Using transcriptomics to identify and validate novel biomarkers of human skeletal muscle cancer cachexia

Nathan A Stephens,Olav Rooyackers,Iain J Gallagher,James A Ross,James A Timmons,Kenneth C Fearon,Richard J Skipworth,Ben H Tan,Troels Marstrand,Lars Lundell,Denis C Guttridge

doi:10.1186/gm122

Abstract

BackgroundCancer cachexia is a multi-organ tissue wasting syndrome that contributes to morbidity and mortality in many cancer patients. Skeletal muscle loss represents an established key feature yet there is no molecular understanding of the disease process. In fact, the postulated molecular regulators of cancer cachexia originate largely from pre-clinical models and it is unclear how these translate to the clinical environment.MethodsRectus abdominis muscle biopsies were obtained from 65 upper gastrointestinal (UGI) cancer patients during open surgery and RNA profiling was performed on a subset of this cohort (n = 21) using the Affymetrix U133+2 platform. Quantitative analysis revealed a gene signature, which underwent technical validation and independent confirmation in a separate clinical cohort.ResultsQuantitative significance analysis of microarrays produced an 83-gene signature that was able to identify patients with greater than 5% weight loss, while this molecular profile was unrelated to markers of systemic inflammation. Selected genes correlating with weight loss were validated using quantitative real-time PCR and independently studied as general cachexia biomarkers in diaphragm and vastus lateralis from a second cohort (n = 13; UGI cancer patients). CaMKIIβ correlated positively with weight loss in all muscle groups and CaMKII protein levels were elevated in rectus abdominis. TIE1 was also positively associated with weight loss in both rectus abdominis and vastus lateralis muscle groups while other biomarkers demonstrated tissue-specific expression patterns. Candidates selected from the pre-clinical literature, including FOXO protein and ubiquitin E3 ligases, were not related to weight loss in this human clinical study. Furthermore, promoter analysis identified that the 83 weight loss-associated genes had fewer FOXO binding sites than expected by chance.ConclusionWe were able to discover and validate new molecular biomarkers of human cancer cachexia. The exercise activated genes CaMKIIβ and TIE1 related positively to weight-loss across muscle groups, indicating that this cachexia signature is not simply due to patient inactivity. Indeed, excessive CaMKIIβ activation is a potential mechanism for reduced muscle protein synthesis. Our genomics analysis also supports the view that the available preclinical models do not accurately reflect the molecular characteristics of human muscle from cancer cachexia patients.

Highlights

Cancer cachexia is a multi-organ tissue wasting syndrome that contributes to morbidity and mortality in many cancer patients
A PubMed analysis indicates that almost one-third of documents discussing cancer cachexia are review articles, highlighting the need for more primary investigations to Muscle mass is maintained by physical activity, reflect ing a balance between protein synthesis and degradation
Human cancer cachexia is a chronic process and weight loss is not as rapid and generally not as severe as the acute muscle wasting observed in animal models

Summary

Introduction

Cancer cachexia is a multi-organ tissue wasting syndrome that contributes to morbidity and mortality in many cancer patients. The postulated molecular regulators of cancer cachexia originate largely from pre-clinical models and it is unclear how these translate to the clinical environment. Many of the molecular signaling pathways that are postulated to contribute to muscle atrophy in preclinical models mediate their effects through activation of the UPP [6]. Identification of two muscle-specific E3 ubiquitin ligases, MuRF-1 and MAFbx/atrogin-1, in a large number of animal models of atrophy [7,8] has been used to provide an argument for a major contribution of the UPP in muscle wasting, such that these genes are measured as surrogate indicators of UPP activation. It should be kept in mind that active tissue remodeling, even with net protein accretion, may well rely partly on the protein degradation pathways and, as such, they may not represent logical surrogates for commenting on net protein degradation

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