Abstract 661: Establishment of transgenic mice with liver-specific BRAF V600E mutation

Abstract

Abstract Diethylnitrosamine (DEN) is often used to induce hepatocarcinogenesis in rodents to investigate the pathogenesis of hepatocellular carcinoma (HCC). BrafV637E mutation, corresponding to the human BRAFV600E mutation, is highly frequent in DEN-induced hepatic tumors depending on mouse strains. To investigate the role of BrafV637E mutation in DEN-induced hepatocarcinogenesis, we established the transgenic mice (Alb-Cre/BRAFV600E), in which human BRAFV600E mutation is specifically expressed in the liver, and compared the properties of DEN-induced hepatic tumors and the livers of the transgenic mice. The hepatic tumors were induced by neonatal treatment with DEN, isolated at various time points, and stored as frozen or paraffin-embedded materials. For production of the transgenic mice, we obtained homozygous male BRAFV600E mice, in which a LoxP-flanked cassette containing the human BRAF exons 15-18 cDNA, mouse polyadenilation sequences and the neo gene was inserted into the mouse Braf intron 14 upstream of the human BRAF exon 15 cDNA that encodes the BRAFV600E mutation, and crossed them with female Alb-Cre mice by in vitro fertilization. RT-PCR and cDNA sequencing revealed that the liver of Alb-Cre/BRAFV600E mice expressed mRNA derived both from normal mouse and mutated human BRAF exon 15. The transgenic mice showed 20% decrease in body weight compared to normal mice, but 5 fold-increase in the liver/body weight. Histological examination revealed that the liver was entirely consisted of basophilic hepatocytes resembling to those in DEN-induced foci of cellular alteration at 8 weeks of birth, and bile duct cells were further increased at 10-12 weeks. The Ki-67 labeling index of hepatocytes in the Alb-Cre/BRAFV600E mice was 3 times higher than normal mice. Immunoblot analysis revealed that the liver of Alb-Cre/BRAFV600E mice expressed the BRAFV600E protein and showed hyperphosphlylation of ERK1 and AktS473 like the DEN-induced hepatic tumors. Furthermore, immunohistochemical staining detected increased C5/C5a expression in the hepatocytes of Alb-Cre/BRAFV600E mice, and by immunoblot analysis C5a, a protease-cleaved form of C5, was specifically detected in Alb-Cre/BRAFV600E liver like DEN-induced tumors. These results indicate that the liver specific expression of mutationally-activated BRAF led to the hepatocytic phenotype mimicking the DEN-induced hepatic tumors. Citation Format: Hiroki Tanaka, Masahiro Yamamoto, Kosuke Yamazaki, Keiko Shimizu, Katsuhiro Ogawa. Establishment of transgenic mice with liver-specific BRAF V600E mutation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 661.