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.

Kinase Suppressor of Ras 1 Promotes YAP/TAZ-Mediated Tumorigenesis in the Liver

( Hyuk Moon ),( Kyungjoo Cho ),( Soonyoung Shin ),( Simon W. Ro ),( Beom Kyung Kim ),( Seung Up Kim ),( Jun Yong Park ),( Sang Hoon Ahn ),( Do Young Kim ),( Kwang-hyub Han ) 대한간학회 2018 춘·추계 학술대회 (KASL) Vol.2018 No.1

Aims: Liver cancer is the second most common cause of cancer-related deaths worldwide. The Hippo signaling pathway is tumor suppressive, as its inactivation leads to tissue overgrowth and tumor formation via YAP- or TAZ-mediated transcriptional activation. YAP is overexpressed in 62% of patients with hepatocellular carcinoma (HCC) and in almost 90% of human cholangiocarcinoma (CCC). Kinase Suppressor of Ras 1 (KSR1) is a scaffold protein for the Ras/Raf/MEK/ERK signaling pathway, promoting activation of MEK and ERK. Pro-tumorigenic roles of KSR1 in Ras-activated cancers have been verified in murine models for lung, skin, and pancreatic cancers. In this study, we have investigated the role of KSR1 in YAP/TAZ-mediated hepatocarcinogenesis. Methods: Transposons were constructed encoding KSR1 and an activated from of TAZ (TAZ^S89A). Transposons were hydrodynamically delivered to livers 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: Analysis of gene expression levels in human HCC and CCC samples deposited in The Cancer Genome Atlas (TCGA) revealed that KSR1 was significantly upregulated both in human HCC and CCC, compared with non-tumoral surrounding livers (P<0.0001 in HCC and P=0.0063 in CCC). Co-expression of KSR1 and an activated form of TAZ (TAZ^S89A) led to the development of both HCC and CCC in the murine livers, while expression of TAZ^S89A alone failed to induce hepatic tumors. Conclusions: KSR1 promotes hepatocarcinogenesis, both in HCC and CCC. Suppression of KSR1 might be an attractive therapeutic option for both types of hepatic malignancies.

Development of Mouse Models for Hepatocellular Carcinoma and Cholangiocarcinoma Induced by Activated TAZ

( Hyuk Moon ),( Kyungjoo Cho ),( Do Young Kim ),( Simon W. Ro ),( Kwang-hyub Han ) 대한간학회 2017 춘·추계 학술대회 (KASL) Vol.2017 No.1

Aims: Hepatocellular carcinoma (HCC) is the second most common cause of cancer-related deaths worldwide. The Hippo signaling pathway is tumor suppressive, as its inactivation leads to tissue overgrowth and tumor formation via YAP- or TAZ-mediated transcriptional activation. YAP is overexpressed in 62% of patients with HCC and in almost 90% of human cholangiocarcinoma (CC). High YAP/TAZ levels correlate with poor prognosis for both types of liver cancer. In this study, we have developed transgenic mouse models for HCC and CC induced by an activated form of TAZ (TAZ^S89A) combined with an activated form of RAS or PI3K, respectively. Methods: Transposons were constructed encoding TAZ^S89A, an activated from of human H-RAS (HRAS^G12V), and an activated form of human PI3K (PI3K^E545K). Transposons were hydrodynamically delivered to livers 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: HCCs were induced by the stable expression of TAZ^S89A and HRAS^G12V, while CCs developed in livers expressing TAZ^S89A and PI3K^E545K. Nuclear accumulation of TAZ was confirmed by im-munohistochemistry in both types of tumors. There were lung metastases found in 20% mice bearing HCCs induced by TAZ^S89A and HRAS^G12V. Conclusions: TAZ can induce tumorigenesis toward either HCC or CC depending on collaborating oncogene. HCCs induced by TAZ^S89A and HRAS^G12V were highly invasive and led to metastasis to the lung in 20% of mice.

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^T58A) 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^T58A and the wild-type (WT) c-Myc in the liver. Methods: Transgenic mouse models expressing c-Myc^T58A 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^T58A 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^T58A or WT c-Myc with 100% penetration. There was no significant difference in animal survivals between the c-Myc^T58A 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^T58A 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^T58A, compared with WT c-Myc.

Autophagy Inhibitor, Chloroquine Did Not Affect Tumor Development in a Transgenic Mouse Model of HCC

( Soonyoung Shin ),( Hyuk Moon ),( Kyungjoo Cho ),( Simon W. Ro ),( Beom Kyung Kim ),( Seung Up Kim ),( Do Young Kim ),( Kwang-hyub Han ) 대한간학회 2018 춘·추계 학술대회 (KASL) Vol.2018 No.1

