NRF2 DLG Domain Mutations Identified in Japanese Liver Cancer Patients Affect the Transcriptional Activity in HCC Cell Lines

Geographically, East Asia had the highest liver cancer burden in 2017. Besides this, liver can-cer-related deaths were high in Japan, accounting for 3.90% of the global deaths. The develop-ment of liver cancer is influenced by several factors and genetic alteration is one of critical fac-tors among them. Therefore, a detailed mechanism driving the oncogenic transformation of liv-er cells needs to be elucidated. Recently, many researchers have focused on investigating the liver cancer genome and identified somatic mutations (MTs) of several transcription factors. In this line, next-generation sequencing of the cancer genome identified that oxidative stress-related transcription factor NRF2 (NFE2L2) is mutated in different cancers, including hepatocellular carcinoma (HCC). Here, we demonstrate NRF2 DLG mutations (NRF2 D29A and L30F), found in Japanese liver cancer patients, upregulate the transcriptional activity of NRF2 in HCC cell lines. Moreover, the transcriptional activity of NRF2 mutations is not suppressed by KEAP1, presumably because NRF2 MTs disturb proper NRF2-KEAP1 binding and block KEAP1-mediated degradation of NRF2. Additionally, we exhibit that both MTs upregulate the transcriptional activity of NRF2 on MMP9 promoter in Hepa1-6 and Huh7 cells, suggesting that MTs derived gain-of-function of NRF2 may be important for liver tumor progression. We also find ectopic overexpression of oncogenic BRAF WT and V600E increased the transcriptional ac-tivity of NRF2 WT on both 3xARE reporter and MMP9 promoter. Interestingly, NRF2 D29A and L30F MTs with oncogenic BRAF V600E MT synergistically upregulate the transcription activity of NRF2 on 3xARE reporter and MMP9 promoter in Hepa1-6 and Huh7 cells. In summary, our findings suggest that MTs in NRF2 have the pathogenic effect, and NRF2 MTs together with on-cogenic BRAF V600E MT synergistically cause more aberrant transcriptional activity. The high activity of NRF2 MTs in HCC with BRAF MT warrants further exploration of this pathway\u27s po-tential diagnostic, prognostic, and therapeutic utility in HCC