The role of Nrf2/Keap1 pathway in a nutritional model of rat hepatocarcinogenesis.

Non-alcoholic fatty liver disease (NAFLD) is becoming an emerging pathology in Western Countries. Although its initial stage, steatosis, is a benign and reversible condition, in some cases it can lead to more severe pathologies, including non-alcoholic steatohepatitis (NASH), that progress to liver fibrosis and cirrhosis and constitute a risk factor for the development of hepatocellular carcinoma (HCC). Although most experimental studies suggest that oxidative stress and antioxidative intracellular balance are crucial for neoplastic transformation , mechanisms leading to the NASH development and its progression to cancer remain unclear. A critical role of the Nrf2/Keap1 pathway, the main regulator of intracellular antioxidant response and detoxification, has been recently proposed in chemically induced rat liver carcinogenesis. To further investigate the role of this pathway in neoplastic transformation in a steatotic environment, I took advantage from a nutritional model of rat hepatocarcinogenesis, which allows to follow the process since the very early phases. In this work, I show that choline-devoid methionine-deficient diet (CMD) feeding of rats, pre-treated with a single diethylnitrosamine (DENA) administration, results in the development of preneoplastic nodules identified by the marker GST-P (placental isoform of gluthatione-S-transferase). Some of these nodules show resistance to steatosis which correlate with higher proliferative capacity. qRT-PCR analysis of two Nrf2 target genes, NQO1 and HMOX1, from single microdissected nodules, together with IHC staining for Nrf2, NQO1 and another Nrf2 target, G6PD, revealed increased pathway activation in preneoplastic lesions. Lesions overexpressing G6PD, a crucial enzyme for pentose phosphate pathway (PPP) and a marker of ‘metabolic switch’, constituted 23% of the GST-P+ population. Notably, these lesions displayed a significantly higher proliferation rate compared to those G6PD-negative. Importantly, we found that 44% of the identified nodules were mutated for Nrf2. However, Nrf2 mutation was not required for pathway activation, which occurred also independently from expression of p62, a protein involved in autophagy and previously correlated with the Nrf2/Keap1 pathway activation. In conclusion, these results suggest that the Nrf2/Keap1 pathway plays an important role in the process of NAFLD-related hepatocarcinogenesis, since the early steps of hepatocarcinogenesis.. Moreover, among preneoplastic nodules we identified a subset of rapidly growing lesions positive for G6PD, that can likely be considered the most aggressive. Our findings, although leaving several open questions, open the path to in depth studies aimed at addressing the exact role of the Nrf2/Keap1 pathway in NASH and preneoplastic development. This information would also have a translational value if inhibition of the Nrf2/Keap1 pathway could interfere with NASH progression to HCC.