Abstract 2927: Inactivation of traf3 promotes intrahepatic cholangiocarcinoma development via hepatocyte trans-differentiation
Authors: Shiode Yuto , Kodama Takahiro , Hikita Hayato , Sakamori Ryotaro , Tatsumi Tomohide , Takehara Tetsuo 
Published: July 2021

Abstract
Aim: Intrahepatic cholangiocarcinoma (ICC) is a deadly cancer but little is known about its carcinogenesis mechanism. Insertional mutagenesis screen using sleeping beauty transposon (SBT) is a versatile tool to identify cancer genes in a high-throughput manner. We aimed to discover genes driving ICC by SBT mutagenesis screen in mice.
Methods: The mice with hundreds copies of movable mutagenic SBT were crossed with the mice knocking out Pten, a recurrently mutated cancer gene in human ICC, in hepatocytes and intrahepatic cholangiocytes using Albumin-Cre (Alb-Cre). They were aged and all the SBT-induced liver tumors were histologically assessed. Among the tumors with CCC component, frequently mutated genes were identified by the sequencing of transposon insertion sites. We then generated the mice with deletion of Traf3 and Pten in the liver or adult hepatocytes or intrahepatic cholangiocytes using Albumin-Cre (L-DKO) or Alb-CreERT2 (H-DKO) or K19-CreERT2 (C-DKO) mice, respectively. Adult hepatocytes-specific Traf3/Pten DKO mice was also generated by hydrodynamic tail vein injection (HTVi-DKO) or adeno-associated virus (AAV-DKO)-mediated Cre recombinase delivery. To identify cell origin of ICC, lineage tracing was performed by crossing H-DKO mice with ROSA26-LacZ reporter mice(H-DKO/LacZ). Single cell RNA-sequencing (scRNA-seq) was performed to clarify differentiation process of liver cells in the L-DKO mice.
Results: Transposon mutagenesis significantly accelerated liver tumorigenesis in the Pten-deficient mice. Among 20 tumors rich in CCC component, Traf3 was most frequently mutated by SBT in a loss-of-function manner. L-DKO mice showed marked biliary hyperplasia at 4 weeks of age and developed ICC at 3 months of age. While H-DKO, HTVi-DKO and AAV-DKO mice also phenocopied L-DKO mice, C-DKO mice did not recapitulate those phenotypes. Proliferating bile ducts and cholangiocarcinoma cells in H-DKO/LacZ mice were positive for β-galactosidase staining, indicating that they were originated from hepatocytes. ScRNA-seq identified unique cell population with both hepatocytes and cholangiocyte properties only in the liver of L-DKO mouse but not in the wild-type mouse. Trajectory analysis suggested that they were transdifferentiated from mature hepatocytes. In vitro experiments showed that Traf3/Pten inhibition significantly upregulated cholangiocyte markers in murine organoid-derived and human immortalized hepatocytes. Both L-DKO and H-DKO mice showed elevation of TNFα in the liver and its deletion significantly ameliorated development of ICC in the L-DKO mice. Human ICC patients with low intra-tumor Traf3 levels had more advanced stages and poorer prognosis than those with its high levels.
Conclusion: Inactivation of Traf3 promotes development of ICC via hepatocyte trans-differentiation in mice and may contribute to ICC pathogenesis in humans.
Citation Format: Yuto Shiode, Takahiro Kodama, Hayato Hikita, Ryotaro Sakamori, Tomohide Tatsumi, Tetsuo Takehara. Inactivation of traf3 promotes intrahepatic cholangiocarcinoma development via hepatocyte trans-differentiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2927.