Molecular biomarker discovery and physiological assessment of skeletal muscle in cancer cachexia

Abstract

Cachexia affects up to two thirds of all cancer patients with progressive disease. It is a syndrome characterised by weight-loss, anorexia, fatigue, asthenia, peripheral oedema, and is responsible for around 20% of cancer deaths. Cachectic patients suffer loss of both muscle mass and adipose tissue (with comparative sparing of visceral protein) and the lean tissue loss appears resistant to nutritional support. Progress in the treatment of cancer cachexia has been hampered due to poor understanding of the molecular mechanisms of skeletal muscle wasting in humans (rather than preclinical models) combined with a lack of accurate phenotyping particularly with respect to loss of skeletal muscle mass and function. The aim of the present thesis was to improve the knowledge and tools available for early intervention studies. The thesis focused on skeletal muscle as a key compartment in cancer cachexia. The experimental model was patients with upper gastrointestinal (UGI) cancer undergoing potentially curative surgery due to the associated higher incidence of cachexia along with the ability to access tissue biopsies. The thesis broadly divides into two sections. Part I reports a series of cancer cachexia biomarker discovery studies based on direct biopsy and analysis of human skeletal muscle. Part II focused on assessment and phenotyping of skeletal muscle mass and function in cachectic UGI cancer patients. In addition, the feasibility of longitudinal clinical studies that utilise such methodology is reported. Intramuscular β-dystroglycan protein content (assessed using Western blot) was identified as a potential biomarker of cancer cachexia whereas changes in the structural elements of muscle (myosin heavy chain or dystrophin) appeared to be survival biomarkers. Using transcriptomics, an 82-gene signature was demonstrated to correlate with weight-loss. Quantitative real-time polymerase chain reaction (qRT-PCR) was carried out to examine the genes from this signature that were most upregulated. The exercise activated genes, CAMk2β and TIE1, correlated positively with weight-loss across different muscle groups (Rectus abdominis, Vastus lateralis, Diaphragma) indicating that cachexia was not simply due to inactivity and suggesting that these genes could be used as biomarkers of cachexia. None of the biomarkers discovered were consistent with pre-clinical models and therefore require further study before progressing to a validation programme. Electron microscopy of muscle biopsies demonstrated that the number and size of intramyocellular lipid droplets was increased in the presence of cancer and increases further with weightloss/ loss of adipose mass in other body compartments. The specific mechanisms and drivers of this phenomenon remain to be elucidated, but could relate to enhanced lipolysis or mitochondrial dysfunction in skeletal muscle as well as influencing muscle mechanical quality. Physiological assessment of patients with cancer cachexia established the negative impact that cachexia can have on muscle mass, function, muscle quality and quality of life, but demonstrated that the degree of impairment varies with sex and between muscle groups. Furthermore, the challenge of longitudinal studies in this patient group where frailty and clinical deterioration limit repeated assessments was highlighted. Such issues emphasise the need for a dual approach to the classification of cancer cachexia: if molecular markers prove difficult to discover or validate, then more specific and robust physiological indices of skeletal muscle mass and function may be the more important route to improve clinical trial design and cachexia classification.