Basic, HCC basic : O-034 ; Pro-oncogenic NM23-H2 modulates MDM2 expression in hepatocarcinogenesis

( Mi Jin Lee ),( Goung Ran Yu ),( Hua Lee ),( Sang Wook Kim ),( Pei Pei Hao ),( In Hee Kim ),( Dae Ghon Kim ) 대한간학회 2012 춘·추계 학술대회 (KASL) Vol.2012 No.-

Background: NM23 is a family of structurally and functionally conserved proteins known as nucleoside diphosphate kinases (NDPK). NM23-H1 and NM23-H2 are expressed abundantly in HCC. NM23-H2 is a basic protein recently identified as the human PuF factor, which is a transcriptional activator of the c-Myc proto-oncogene. Although the NM23-H1 protein is implicated as a metastasis suppressor, the role of NM23-H2 appears to be less understood. Thus, the aim of this study is to examine functional role and mechanism of NM23-H2 involved tumorigenesis in HCC. Methods: We examined the NM23-H1, H2 and LV mRNA expression in HCC by Realtime-PCR analysis and NM23-H2 protein expression in HCC by immunoblot and immunohistochemistry. Focus formation and anchorage-independent growth were examined in stable cell lines expressing NM23-H2 using soft agar. Using overexpression of NM23-H2 by adenoviral system, the molecular mechanism of NM23-H2 mediated tumor cell growth was assessed in experimental cell culture and in vivo animal model. Results: The level of NM23-H2 expression in tumor tissues and the surrounding matrix appeared to be independent of etiology and tumor differentiation. Ectopic expression of NM23-H2 in NIH3T3 fibroblasts and HLK3 hepatocytes enhanced focus formation, and allowed anchorage-independent growth. Ectopic expression of NM23-H2 induced MDM2 expression. However, MDM2 mRNA and promoter activity was not changed by ectopic expression of NM23-H2, but NM-23H2 interacted with MDM2. The NIH3T3 fibroblasts and HLK3 hepatocytes stably expressing NM23-H2 produced tumors in athymic mice. Lentiviral delivery of NM23-H2 shRNA inhibited tumor growth of xenotransplanted tumors produced from HLK3 cells stably expressing NM23-H2. Conclusions: These results indicate that NM23-H2 may be pro-oncogenic and regulate MDM2 expression in hepatocarcinogenesis. Therefore, this pathway may be an useful target for HCC treatment.

Slide Session : K-GIO-03 ; GI Oncology : Pro-Oncogenic NM23 Contributes to Hepatocarcinogenesis Through the AKT/ Extracellular Signal-Regulated Kinase (ERK)1/2 Pathway

( Mi Jin Lee ),( Goung Ran Yu ),( Hua Lee ),( Jun Zhang ),( Yun A Kim ),( Dae Ghon Kim ) 대한내과학회 2014 대한내과학회 추계학술대회 Vol.2014 No.1

Background: NM23 is a family of structurally and functionally conserved proteins known as nucleoside diphosphate kinases (NDPK). NM23-H1, NM23-H2 and NM23- LV are expressed abundantly in HCC. NM23-H2 is a basic protein recently identifi ed as the human PuF factor, which is a transcriptional activator of the c-Myc proto-oncogene. Although the NM23 protein is implicated as a metastasis suppressor, the role of NM23 appears to be less understood. Thus, the aim of this study is to examine functional role and mechanism of NM23 involved tumorigenesis in HCC. Methods: We examined the NM23-H1, H2 and LV mRNA expression in HCC by Realtime- PCR analysis and NM23-H1, H2 and LV protein expression in HCC by immunoblot and immunohistochemistry. Focus formation and anchorage-independent growth were examined in stable cell lines expressing NM23 using soft agar. Using overexpression of NM23 by adenoviral system, the molecular mechanism of NM23 mediated tumor cell growth was assessed in experimental cell culture and in vivo animal model. Results: The level of NM23 expression in tumor tissues and the surrounding matrix appeared to be independent of etiology and tumor differentiation. Ectopic expression of NM23-H2 in NIH3T3 fi broblasts and HLK3 hepatocytes enhanced focus formation, and allowed anchorage-independent growth. Overexpression of NM23 results in increased tumor cell proliferation, which is relevant to activation of AKT and extracellular signal-regulated kinase (ERK)1/2 phosphorylation. The NIH3T3 fi broblasts and HLK3 hepatocytes stably expressing NM23-H2 produced tumors in athymic mice. Lentiviral delivery of NM23-H2 shRNA inhibited tumor growth of xenotransplanted tumors produced from HLK3 cells stably expressing NM23-H2. Conclusions: These results indicate that NM23 may be pro-oncogenic and involved in hepatocarcinogenesis through AKT/ERK signaling pathway. Therefore, this pathway may be an useful target for HCC treatment.

