Relevancia biológica del complejo enzimático NatB en el desarrollo de hepatocarcinoma y la función hepática

Resumen

NatB enzymatic complex, composed by Naa20 catalytic and Naa25 accessory subunits, acetylates eukaryotic proteins at their initial methionine α-amino group when it is followed by Asp, Glu, Asn, or Gln. This function has been conserved from yeast to humans; however it is not known its physiological relevance. We have recently observed that hNatB enzyme catalyzes N-terminal acetylation of 10% human proteome, being necessary for a proper activity of actin cytoskeleton, organelle with relevant implications in tumor development. Nowadays the identification of new targets for systemic therapy in the treatment of hepatocellular carcinoma (HCC) is one of the main needs in HCC treatment. Therefore, the aim of this study was to explore the therapeutic potential of NatB inhibition in HCC and its implication in HCC development and HCC patient prognosis. Expression of hNatB was inhibited in HCC human cell lines, Huh7 and PLC/PRF/5, and the effects on cellular proliferation, signaling pathways and tumoral development were analyzed in xenotransplant mouse model were analyzed. We observed hNatB expression inhibition resulted in the blockade of Huh7 and PLC/PRF/5 cell lines proliferation and in the impairment of their tumoral properties. This effect was associated with actin cytoskeleton disorganization that results in YAP oncogene inactivation and in a reduced Epidermal Growth Factor (EGF)-mediated ERK activation. Besides, hNatB subunits expression was analyzed in paired tumor and non-tumoral samples from 31 HCC patients with liver resection or transplantation. The association of NATB expression and clinical, radiological and histological variables related to HCC prognosis was analyzed. An overexpression of hNatB in tumor compared to non-tumor samples was observed in more than half of HCC patients (51%). Moreover, the overexpression of both NatB subunits was associated with microscopic vascular invasion. So, hNatB mediated protein N-terminal acetylation could be a new therapeutic target for the treatment of HCC and its overexpression could be related to more aggressive phenotype of HCC as it is associated with microvascular invasion. The other goal of this project was the generation of Naa25 hepatocyte specific knockout mice, inactivating NatB enzymatic activity in these cells. We observed a clear elevation of alkaline phosphatase, bilirubin and biliary salts plasma levels indicating bile duct obstruction and intrahepatic cholestasis. These effects are associated with a deficient bile canaliculi structure and disrupted hepatocyte polarization in the vicinity of central veins. Moreover, there are an increased number of pericentral hepatocytes that present less membranous b-catenin and display cytoplasmic localization of b-catenin. This rearrangement of b-catenin promotes an aberrant liver metabolic zonation, presenting an increased number of pericentral hepatocytes with activated glutamine synthetase and inhibited arginase-1 expression. Therefore, these animals show low serum urea as consequence of deficient urea cycle. In addition, mNaa25 knockout mice have hepatocytes with big nuclei, generated as a result of deficient mitosis that promotes the presence of an increased number of polyploidy hepatocytes. To conclude, NatB mediated regulation of actin cytoskeleton structure and activity is necessary for arrangement of b-catenin subcellular localization and function, proper liver metabolic zonation, bile acids secretion and hepatocyte division.