Nrf2 targeting: a potential therapeutic strategy in hepatocellular carcinoma

The NRF2-KEAP1 pathway is the main intracellular defence against environmental stress, but its dysregulation has been observed in several human cancers, including hepatocellular carcinoma (HCC), one of the most aggressive cancer. However, how exactly aberrant NRF2 activation contributes to malignancy remains elusive. Aim of the present work is to assess i) whether the activation of the Nrf2-Keap1 pathway is an early event in rat hepatocarcinogenesis and is maintained all along the process, and ii) the molecular alterations underlying the activation of the pathway. Moreover, to directly establish the role of Nrf2 in hepatocarcinogenesis we used the recently developed Nrf2-Knockout (Nrf2-KO) rats. To this aim, we adopted a nutritional model of rat hepatocarcinogenesis consisting of a single dose of diethylnitrosamine (DENA) followed by a choline-methionine-deficient (CMD) diet for 4 months. This regimen causes extensive triglyceride accumulation, oxidative damage and the onset of preneoplastic lesions. Rats were then placed on basal diet and sacrificed after 6,10 and 13 months from DENA injection. When using Nrf2-KO rats, the animals were subjected to the CMD diet for 6 and 10 weeks following a single injection of DENA. The results showed that Nrf2 activation occurs since the early steps of hepatocarcinogenesis and persists all along the tumorigenic process even in the absence of the promoting environment generated by the CMD diet. Nrf2 mutations were very frequent at early steps of hepatocarcinogenesis (90%), but their number diminished with the progression of malignancy (25%). Interestingly, while very few mutations of Keap1 and no methylation of its promoter could be found at any stage of the process, accumulation of P62 occurred in HCCs suggesting that activation of Nrf2 at late stages of the process could be a consequence of Keap1 sequestration by p62. To directly assess the role of Nrf2 in hepatocarcinogenesis, we used a genetic approach consisting of Nrf2-KO rats. The results showed that Nrf2-KO rats, unlike wild-type animals, did not develop preneoplastic lesions after treatment with DENA followed by CMD diet for 6 or 10 weeks. Interestingly, no difference in biotransformation of DENA, DNA alkylation, hepatocellular necrosis or compensatory regeneration was found between WT and KO rats. The latter result demonstrates that while loss of Nrf2 does not inhibit the initiation step of hepatocarcinogenesis, it completely impairs the clonal expansion of initiated cells, suggesting that Nrf2 is critical in the onset of HCC.