Abstract A02: OXPHOS inhibition and pentose phosphate pathway induction are early events priming preneoplastic lesions toward HCC development
Authors: Kowalik Marta A. , Guzzo Giulia , Morandi Andrea , Perra Andrea , Menegon Silvia , Angioni Maria M. , Giordano Silvia , Chiarugi Paola , Rasola Andrea , Columbano Amedeo 
Published: May 2016

Abstract
Introduction: A shift towards a Warburg metabolism in which aerobic glycolysis is increased has long been associated to cancer cell transformation. However, whether the switch from oxidative phosphorylation to glycolysis can occur at early stages of cancer development, particularly in hepatocellular carcinoma (HCC), remains elusive.
Materials and Methods: Preneoplastic hepatic lesions and Hepatocellular carcinomas were induced in rats subjected to the Resistant-Hepatocyte (RH) model, consisting of a single dose of dietthylnitrosamine (DENA) and a 2-week feeding a diet supplemented with 2-acetylaminoaminofluorene (2-AAF). In vitro experiments were performed in HCC cells obtained by perfusion of HCC-bearing rats or immortalized rat hepatocytes.
Results and discussion: Using the Resistant-Hepatocyte (R-H) model, we show that the acquisition of the Warburg phenotype is a very early event in rat HCC development as demonstrated by concomitant MCT4 expression and oxidation/inhibition of pyruvate kinase M2 (PKM2). In keeping with this, we also observed inhibition of succinate dehydrogenase (SDH) by the chaperone tumor necrosis factor receptor-associated protein 1 (TRAP1) and an increase in the expression and activity of citrate synthase (CS). In these preneoplastic lesions, metabolic reprogramming towards the Pentose Phosphate Pathway (PPP) was indicated by a strong increase in the expression and activity of glucose-6-phosphate dehydrogenase (G6PD). G6PD increased expression was observed exclusively in the highly proliferating KRT-19 positive preneoplastic lesions, considered the HCC precursor lesions in the R-H model, and was associated with low levels of miR-1, a miRNA known to target G6PD. Accordingly, forced expression of miR-1 down-regulated G6PD expression in HCC cells. PPP induction has been suggested to be one of the mechanisms by which deregulated NRF2-KEAP1 signaling promotes cellular proliferation and tumorigenesis. Since in the R-H rat model a sustained activation of the NRF2/KEAP1 pathway occurs in KRT-19+ nodules, we investigated the effect of impairing NRF2 in cells derived from R-H rat HCC. Notably, NRF2 silencing decreases G6PD and increases miR-1 expression, consequently inhibiting PPP and PKM2 oxidation. Finally, an inverse correlation between miR-1 and its target gene G6PD was found in human HCC patients.
Conclusion: Our results demonstrate that Warburg metabolic deregulation and PPP induction are early events in HCC development. Crucially, TRAP1 and NRF2 are key regulators of this metabolic reprogramming in preneoplastic hepatocytes
Citation Format: Marta A. Kowalik, Giulia Guzzo, Andrea Morandi, Andrea Perra, Silvia Menegon, Maria M. Angioni, Silvia Giordano, Paola Chiarugi, Andrea Rasola, Amedeo Columbano. OXPHOS inhibition and pentose phosphate pathway induction are early events priming preneoplastic lesions toward HCC development. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A02.