C-terminal-truncated HBV X promotes hepato-oncogenesis through inhibition of tumor-suppressive β-catenin/BAMBI signaling

이석,이미진,Jun Zhang,Goung-Ran Yu,김대곤 생화학분자생물학회 2016 Experimental and molecular medicine Vol.48 No.-

C-terminal-truncated hepatitis B virus (HBV) X (HBx) (ctHBX) is frequently detected in hepatocellular carcinoma (HCC) through HBV integration into the host genome. However, the molecular mechanisms underlying ctHBx-associated oncogenic signaling have not yet been clarified. To elucidate the biological role of ctHBx in hepato-oncogenesis, we functionally analyzed ctHBx-mediated regulation of the activin membrane-bound inhibitor bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) through transforming growth factor-β (TGF-β) or β-catenin (CTNNB1) in HCC cells and in an animal model, and we compared its role to that of the full-length HBx protein. Ectopic ctHBx expression generated more colonies in anchorage-dependent and -independent growth assays than did HBx expression alone. ctHBx downregulated BAMBI to a greater degree than did HBx in HCC cells. HBx activated the Wnt/β-catenin pathway, which positively regulated the BAMBI expression through T-cell factor 1 signaling, whereas ctHBx negatively regulated the Wnt/β-catenin pathway. BAMBI downregulated the β-catenin and TGF-β1 signaling pathways. TGF-β1 positively regulated BAMBI expression thorough Smad3 signaling. Furthermore, knockdown of BAMBI was more tumorigenic in HCC cells. Therefore, downregulation of both β-catenin and TGF-β1 signaling by BAMBI might contribute to tumor suppression in mice xenotransplanted with HepG2 or SH-J1 cells. Taken together, ctHBx may have a more oncogenic role than HBx through its inhibition of tumor-suppressive β-catenin/BAMBI signaling.

Basic, HCC basic : O-034 ; Pro-oncogenic NM23-H2 modulates MDM2 expression in hepatocarcinogenesis

( Mi Jin Lee ),( Goung Ran Yu ),( Hua Lee ),( Sang Wook Kim ),( Pei Pei Hao ),( In Hee Kim ),( Dae Ghon Kim ) 대한간학회 2012 춘·추계 학술대회 (KASL) Vol.2012 No.1

Background: NM23 is a family of structurally and functionally conserved proteins known as nucleoside diphosphate kinases (NDPK). NM23-H1 and NM23-H2 are expressed abundantly in HCC. NM23-H2 is a basic protein recently identified as the human PuF factor, which is a transcriptional activator of the c-Myc proto-oncogene. Although the NM23-H1 protein is implicated as a metastasis suppressor, the role of NM23-H2 appears to be less understood. Thus, the aim of this study is to examine functional role and mechanism of NM23-H2 involved tumorigenesis in HCC. Methods: We examined the NM23-H1, H2 and LV mRNA expression in HCC by Realtime-PCR analysis and NM23-H2 protein expression in HCC by immunoblot and immunohistochemistry. Focus formation and anchorage-independent growth were examined in stable cell lines expressing NM23-H2 using soft agar. Using overexpression of NM23-H2 by adenoviral system, the molecular mechanism of NM23-H2 mediated tumor cell growth was assessed in experimental cell culture and in vivo animal model. Results: The level of NM23-H2 expression in tumor tissues and the surrounding matrix appeared to be independent of etiology and tumor differentiation. Ectopic expression of NM23-H2 in NIH3T3 fibroblasts and HLK3 hepatocytes enhanced focus formation, and allowed anchorage-independent growth. Ectopic expression of NM23-H2 induced MDM2 expression. However, MDM2 mRNA and promoter activity was not changed by ectopic expression of NM23-H2, but NM-23H2 interacted with MDM2. The NIH3T3 fibroblasts and HLK3 hepatocytes stably expressing NM23-H2 produced tumors in athymic mice. Lentiviral delivery of NM23-H2 shRNA inhibited tumor growth of xenotransplanted tumors produced from HLK3 cells stably expressing NM23-H2. Conclusions: These results indicate that NM23-H2 may be pro-oncogenic and regulate MDM2 expression in hepatocarcinogenesis. Therefore, this pathway may be an useful target for HCC treatment.

