The involvement of the Nrf2 / ß-catenin axis in hepatocarcinogenesis

Nuclear (erythroid-derived 2)-like 2 factor (Nrf2) protects against carcinogenesis and many other chronic diseases thought activating the cellular defence mechanisms. Moreover, it is also involved in proliferation and differentiation processes during liver regeneration. However, Nrf2 acts as a double-edged sword: Nrf2 activity in tumour cells supports cancer cell survival and progression. Current research articles describe various gain of function mutations in genes of the Keap1-Nrf2 pathway that lead to continuous Nrf2 activation as an early event in primary liver cancers (PLC). Therefore, it seems to be obvious that Nrf2 might be involved in tumour initiation processes. To examine the influence of continuous Nrf2 activity in hepatic cells the Keap1::Alb-Cre mouse model was used. Hepatic cells of these mice display continuous Nrf2 activity as a consequence of an albumin promoter driven Keap1 gene deletion, the inhibitor of Nrf2. After 90 weeks the liver of the Keap1::Alb-Cre mice were analysed. The livers showed various forms of primary liver cancer inter alia hepatoblastoma (HB), portal field dysplasia, and hepatocarcinomas (HCC). The malignancy of these tumours was confirmed by the presence of lung metastasis in these animals. Continuous Nrf2 activity creates a pro-regenerative milieu that is reflected by proliferation and hypertrophy of adult hepatic cells and liver progenitor cell (LPC) activation. It is already known that the activation and transformation of LPC into cancer stem cells (CSC) is an early event in hepatocarcinogenesis. The Keap1::Alb-Cre liver showed an increased number of activated LPC that finally resulted in HB development around the central vein and oval cell dysplasia in the portal field area. ß-catenin activation is a hallmark of HB and appears to play a crucial role in its pathogenesis. Therefore, we hypothesised that Nrf2 induced ß-catenin activity leads to CSC formation. In the Keap1::Alb-Cre livers we determine an increased ß-catenin expression and nuclear ß-catenin localisation in tumour lesions. In silico analyses of the ß-catenin promoter of various species confirmed the presence of a consensus sequence of the antioxidant response element (ARE), the Nrf2 binding site. The functionality of this ARE-sequence in the murine ß-catenin promoter was validated by reporter-gene assay, EMSA and ChIP experiments. To determine the involvement of Nrf2 induced ß-catenin expression in hepatocarcinogenesis a tumour screening in 28 weeks old Keap::Alb-Cre and Keap1::ß-Catenin::Alb-Cre double-knockout mice was preformed and showed that HB development is 50 % decreased in the Keap1::ß-cat::Alb-Cre livers in comparison to the Keap::Alb-Cre livers. These findings indicate the proto-oncogenic character of gain of function mutations in the genes of the Nrf2/Keap1-system. Continuous Nrf2 activation directly up-regulates ß-catenin expression a prerequisite to induce HB formation. In addition, we showed that an Nrf2 knockdown in hepatic tumour cells leads to decreased ß-catenin expression and inhibits the proliferation of primary hepatic tumour cells. Therefore, targeting the Nrf2/Keap1 pathway has a great potential to inhibit hepatocarcinogenesis in an early stage