Participation of the MAPK pathway in the acceleration of hepatocellular carcinogenesis induced by the Raptor/mTORC1 deletion in hepatocytes

• Grant number: 20/16656-6
• Support Opportunities: Scholarships in Brazil - Post-Doctorate
• Effective date (Start): August 01, 2021
• Effective date (End): July 31, 2023
• Field of knowledge: Biological Sciences - Physiology - Physiology of Organs and Systems
• Principal Investigator: William Tadeu Lara Festuccia
• Grantee: Érique de Castro
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Non-alcoholic liver disease associated with metabolic dysfunction (MAFLD) is a heterogenous group of diseases that comprise from a simple liver steatosis (NAFL) to more severe conditions such as Steatohepatitis (NASH), Cirrhosis and Hepatocellular Carcinoma (HCC). The PI3K-mTORC2-Akt-mTORC1 signaling pathway is directly involved in the NAFL-NASH-HCC progression and overactivated in 40-50% of HCC. Inhibition or deletion of PTEN in hepatocytes promotes a constitutive activation of PI3K-mTORC2-Akt-mTORC1 signaling pathway. Mechanistic target of rapamycin complex 1 (mTORC1) is a molecular sensor of nutrients and growth factors that acts as a central component of signaling pathways that regulated cell metabolism and survival by modulating processes such as protein and lipid synthesis, autophagy, as well as mitochondrial morphology and function. The involvement of mTORC1 in the genomic instability associated to lipotoxicity and oxidative stress that characterize NAFL progression to HCC is unknown. To investigate the role of mTORC1 in this progression, we will use mice with deletion of PTEN exclusively in hepatocytes (L-PtenKO) that spontaneously develop NAFL, NASH and HCC in well-defined moments (8, 24, 48 weeks old, respectively) associated or not with the deletion of Raptor, a protein that is essential for mTORC1 activity (double knockout, L-Pten+Raptor KO). Partial results indicate that deletion of Raptor in hepatocytes exacerbates the Hepatomegaly, accelerates HCC development and increases ERK phosphorylation induced by PTEN deletion. We propose herein to test the hypothesis that hepatocyte mTORC1 deficiency accelerates HCC by impairing triacylglycerol synthesis, which results in lipotoxicity, mitochondrial dysfunction, oxidative stress, inflammation and DNA damage through a mechanism that involves MAPK activation. To test this hypothesis, we will conduct in vitro experiments with primary hepatocytes isolated from 6 weeks old mice with deletion of PTEN associated or not to the deletion of Raptor. These cells will be evaluated for intracellular signaling through PI3K-mTORC2-Akt-mTORC1 (p-Akt T308 and S473, p-S6K-1 and p-S6), MAPK (p-Erk and p-Raf), and TLR-NFºB (p-IKK and p-NFºB), fatty acid metabolism (incorporation in triacylglycerol and oxidation), acylcarnitine content, glycerol 3-phosphate synthesis (glyceroneogenesis, glycerol kinase and short glycolysis), mitochondrial respiration (coupled, uncoupled and maximal), H2O2 production via electron transport chain and glycerol 3-phosphate dehydrogenase, autophagy, mitophagy, among other processes, in the absence or presence of PD0325901, an selective inhibitor of MAPKs. These variables will be also evaluated in vivo in (1) mice with Pten and Pten+Raptor deletion in hepatocytes of 8- and 24-weeks old mice treated or not with PD0325901 and (2) mice with Raptor deletion in hepatocytes fed a high- fat, sucrose and cholesterol diet combined with the carcinogenic CCl4. They will be evaluated for liver ferroptosis, inflammatory profile (leukocyte infiltration and content of cytokines and lipid mediators as ceramides and prostaglandins), isolated mitochondria respiration, lipid composition, redox state (H2O2 production, malondialdehyde (MDA), 4-hydroxy-2-nonenal (4-HNE) and GSH/GSSG contents and SOD1, catalase and GPX activities). DNA damage will be evaluated by comet assay, immunohistochemistry for p53 and p-H2A.X and content of 8-oxodG. Depending on the results obtained, a phosphproteomic analysis will be realized to identify novel mTORC1 and MAPK targets related to the investigated processes. Our major goal is therefore to unveil novel therapeutic targets for HCC prevention and treatment. (AU)