Targeting LGALS3 to treat steatotic liver disease and hepatocarcinogenesis

Non-alcoholic fatty liver disease (NAFLD), recently renamed as Metabolic dysfunction-associated steatotic liver disease (MASLD), is the leading cause of chronic liver disease (CLD) and liver-related morbidity and mortality worldwide. NAFLD, characterized by hepatic fat accumulation and coexisting cardiometabolic risk factors, is often clinically associated with liver fibrosis, with a massive activation of the hepatic stellate cells (HSCs) into myofibroblasts-like cells that secrete large amount of extracellular matrix (ECM) proteins. ECM remodelling towards a fibrotic phenotype represents an important risk factor for hepatocellular carcinoma (HCC), which incidence and mortality have been continuously increasing, and, unfortunately, the approved pharmacological therapies are still poorly effective. Thus, there is an urgent need for new early biomarkers of disease and new therapeutic targets. In the last five years, several molecules have been identified as potential targets or biomarkers, and among them, Galectin 3 (Gal-3) appears to be one of the most promising. While Gal-3 is not expressed by hepatocytes in the healthy liver, its elevated serum levels have been observed in individuals with steatotic liver disease, non-alcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and HCC. Galectin 3, previously known as MAC2, is a chimera type galectin, that belong to the family of β-galactoside-binding proteins that are involved in mRNA splicing, cell cycle, cell growth and cell apoptosis. Considering the involvement of Gal-3 in liver physiology, as well as the clinical evidence linking this protein to chronic liver disease, the first aim of my project was to validate its overexpression in in vivo and in vitro models of steatosis and hepatocellular carcinoma. These models were used to study the role of T3, which is able to improve steatosis and to reduce the number and size of tumours. Together with these beneficial effects, we investigated the response of Gal-3. Once validated the models, the focus has shifted to the study of the molecular mechanisms regulated by Gal-3, by the employment of the gene silencing in a 3D in vitro model of steatosis. The same model was also used to verify the in vitro effect of T3 and MGL-3196 (a selective agonist of the thyroid hormone receptor β-isoform) on neutral lipid content and collagen accumulation. The gene silencing was then combined with the administration of either T3 or MGL-3196, to evaluate the possible involvement of Gal-3 in the effects mediated by the two compounds. The results of this study show that the role of Gal-3 can be efficiently studied with the employment of steatosis or hepatocarcinogenic in vitro and in vivo models. Gal-3 depletion causes a reduction of neutral lipid content and type I collagen accumulation in 3D spheroids. Analysis of genes involved in lipid metabolism demonstrated significant changes in genes involved in β-oxidation and triglyceride synthesis. The combination of T3/MGL-3196 treatment and gene silencing did not show worse or better outcomes, suggesting a possible involvement of Gal-3 in the T3 pathway.