Abstract 981: Loss of TGFβ signaling leads to increased susceptibility to HCC in mice: Evidences of lncRNA H19's effects in HCC promotion

The output of a TGF-β response is highly contextual depending on specific tissue types and diseases. This is especially true in hepatocellular carcinoma (HCC) initiation and progression, where the tumor-initialing and malignant cells are highly influenced by the background liver environments. To delineate the role of TGF-β in the process of hepatocarcinogenesis, we employed a novel tumor-initiating cell transplantation system that avoids the possible effects incurred from genetic manipulation of the background liver. Specifically, TGF-β receptor II (Tgfbr2) flox/flox mice at 14 days of age were given one dose (25μg/g bodyweight) of the hepatic carcinogen diethylnitrosamine (DEN) via intraperitoneal injection. Three months later, the initiated hepatocytes from Tgfbr2 flox/flox mice were isolated and transplanted to the same strain (C57Bl/6) wild type recipient mice. Four weeks after transplantation, the recipient mice received one does of Cre recombinase adenovirus (Ad-Cre) through tail vein injection to delete Tgfbr2 in the transplanted tumor-initiating hepatocytes; separate group of the recipient mice were injected with the control adenovirus (Ad-GFP). The recipient mice were closely monitored for liver tumor burden. We observed that deletion of Tgfbr2 in tumor-initiating hepatocytes by tail vein injection of Ad-Cre led to formation of more and bigger liver tumors (compared to Ad-GFP group). This finding suggests that TGF-β inhibits the malignant potential of tumor-initiating hepatocytes. In parallel, we further analyzed the effect of TGF-β in tumor initiating hepatocytes in vitro. Specifically, tumor-initiating hepatocytes isolated from DEN-treated Tgfbr2 flox/flox mice were infected with Ad-Cre or Ad-GFP prior to TGF-β1 treatment in vitro; RNAs were then isolated for transcriptome sequencing analysis. We identified a group of lncRNAs that were noticeably regulated by TGF-β, including the lncRNA H19. We found that deletion of Tgfbr2 by Ad-Cre in tumor initiating hepatocytes led to a 5-fold increase of H19 expression. We observed that deletion of H19 by siRNA decreased the tumor-initiating cell property, whereas forced overexpression of H19 enhanced it. Chromatin immunoprecipitation assay showed that SOX2 bound to the promoter region of H19 gene. While SOX2 overexpression in tumor-initiating hepatocytes enhanced H19 transcription, SOX2 knockdown reduced it. Furthermore, TGF-β treatment reduced the protein level of SOX2 in tumor-initiating hepatocytes. Taken together, our findings disclose a novel mechanism that importantly regulates hepatocarcinogenesis — TGF-β inhibits H19 expression via SOX2 in tumor-initiating hepatocytes and this effect leads to inhibition of HCC development. Citation Format: jinqiang Zhang, Chang Han, Weina Chen, Kyoungsub Song, Ying Wang, Lu Yao, Nathan Ungerleider, Hyunjoo Kwon, Tong Wu. Loss of TGFβ signaling leads to increased susceptibility to HCC in mice: Evidences of lncRNA H199s effects in HCC promotion. [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 981.