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PE-096: An Increased Incidence of Hepatocellular Carcinoma in Fibrotic Livers
( Kyungjoo Cho ),( Sook In Chung ),( Hyuk Moon ),( Simon W. Ro ),( Kwang-hyub Han ) 대한간학회 2016 춘·추계 학술대회 (KASL) Vol.2016 No.1
Aims: Liver fibrosis and its end-stage disease, cirrhosis, are major risk factors for hepatocellular carcinoma (HCC) and present in 80 to 90% of patients with HCC. Current genetically engineered mouse models for HCC, however, generally do not feature liver fibrosis, which is a critical discrepancy between human HCC and murine models thereof. In this study, we developed a transgenic mouse model of HCC with concurrent liver fibrosis induced by the treatment with carbon tetrarchloride. Methods: Employing hydrodynamic transfection (HT), coupled with the Sleeping Beauty (SB) transposon system, liver was stably transfected with transposons expressing cMyc and a short hairpin RNA down-regulating p53 (shp53). A chronic liver injury model, induced by hepatotoxic carbon tetrachloride (CCl4), was applied to the transgenic mice, allowing cells expressing cMyc plus shp53 to become malignant in the background of liver fibrosis. Results: Livers harvested about 3 months after HT had excessive collagen deposition and activated hepatic stellate cells surrounding the tumors. Hepatocarcinogenesis was significantly accelerated in the fibrotic livers compared to those of the control, significantly decreasing the life span of the mice. The tumor incidence and average number of tumors per mouse were significantly higher in the group treated with CCl4 compared to the vehicle-treated control mice, following HT (p < 0.01). Conclusions: Transgenic model for HCC was successfully developed in fibrotic liver background. Liver fibrosis significantly accelerated tumor development in the liver.
( Kyungjoo Cho ),( Hyuk Moon ),( Soonyoung Shin ),( Simon W. Ro ),( Hye Won Lee ),( Beom Kyung Kim ),( Do Young Kim ),( Kwang-hyub Han ) 대한간학회 2018 춘·추계 학술대회 (KASL) Vol.2018 No.1
Aims: Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer in adults and the second leading cause of cancer-related deaths worldwide. Tumor microenvironment is composed of myofibroblasts, fibroblasts, immune-inflammatory cells, extracellular matrix, blood vessels, etc. and closely involved in multiple facets of tumorigenesis. Studies have shown that response to chemotherapy is highly affected by drug penetration through tumor tissue, highlighting the role of tumor microenvironment in cancer chemotherapy. Compared with tumor cell monolayer culture, multi-cellular tumor spheroid (MCTS) is superior in mimicking tumor microenvironment and thus a suitable model for studying drug penetration into tumor. The purpose of this study is to establish the MCTS model to investigate the interaction with the microenvironment in tumor and to investigate the effect of microenvironment on drug permeability. Methods: To generate multicellular tumor spheroids, HCC cells were seeded at a density of 6 × 103 cells/well in 96-well round-bottom ultra-low attachment microplates. The plates were incubated for 3 days at 37 °C in a humidified atmosphere of 5% CO2. To generate MCTS, HCC cells and stromal cells (LX2, WI38, and HUVECs) were mixed at a 1:1 ratio. Diameter was measured using a confocal microscope and an image analyzer to determine the compactness of spheroid. Protein expression levels in MCTS were determined by immunoblots. For drug penetration study, fluorescent chemicals (e.g., verteporfin) were used and the distribution of drug within MCTS was determined by a LSM700 confocal microscope with a 425 to 440nm excitation and a 700 to 730nm emission filter set. Results: Various multi-cellular tumor spheroid (MCTS) models were developed using HCC cell lines with various degrees of differentiation such as SNU449 (Well differentiated), SNU3059 and SNU3160 (moderately differentiated), and Hep3B (poorly differentiated). The volume, shape, and compactness of HCC MCTS were heterogeneous depending on the differentiation degree. Well differentiated SNU449 MCTS showed the least compactness of tumor spheroids, while Hep3B MCTS had the highest compactness. Of note, YAP/TAZ levels in HCC MCTS were significantly different. SNU449 MCTS model had a low level of YAP/TAZ, while Hep3B MCTS model had the highest level of YAP/TAZ expression. Drug penetration into tumor spheroids was significantly retarded due to the multi-cellular components within HCC MCTS. HCC MCTS with higher YAP/TAZ levels increased the compactness and inhibited drug penetration. Conclusions: In this study, diverse MCTS models have been developed using HCC of different degrees of differentiation and stromal cells such as HSCs, fibroblasts, and endothelial cells. MCTS with poorly differentiated HCC showed an increased compactness of spheroids, an elevated level of YAP/TAZ and a limited drug penetration. Reducing tumor compactness or stromal activation should be considered to improve a response to chemotherapy in patients with advanced HCC.
T58A Mutation Does Not Enhance Tumorigenic Potentials of C-MYC in the Liver
( Kyungjoo Cho ),( Sook In Chung ),( Hyuk Moon ),( Daeyoung Kim ),( Do Young Kim ),( Simon W. Ro ),( Kwang-hyub Han ) 대한간학회 2017 춘·추계 학술대회 (KASL) Vol.2017 No.1
Aims: Liver cancer is a major health concern worldwide, ranking the second among cancer-related mortality. The c-Myc gene is epigenetically altered in almost 50% of human liver cancers, leading to persistent over-expression of c-Myc. Mutation at codon 58 of c-Myc (c-Myc<sup>T58A</sup>) can enhance oncogenic potentials of c-Myc through suppressing apoptotic signaling cascades or stabilizing the oncoprotein. In this study, we compared tumorigenic potentials between c-Myc<sup>T58A</sup> and the wild-type (WT) c-Myc in the liver. Methods: Transgenic mouse models expressing c-Myc<sup>T58A</sup> and WT c-Myc were developed using hydrodynamic transfection. Transposons encoding an activated from of human H-RAS were mixed with transposons encoding either c-Myc<sup>T58A</sup> or WT c-Myc. The DNA mixtures were injected into the lateral tail veins of 6-week-old C57BL/6 mice. Mice were monitored at least twice per week and sacrificed when moribund. Tumor-bearing livers were formalin fixed for hematoxylin- eosin staining and immunohistochemistry. Results: Hepatocellular carcinomas (HCC) were induced by co- expression of HRAS with either c-Myc<sup>T58A</sup> or WT c-Myc with 100% penetration. There was no significant difference in animal survivals between the c-Myc<sup>T58A</sup> and WT c-Myc groups. The numbers and sizes of tumors were similar between the two groups. Cellular proliferation (determined by Ki-67 staining) and apoptosis levels (by TUNEL assay) were also similar between c-Myc<sup>T58A</sup> and WT c-Myc groups. Finally, there was no difference in phenotypes of malignant hepatocytes between the two groups. Conclusions: T58A mutation does not enhance tumorigenic potentials of c-MYC in our transgenic mouse models. No downregulation of apoptosis was detected in c-Myc<sup>T58A</sup>, compared with WT c-Myc.