Aims: Autophagy is an intracellular recycling process by which damaged or superfluous proteins are delivered to lysosomes for degradation, and then utilized as energy resources and macromolecular precursors. Autophagy in cancer is a highly debated subject. Research has shown that autophagy can become either tumor-promoting or tumor-suppressive depending on cellular or genetic context. Here, we investigated the role of autophagy in hepatocellular carcinoma (HCC) by applying an autophagy inhibitor, chloroquine to a transgenic mouse model of HCC. Methods: Transposons were constructed encoding an activated from of RAS (HRASG12V) and short hairpin suppressing P53 (shp53). Transposons were hydrodynamically delivered to livers of 6-week-old C57BL/6 mice. Mice were administered intraperitoneally with chloroquine at a daily dose of 60mg/kg for five weeks. Control mice were given a phosphate buffered saline (PBS). Mice were monitored at least twice per week. Results: The sizes and numbers of tumor nodules were similar between chloroquine group and control when livers were harvested at 5 weeks after the delivery of oncogenes. Animal survivals were not significantly different between the two groups, suggesting that the treatment with chloroquine does not affect liver tumorigenesis induced by HRASG12V plus shp53. Conclusions: Our study suggests that autophagy inhibition has a minimal role in HCC under the genetic context of RAS signaling activation and P53 downregulation.

Differential Hepatocarcinogenic Potentials between KRAS Splicing Variants

( Hyuk Moon ),( Sook In Chung ),( Simon W. Ro ),( Kwang-hyub Han ) 대한간학회 2016 춘·추계 학술대회 (KASL) Vol.2016 No.1

Aims: In humans, three RAS genes encode four RAS proteins with a high degree of sequence homology: HRAS, NRAS, KRAS4A and KRAS4B, with the latter two resulting from alternative splicing of exon 4 of the KRAS gene. Activation of RAS signaling pathways is considered a key oncogenic event in human carcinogenesis. One unresolved question is whether there are differential oncogenic potentials among activated RAS isoforms. Methods: Hydrodynamic transfection was performed with transposons expressing short hairpin RNA down-regulating p53 and each of activated RAS isoforms, and livers were harvested at 23 days after gene delivery to investigate the presence of tumors. Also, survivals of mice expressing different RAS isoforms were compared following hydrodynamic transfection. Results: No differences were found in hepatocarcinogenic potentials among RAS isoforms as determined by both gross examination of livers and liver weight per body weight ratios (LW/BW) of mice expressing HRASQ61L, KRAS4BG12V, and HRASQ61L, respectively. However, tumorigenic potentials were significantly different between KRAS splicing variants. The LW/BW ratio in KRAS4AG12V mice was significantly lower than that in KRAS4BG12V group (p < 0.001) and KRAS4AG12V mice lived significantly longer than KRRAS4BG12V mice (p < 0.0001). Immunoblotting revealed that tumors from KRAS4AG12V mice had an elevated expression of p16INK4A compared with KRAS4BG12V tumors. Co-expression of p16INK4A with KRAS4BG12V significantly reduced tumor growth, suggesting that the up-regulation of p16INK4A by KRAS4AG12V likely retarded tumor development driven by KRAS4AG12V. Conclusions: Oncogenic potentials differed significantly between the two KRAS splicing variants; KRAS4B being more tumorigenic than KRAS4A in the liver. Thus, it is presumed that when an activating mutation arises in KRAS, KRAS4B will predominantly lead the tumorigenic processes.

Establishment of 3D Multicellular Tumor Spheroids (MCTS) using Hepatocellular Carcinoma and Stromal cells for Screening Anti-cancer Therapeutic Agents

( 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.

Differential Tumorigenic Effects by C-Myc Mutants in Liver Cancer

( Daeyoung Kim ),( Hyuk Moon ),( Sook In Chung ),( Simon W. Ro ),( Kwang-hyub Han ) 대한간학회 2016 춘·추계 학술대회 (KASL) Vol.2016 No.1

Aims: Liver cancer is a major health concern worldwide, ranking third in terms of cancer-related mortality. The c-Myc gene is epigenetically altered in almost 50% of human liver cancers, leading to persistent over-expression of cMyc. In addition to quantitative changes of cMyc protein in cancers, mutation leading to amino acid substitution of cMyc has been found in a certain type of cancers. In this study, we compared tumorigenic potentials among c-Myc mutants in the liver. Methods: Transgenic liver cancer mouse models expressing different c-Myc mutants were developed using hydrodynamic transfection. Transposon vectors encoding the wild-type c-Myc, c-MycT58A, and c-MycS71F were constructed. To induce liver cancer, 20 μg of transposons were mixed with plasmids expressing the Sleeping Beauty transposase and then diluted in 2.5 ml of 0.9% saline. 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. Results: Hepatocellular carcinomas (HCC) were induced by the stable expression of c-Myc and shp53. Wild type c-Myc was less tumorigenic than c-Myc2T58A or c-Myc2S71F when co-expressed with shp53. The c-Myc mutant groups, c-Myc2T58A or c-Myc2S71F died earlier than the c-Myc wild type group (p< 0.05). There was no difference in phenotypes of malignant hepatocytes among tumors induced by cMyc mutants and wild-type. Conclusions: Co-expression of c-Myc and shp53 in the mouse liver promoted hepatocarcinogenesis. Wild type c-Myc was less tumorigenic than c-Myc2T58A or c-Myc2S71F under the condition that P53 was down-regulated.