of muscle oxidative phenotype (OXPHEN) stimulates cancer-induced muscle wasting. The aim of the current proof of concept study is to validate the presence of muscle OXPHEN loss in newly diagnosed patients with lung cancer, especially in those with cachexia.

Methods: Quadriceps muscle biopsies of comprehensively phenotyped pre-cachectic (n = 10) and cachectic (n = 16) patients with non-small cell lung cancer prior to treatment were compared with healthy age-matched controls (n = 22). OXPHEN was determined by assessing muscle fibre type distribution (immunohistochemistry), enzyme activity (spectrophotometry), and protein expression levels of mitochondrial complexes (western blot) as well as transcript levels of (regulatory) oxidative genes (quantitative real-time PCR). Additionally, muscle fibre cross-sectional area (immunohistochemistry) and systemic inflammation (multiplex analysis) were assessed.

Results: Muscle fibre cross-sectional area was smaller, and plasma levels of interleukin 6 were significantly higher in cachectic patients compared with non-cachectic patients and healthy controls. No differences in muscle fibre type distribution or oxidative and glycolytic enzyme activities were observed between the groups. Mitochondrial protein expression and gene expression levels of their regulators were also not different.

Conclusion: Muscle OXPHEN is preserved in newly diagnosed non-small cell lung cancer and therefore not a primary trigger of cachexia in these patients.

Keywords: Cancer cachexia; Non‐small cell lung cancer; Oxidative phenotype; Skeletal muscle; Systemic inflammation.

Figure 1

Normal OXPHEN in patients with lung cancer pre-cachexia and cachexia. Quadriceps muscle biopsies were processed for analysis of muscle fibre subtypes, enzyme activity, and protein expression. (A) Distribution of oxidative type I and glycolytic type II muscle fibre types in quadriceps muscle. Assessment of fibres expressing different myosin heavy chain isoforms was performed using immunohistochemistry and myosin adenosine 5′-triphosphatase staining. (B) Muscle oxidative and glycolytic enzyme activity. Activity of oxidative (β-hydroxyacyl-CoA dehydrogenase and citrate synthase) and glycolytic (phosphofructokinase) enzymes was assessed. Ratios of oxidative to glycolytic were calculated for the different enzymes. (C) Protein expression of Oxphos proteins. Expression of ATP synthase and I–IV Oxphos protein complexes was assessed using western blot analysis. Glyceraldehyde 3-phosphate dehydrogenase was used as a loading control. Co, healthy controls; Pre, pre-cachectic patients, Cach, cachectic patients. * Significant difference between indicated groups, P < 0.05.

Figure 2

Muscle fibre atrophy is independent of fibre type in lung cancer cachexia. Cross-sectional of individual muscle fibres was assessed using immunohistochemical staining of laminin. Assessment of fibres expressing different myosin heavy chain isoforms was performed using immunohistochemistry and myosin adenosine 5′-triphosphatase staining.

Figure 3

Correlation between weight loss and protein expression of Oxphos complex II in healthy controls, pre-cachectic, and cachectic patients. A significant correlation between weight loss and Oxphos complex II protein expression was found in cachectic patients (R = 0.826, P < 0.05) but not in healthy control subjects or pre-cachectic patients.