| Summary: | Cachexia is a syndrome of progressive nutritional depletion that occurs in up to one-half of all patients with cancer. In particular, cachexia involves a severe and specific loss of skeletal muscle, which is associated with worsened patient morbidity, reduced quality of life (QoL), and increased mortality. The aims of this thesis were to investigate specific mediators, mechanisms and biomarkers of muscle wasting and function in cancer cachexia. Models included human cancer cell lines and tissue samples, and patients with different types of upper gastrointestinal (GI) cancer. Upper GI cancer was chosen as the model for investigation as such patients are known to have a high prevalence of cachexia. Tumour-derived cachectic mediators were investigated initially (Chapters 3 to 5). There has been much speculation about whether the murine tumour-derived cachectic mediator known as proteolysis-inducing factor (PIF) has a human homologue, and how this may be derived from a larger gene known to encode for dermcidin, an anti-microbial peptide found in sweat. Messenger ribonucleic acid (mRNA) expression levels of dermcidin were analysed in human tumour tissue and human cancer cell lines. Expression of dermcidin was highly variable in human tumours, but appeared to be largely absent or expressed at only very low levels in oesophago-gastric cancer and prostate cancer (Chapter 3). Additionally, although human cell lines did express dermcidin mRNA, sequence analysis of complementary DNA (cDNA) derived from this mRNA demonstrated that transcripts lacked a site for the glycosylation critical to the proteolysis-inducing activity of murine PIF, thus raising doubt about the existence of human PIF (Chapter 4). Attention was then focused on plasma concentrations of macrophage inhibitory cytokine-1 (MIC-1), another potential tumour-derived mediator of cachexia in humans (Chapter 5). In a cohort of patients with oesophago-gastric cancer (n=293), although MIC-1 concentrations correlated with systemic inflammation (a key component of the cachexia syndrome), there was no independent link between plasma MIC-1 and nutritional status or survival. Thus, further investigations are required before MIC-1 can be determined as a key mediator of cancer cachexia in humans. Next, the role of circulating neuroendocrine mediators, specifically sex steroids and gonadotropins, were investigated in a cohort of patients with advanced pancreatic cancer (n=175) (Chapter 6). Testosterone is known to be a significant determinant of muscle mass. However, 73% of male cancer patients were hypogonadal (as defined by calculated serum free testosterone). Hypogonadal males demonstrated shortened survival compared with eugonadal males. Furthermore, male opioid use (an iatrogenic cause of male hypogonadism) was associated with shortened survival. In contrast, 18% of postmenopausal females exhibited premenopausal or 'hyperoestrogenic' serum oestradiol levels. Hyperoestrogenic females demonstrated shortened survival compared with eugonadal females. Thus, clinical trials of androgen replacement therapy (particularly in the presence of opioid analgesia) may be a potential route forwards in males with non-hormone sensitive types of cancer. Regulatory mechanisms of protein degradation within skeletal muscle were then examined (Chapters 7 and 8). It has been suggested that there may be parallels in the mechanisms of muscle wasting observed in genetic disorders, such as the muscular dystrophies, and the mechanisms observed in acquired conditions such as cancer cachexia. In rectus abdominis muscle samples from patients with oesophago-gastric cancer (n=27), deregulation of the sarcolemma-bound, muscular dystrophy-associated dystrophin glycoprotein complex (DGC) (involving reduced expression of dystrophin and hyperglycosylation of key DGC proteins) was found in 17/27 (63%) samples (Chapter 7). Moreover, the presence of DGC deregulation was associated with worsened patient function and shortened patient survival. These results suggest a possible common pathway between genetic and acquired forms of muscle wasting. The mass of a muscle is determined by the balance between protein synthesis and degradation. Pathways that can both increase protein degradation and suppress synthesis could have a profound influence on the rate of muscle wasting. Levels of the activated (phosphorylated) forms of the dsRNA-dependent protein kinase (PKR) and eukaryotic translation initiation factor 2a (eEF2a), intracellular mediators in pathways leading to increased muscle protein degradation and reduced protein synthesis, were determined in rectus abdominis samples from oesophago-gastric cancer patients (n=15) (Chapter 8). Levels of both phospho PKR and phospho eIF2a were significantly enhanced in muscle from cachectic patients, and there was a linear relationship between myosin expression and the extent of eIF2a phosphorylation. These results suggest that phosphorylation of PKR may be an important initiator of muscle wasting in cancer cachexia. Next, the presence of urinary biomarkers of skeletal muscle wasting were analysed using mass spectrometry in urine samples from cachectic oesophago-gastric cancer patients (n=8), weight-stable cancer patients (n=8) and healthy controls (n=8) (Chapter 9). The identification of early biomarkers of cachexia, the clinical detection of which would allow the institution of prophylactic therapeutic measures, is one of the key areas of need in cancer cachexia research. The number of protein species identified in urine samples from cachectic cancer patients (n=199) was significantly greater than that identified in samples from weight-stable cancer patients (n=79) and controls (n=49). Proteins identified specifically in cachectic samples, and thus potential biomarkers of muscle wasting that could be utilised in future clinical trials, included muscle (myosin species), cytoskeletal, and microtubule-associated proteins. Lastly, the use of objective assessment of physical activity as a potential biomarker of skeletal muscle function, and thus a novel outcome measure in the clinical management of advanced cancer patients, was explored (Chapters 10 and 11). Criterion-based validation of estimates of energy expenditure using an accelerometer-based activity-monitoring system (activPAL™) was performed using a combination of doubly labelled water and indirect calorimetry in cancer patients (n=6) and healthy controls (n=9) (Chapter 10). Although absolute errors for activPAL™-derived estimates of mean energy expenditure of activity (1.4%) and mean total energy expenditure (0.4%) were low, there was considerable variability in individual patient results, suggesting that further prospective validation studies of energy expenditure are required. (Step count and other PA measures have been validated previously). The activPAL™ meter was also used to take sequential measurements of objective PA in a cohort of patients with upper GI cancer undergoing palliative chemotherapy (n=16) (Chapter 11). Recruited patients demonstrated a complex "journey" of PA during treatment, with individuals describing worsened, improved or unchanged PA. However, the overall trend was one of deterioration in PA during chemotherapy. Importantly, objective measures of PA correlated with subjective scores of QoL, fatigue and performance status, supporting the use of objective PA as a patient-centred outcome. In summary, certain circulating mediators (sex steroids) and intracellular mechanisms (DGC deregulation and PKR phosphorylation) have been shown to be important in the aetiology of cachexia/skeletal muscle wasting in cancer, and the determination of patient outcome. Urinary biomarkers of muscle protein degradation (myosin species) and biomarkers of muscle function (physical activity) may be useful as inclusion criteria and/or outcome measures in clinical trials of anti-cachexia therapies.
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