EKI1 (Epha2 Kinase Inhibitor 1) Suppresses Tumor Growth in Hepatocellular Carcinoma and Cholangiocarcinoma by Inducing Autophage and Apoptosis

( Mi-jin Lee ),( Goung-ran Yu ),( Hua Lee ),( Yun-a Kim ),( Jun Zhang ),( Dae-ghon Kim ) 대한간학회 2016 춘·추계 학술대회 (KASL) Vol.2016 No.1

Aims: Erythropoietin-producing hepatocellular receptor tyrosine kinase subtype A2 (EphA2) is an attractive therapeutic target for suppressing tumor progression. EKI1 is coordination complex of zinc and known for its use in treating dandruff and seborrhoeic dermatitis. The aim of this study is to discover novel small molecules to inhibit EphA2 for the treatment of hepatocellular carcinoma and cholangiocarcinoma. Methods: To discover novel and potent EphA2 inhibitors, we performed HTRF (Homogeneous time resolved fluorescence) kinase assay using the chemical library of Korea Chemical Bank and primary screened novel hit compounds. The enhancement of EKI1-mediated apoptosis and autophage were examined using immunoblotting and FACS analysis. Additionally, the antitumor effect of EKI1 was assessed using a mouse model. Results: Thirty-six compounds screened as EphA2 kinase inhibitor by HTRF assay. We validated these substances related to inhibit cell proliferation and cell death. We identified EKI1, a effective theranostics based small molecules. Human hepatocellular carcinoma cell line and cholangiocarcinoma cell lines were treated various concentration of EKI1 for 12h. At low concentration of EKI1, proliferation of these cells was inhibited. At high concentration of EKI1, cell death was induced in these cells. As quantitatively assessed by flow cytometry, apoptosis was induced by EKI1 in cells. We investigated apoptotic signaling by Western blot and observed cleavage or overexpression of pro-caspase-7 and PARP in EKI1 treated cells compared with vehicle. The anti-proliferation effect of EKI was due to an increased autophage, which was confirmed by up-regulation of Autophagy protein 5 (Atg5), BECN and LC3 (autophagosome marker). In addition, EKI1 induced reactive oxygen species (ROS) in JCK and Huh7. We examined EKI1 up-or down-signal transduction pathways to use therapeutic target for HCC and CC. In vivo mouse model, tumor growth was suppressed in EKI1 injected mouse group compared with control group. Conclusions: Our results revealed that autophage and apoptosis are involved in EKI1-mediated tumor cell death. Therefore, the EphA2 kinase inhibitor EKI1 is therapeutic target for hepatocellular carcinoma and cholangiocarcinoma.

Basic, Research : Lipocalin-2 Negatively Modulates the EMT in Hepatocellular Carcinoma through the EGF (TGFbeta1)/Lcn2/Twist1 Pathway

( Yun Peng Wang ),( Goung Ran Yu ),( Mi Jin Lee ),( Sang Yeop Lee ),( In Sun Chu ),( Sun Hee Leem ),( Dae Ghon Kim ) 대한간학회 2013 춘·추계 학술대회 (KASL) Vol.2013 No.1

