Concomitant NFE2L2 and CTNNB1 mutations in a subset of HCC patients: Synergy between Nrf2 and Wnt pathway in hepatocarcinogenesis

Background Hepatocellular cancer (HCC) remains a major clinical need with gradually increasing incidence and currently lacks precision therapy. Thus, there is a need to better characterize molecular drivers in HCC and generate relevant models to test biology and therapies. In the USA, CTNNB1 mutations occur in 26% of all HCCs, however they alone are insufficient to lead to tumors as shown by preclinical studies suggesting cooperation with other pathways. Methods We assessed TCGA and other publically available databases for aberrations that co‐occur with CTNNB1 in HCC cases. We used sleeping beauty transposon/transposase (SBT) to express such clinically relevant mutants in a subset of hepatocytes in vivo in mice using hydrodynamic tail vein injection (HTVI). Specifically, we co‐delivered T41A‐CTNNB1 and wild type (WT)‐NFE2L2, G31A‐NFE2L2 or T80K‐NFE2L2 into 6–8 weeks old FVB mice via SBT‐HTVI and assessed tumorigenesis over time. WGS was performed on mouse tumors to identify differentially expressed genes and pathways. Similarity between the mouse model and human HCC subset was addressed by overlap between the most significantly altered pathways. Results Of the 377 HCC patients in TCGA, 99 patients showed CTNNB1 mutations. Analysis for Nrf2 pathway showed 15 HCCs with NFE2L2 and 18 with KEAP1 mutations. 12 patients showed coexistence of CTNNB1 mutations with wither NFE2L2 (n=6) or KEAP1 (n=6). Co‐expression of T41A‐CTNNB1 with G31A‐NFE2L2 or T80K‐NFE2L2 but not WT‐NFE2L2 led to HCC by 13–14 weeks. H&E staining confirmed the presence of well‐differentiated HCC. Immunostaining and WB showed HCCs to be strongly positive for b‐catenin targets (GS and pmTOR) and also for Nrf2 targets including NAD(P)H dehydrogenase [quinone] 1 and peroxiredoxin‐1. Next RNA‐Seq analysis on tumor‐bearing livers in mice using FDR=0.1 as cutoff showed many significantly altered pathways. Analysis of Nrf2‐CTNNB1 mutant HCC in TCGA also identified 64 pathways that were significantly altered using the FDR=0.1 as cutoff. 21 common and significant pathways were identified between our mouse model and patients, which have well known correlation with HCC development. Conclusions Thus, Nrf2 and Wnt pathway cooperate in HCC pathogenesis in a subset of patients. Co‐expression of mutant NFE2L2 and mutant CTNNB1 leads to HCC in mice with notable similarity in pathway alterations to the human HCC subset. This will be a valuable model to study biology and treatment for this subset of HCC cases. Support or Funding Information This work was supported by NIH grants R01CA204586 to S.P.M