Dottorato in TECNOLOGIE BIOMEDICHE INNOVATIVE IN MEDICINA CLINICA

Titolo della tesi: MicroRNAs profiling in cancer-related muscle wasting and evaluation of the role of ANGPTL3 on muscle metabolism

Skeletal muscle is the most abundant tissue in human body involved in many physiological functions, such as locomotion and metabolism. It constitutes ~40% of total body mass, encloses 50%–75% of all body proteins and accounts for about 30%–50% of total protein turnover. The morpho-functional proprieties of skeletal muscle are guaranteed by the dynamic equilibrium that regulates the body composition and homeostasis (i.e. protein turnover). Whenever this dynamic equilibrium is altered, muscle wasting ensues. Two common but distinct conditions characterized by muscle wasting are sarcopenia and cachexia since both present several analogies in the pathogenic mechanisms and the clinical picture. Cachexia is a common complication in patients with cancer, defined as complex multifactorial syndrome associated with metabolic abnormalities and reduced food intake, which result in disordered protein and lipid energy balance, and insulin resistance. It is primarily characterized by ongoing depletion of skeletal muscle and uncontrolled weight loss. Low muscle mass in cancer is associated with poor prognosis. In patients with cancer cachexia survival is directly related to both total weight loss and rate of weight loss. The incidence and prevalence vary depending on tumor type and stage. The development of cancer cachexia is mostly driven by an aberrant tumor-induced inflammatory response, which triggers the catabolic pathways responsible for the high proteolysis rate observed in muscle of cachectic patients. Skeletal muscle catabolism is finely regulated by the insulin signaling pathway which overlaps with the insulin-like growth factor-1 (IGF-1) signaling pathway to maintain muscle homeostasis. Since that, insulin resistance might play a consistent role in muscle wasting. Insulin resistance is present in many cancer patients and may be one mechanism through which muscle wasting occurs. Despite the devasting toll that cancer cachexia has on patients, it is often underdiagnosed and therefore untreated or undertreated. The current treatment strategies are inadequate and are mainly based on nutrition and exercise-based interventions in addition to therapeutic agents available, such as anti-inflammatory drugs. However, the effectiveness of these treatments remains unclear, as clinical outcomes and long-term efficacy reports are insufficient. Therefore, novel early diagnostic biomarkers and therapeutic targets are needed. Nowadays, it has been suggested that some cellular mediators released by host tissue or by tumor cells, such as microRNAs (miRNAs), the small non-coding RNAs crucial regulators of gene expression, might contribute to the development of cachexia or for the systemic inflammation associated with this conditions. Recently, changes in miRNAs and in muscle specific miRNAs (i.e. myomiRs) levels of cachectic patients or in mouse models of cancer cachexia have been revelry reported. Moreover, given the metabolic nature of cancer cachexia, the study of its mechanisms should also include an evaluation of the influence of metabolic pathways, such as those involved in skeletal muscle insulin resistance. Angiopoietin-like (ANGPTL) 3 is an hepatokine emerged as important regulators of energy homoeostasis and lipoprotein metabolism by promoting the inhibition of lipoprotein lipase (LPL), the enzyme responsible for the hydrolysis of circulating triglycerides (TGs) into free fatty acids (FFAs) in the capillaries of fat and muscle tissue in both human and animals. FFAs are a vital energy source for many oxidative tissues, such as heart and skeletal muscle. The tissue distribution of FFAs, often referred as FFAs portioning, shifts according to metabolic state and is important for maintaining metabolic homeostasis. In the fed state, ANGPTL3 act as an endocrine factor to inhibit LPL activity in oxidative tissues, thus funneling TGs to adipose tissue for storage. Conversely, during fasting, LPL down-regulation in adipose tissue direct FFAs to muscle tissue for FFAs oxidation. However, the molecular mechanism by which ANGPTL3 exert this feeding-state induced shifts in LPL activity and fat partitioning remain a work in progress. Importantly, multiple studies found that individuals deficient in ANGPTL3 genes have a lower risk of cardiovascular disease. Moreover, the protective effects of ANGPTL3 loss of function mutations have prompted studies into the development of target therapies to treat dyslipidemia and metabolic disease. Interestingly, there is evidence that ANGPTL3 loss of function can also improve glucose homeostasis, although the pathways have not been yet completely elucidated. Healthy skeletal muscle carefully balances between utilization of glucose and FFAs, depending on fed state and muscle activity. For instance, under fed state, increasing levels of insulin allow skeletal muscle to uptake glucose and store it as glycogen. However, influx of FFAs into skeletal muscle observed during fasting may cause elevated intramuscular lipid content might contribute to muscle insulin resistance. It is thought that alterations in immune-mediated inflammation and FFAs levels trigged during cancer cachexia led to insulin resistance in muscle tissues and ectopic deposits of FFAs. Whether ANGPLT3 have effects on these metabolic alterations observed during muscle-related cancer wasting remain to be elucidate. In this regard, it will be important to unveil the molecular mechanism by which ANGPTL3 either directly, by affecting glucose metabolism, or indirectly, by change lipid metabolism, can modulate energy homeostasis. The hypothesis of the present studies were that i) differentially expressed miRNAs in cancer-related muscle wasting can describe the underlying mechanisms of negatively impact on muscle homeostasis, as well as putative to early diagnosis and response to an intervention; ii) ANGPTL3 can affect the metabolic abnormalities observed during cancer cachexia. To address these premises, i) we profile miRNAs from skeletal muscle and plasma of gastrointestinal cancer patients and investigate their relationship(s) with body compositions; (ii) we evaluate in vitro the effects that ANGPTL3 has on insulin-mediate response in muscle metabolism; iii) we analyzed circulating levels of ANPTL3 from plasma of gastrointestinal cancer patients in association with differentially expressed miRNAs.