The genetic event associated to the initiation process of hepatocarcinogenesis has not been well elucidated. By whole-exome analysis, we identified BrafV637E mutation, which is equivalent to human BrafV600E mutation, in all the four DEN-induced mouse hepatic tumors examined. By Sanger sequencing we validated that the BrafV637E mutation is highly frequent in the hepatic tumors [17/18 (94.4%)] in 1 year-old DEN-treated mice. Using DNA isolated by microscopic dissection we detected the BrafV637E mutation in 14/16 (87.5%) and 7/9 (77.8%) small preneoplastic hepatocyte foci respectively at 5 and 8 months after DEN treatment, indicating that the BrafV637E mutation occurs in the early stage. On the other hand, although the BrafV637E mutation was frequent in hepatic tumors induced by neonatal DEN treatment followed by weekly-administration of CCl4 [5/7 (71.4%)], none of 6 spontaneous and 24 CCl4-induced liver tumors showed the BrafV637E mutation, indicating that the BrafV637E mutation is specific for DEN-induced tumors. Histological analysis revealed that BrafV637E mutation was quite common in the lesions consisted of basophilic cells, while it was much less frequent in the lesions consisted of eosinophilic cells. We then investigated how the BrafV637E mutation influences the properties of preneoplastic/neoplastic hepatocytes. Firstly, the qPCR analysis revealed that p15Ink4b and p19ARF were upregulated in the DEN-induced hepatic tumors as compared to the surrounding non-tumor tissues, indicating that the Braf mutation leads to oncogene induced senescence (OIS), which may be related to the dormancy of preneoplastic lesions in the early stage. Secondly, when investigated the intracellular signaling pathways, Akt phosphorylation was much increased in the hepatic tumors as compared to the surrounding hepatic tissue, while ERK1/2 phosphorylation was not increased, suggesting the possible link between BrafV637E mutation and Akt activation. Thirdly, vemurafenib suppressed proliferation of the BrafV637E-mutated liver tumor cells both in vitro and in vivo, but not the proliferation of the BrafV637E mutation negative cells, indicating that BrafV637E mutation is related to the growth capacity. In summary, our results demonstrated that BrafV637E mutation is an early event in DEN-induced hepatocarcinogenesis, and may be related both to growth suppression via OIS and proliferation through activation of intracellular signaling. (Supported by the grant from the Japanese Ministry of Education, Culture, Sports, Science and Technology)

Citation Format: Masahiro Yamamoto, Hiroki Tanaka, Yuji Nishikawa, Keiko Shimizu, Katsuhiro Ogawa. Frequent BrafV637E mutation in hepatocarcinogenesis induced by neonatal treatment with diethylnitrosamine (DEN) in B6C3F1 mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1592. doi:10.1158/1538-7445.AM2014-1592