Backgrounds/Aims: Lipocalin-2 is preferentially expressed in hepatocellular carcinoma. However, the functional role of Lcn2 in HCC progression is still poorly understood, particularly with respect to its involvement in invasion and metastasis. The purpose of this study was to investigate whether Lcn2 is associated with the EMT change of HCC and to elucidate its signaling pathway. Methods: Lcn2 mRNA expression was confirmed by RT-PCR analysis. Lcn2 protein expression was investigated by immunohistochemistry or Western blot analyses. The proliferation, migration, and invasion ability were measured by MTT, wound healing and invasion assays in HCC cells (SH-J1) stably expressing Lcn2. Tumor growing and metastasis by Lcn2 was tested in vivo animal model. Results: Lcn2 is preferentially expressed in well differentiated HCC versus liver cirrhosis tissues, and its expression is positively correlated with the Edmondson differentiation grade of HCC. The characteristics of EMT are reversed by adenoviral transduction of Lcn2 into SH-J1 cells, including the down-regulation of N-cadherin, vimentin, a-SMA, and fibronectin, and the concomitant up-regulation of CK8, CK18, and desmoplakin I/II. Knock-down of Lcn2 by shRNA in HKK-2 cells highly expressing Lcn2 was associated with EMT change. EGF or TGF-β1 treatment resulted in down-regulation of Lcn2, subsequently accompanied by Twist1 expression and EMT change in HCC cells. Stable Lcn2 expression in HCC cells reduced Twist1 expression, led to inhibition of cell proliferation and invasion in vitro, and suppressed tumor growth and metastatic ability in a mouse model. Furthermore, EGF or TGF-β1 treatment barely changed EMT marker expression in the SH-J1 cells ectopically expressing Lcn2. Ectopic expression of Twist1 induced EMT marker expression even in cells expressing Lcn2, indicating that Lcn2 functions downstream of growth factors and upstream of Twist1. Conclusions: These findings suggest that Lcn2 can negatively modulate the EMT change in HCC cells through the EGF (or TGF-β1)/Lcn2/Twist1 pathway. Thus, Lcn2 may be a candidate metastasis suppressor and a potential therapeutic target in HCC.

Pro-oncogenic potential of NM23-H2 in hepatocellular carcinoma

이미진,Dong-Yuan Xu,Hua Li,Goung-Ran Yu,임선희,추인선,김인희,김대곤 생화학분자생물학회 2012 Experimental and molecular medicine Vol.44 No.3

NM23 is a family of structurally and functionally conserved proteins known as nucleoside diphosphate kinases (NDPK). There is abundant mRNA expression of NM23-H1, NM23-H2, or a read through transcript (NM23-LV) in the primary sites of hepatocellular carcinoma (HCC). Although the NM23-H1 protein is implicated as a metastasis suppressor, the role of NM23-H2 appears to be less understood. Thus, the aim of this study was to examine whether NM23-H2 is associated with hepatocarcinogenesis. The level of NM23-H2 expression in tumor tissues and the surrounding matrix appeared to be independent of etiology and tumor differentiation. Its subcellular localization was confined to mainly the cytoplasm and to a lesser extent in the nucleus. Ectopic expression of NM23-H2 in NIH3T3 fibroblasts and HLK3 hepatocytes showed a transformed morphology, enhanced focus formation, and allowed anchorage-independent growth. Finally, NIH3T3 fibroblasts and HLK3 hepatocytes stably expressing NM23-H2 produced tumors in athymic mice and showed c-Myc over-expression. In addition,NF-κB and cyclin D1 expression were also increased by NM23-H2. Lentiviral delivery of NM23-H2 shRNA inhibited tumor growth of xenotransplanted tumors produced from HLK3 cells stably expressing NM23-H2. Collectively, these results indicate that NM23-H2 may be pro-oncogenic in hepatocarcinogenesis.

Opa Interacting Protein 5 (OIP5) is Involved in Hepatocellular Carcinoma Growth and Metastasis Through mtorc1 and Β-catenin Signaling Pathways

Hua Li,Mi-Jin Lee,Goung-Ran Yu,Xue-Ji Han,Dae-Ghon Kim 한국간담췌외과학회 2014 한국간담췌외과학회 학술대회지 Vol.2014 No.4

ERTC Involved in HCC Growth and Metastasis through p53 and WNK1 Signaling Pathway

( Hua Li ),( Mi-jin Lee ),( Goung-ran Yu ),( Lan Liu ),( Xue-ji Han ),( Dae-ghon Kim ) 대한간학회 2016 춘·추계 학술대회 (KASL) Vol.2016 No.1

Aims: ERTC has been shown to be an important player in the regulation of centrosome/microtubule dynamics during mitosis and found to be deregulated in a variety of human malignancies. But, the functional role and signaling pathway of ERTC in hepatocellular carcinoma (HCC) remains elucidative. Methods: ERTC mRNA and miR-1324 expression was confirmed by real-time PCR analysis in human liver sample. ERTC protein expression was investigated by immunoblotting analysis in HCC cell lines and HCC tissues. The ERTC expression was infected with adenovirus, or knockdown by a delivery with short hairpin RNA (shRNA) or treatment with the potential ERTC inhibitor KHS101 hydrochloride, in Huh7, HepG2, SH-J1 and Alexander cell lines; cells were analyzed for proliferation, migration, and invasion. Tumor metastasis by ERTC and WNK1 was tested in vivo mouse model. Moreover, signaling pathways involved in invasion and metastasis were analyzed by Western blot. Results: ERTC was abundantly expressed in HCC cell lines and HCC tissues compared with non-tumor HCC tissues. ERTC mRNA was positively correlated with the tumor size, worsening differentiation status, lack of fibrous capsule formation, microvessel invasion, intrahepatic metastasis, AFP level and advanced stage of HCC. Knockdown of ERTC in SH-J1 and Alexander cells suppressed migratory and invasive behavior as well as the expression of EMT related markers. Silencing ERTC enhanced p53 expression and decrease phosphorylation of WNK1 in SH-J1 and Alexander cells. The cells with knockdown of ERTC using target shRNA reduced tumor metastasis in a lung metastasis mouse model. Moreover, knockdown of WNK1 also inhibit tumorigenicity and metastatic ability were examined in an orthotopic animal model. miR-1324 interacted with the 3’ untranslated region (3’ UTR) of ERTC. Levels of miR-1324 were correlated inversely with ERTC mRNA and in human HCC samples. Conclusions: Therefore, ERTC may be involved in HCC growth and metastasis through p53 and WNK1 signaling pathway, which may be useful therapeutic targets.

Basic, HCC basic : PE-108 ; Progenitor cell-derived hepatocytes and their characteristics in human

( Pei Pei Hao ),( Mi Jin Lee ),( Goung Ran Yu ),( In Hee Kim ),( Dae Ghon Kim ) 대한간학회 2012 춘·추계 학술대회 (KASL) Vol.2012 No.-

Background: Hepatic progenitor cells (HPCs) are capable of differentiating along the hepatic lineage into hepatocytes or cholangiocytes (bile duct cells), hence play a critical role in the process of liver regeneration. Their biological discrimination and characterization are critical for therapeutic potential. Aims of this study is to establish progenitor cell-derived hepatocytes and to characterize their specific markers. Methods: Potential liver progenitor cells (HNK-1) were established and their various HPC protein expressions were investigated by immunoblotting, immunofluorescence and fluorescence-activated cell sorting (FACS) analyses, compared with those of other HCC cells. Immunohistochemistry was performed to detect these HPC antigen expression in the tissues of hepatic cirrhosis. Anchorage-independent growth and tumorigenicity were determined using soft agar and xenograft assay. Results: The HNK-1 cells highly expressed HPC markers such as EpCAM, CK7, CK19, AFP, CK8, CK18, EFNA1, and Thy1. Whereas, CD133 was barely expressed. In contrast, malignant Hep3B cells were positive in both EpCAM and CD133. Ductular reactions at the periphery of the cirrhotic nodules were immunohistochemically positive for these HPC markers. Sodium butyrate could induce hepatocyte-like morphological changes in HNK-1 cells, accompanying down-regulation of the hepatic progenitor cell markers (EpCAM, CK7, CK19, and EFNA1) and up-regulation of mature hepatocyte markers (albumin, CK8, and CK18) in both dose-dependent and time- dependent manners. Colony formation in vitro and tumorigenesis in vivo showed that there were no tumorigenesis capacity in EpCAM (+)/CD133(-) HNK1cells at the 0-2nd, 10th, 25th, and 50th passages, while the positive control EpCAM (+)/CD133(+) Hep3B cells could induce tumor in the mice model. Conclusions: Taken together, our results suggest that HNK1 cells are progenitor cell-derived hepatocytes and their stemmness- related markers EpCAM (+)/CD133(-) may be a distinguished marker for nonmalignant, progenitor cell-derived hepatocytes.