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1.
Oncotarget ; 8(14): 23507-23516, 2017 Apr 04.
Article in English | MEDLINE | ID: mdl-28186991

ABSTRACT

Hepatitis B virus (HBV) is a major risk factor of chronic liver disease and hepatocellular carcinoma (HCC). Random integration of HBV DNA into the host genome is frequent in HCC leading to truncation of the HBV DNA, particularly at the C-terminal end of the HBV X protein (HBx). C-terminally truncated HBx (HBx-ΔC) has been implicated in playing a pro-oncogenic role in hepatocarcinogenesis. However, the mechanism whereby HBx-ΔC1 contributes to hepatocarcinogenesis remains unclear. In this study, we investigated the functional role of HBx-ΔC1 in regulating liver cancer stem cell (CSC) properties. Using Tet-on inducible system, we found that HBx-ΔC1 enhanced CSC properties including self-renewal, tumorigenicity, chemoresistance, migration and expression of liver CSC markers, when compared with the full-length HBx counterpart and vector control. Interestingly, HBx-ΔC1 conferred resistance in HCC cells towards sorafenib treatment through suppression of apoptotic cascade. In addition, HBx-ΔC1 upregulated a panel of stemness genes, in which Nanog was found to be among the most significant one in both trasnfected cell lines. Consistently, Nanog was upregulated in human HCC samples which had HBx-ΔC1 expression. Furthermore, the induction of CSC properties by HBx-ΔC1 was via the Stat3/Nanog pathway, as administration of Stat3 inhibitor abolished the HBx-ΔC1-induced self-renewing capacity. In conclusion, our data suggest that HBx-ΔC1 enhances liver CSCs properties through Stat3/Nanog cascade, and provide a new insight for the therapeutic intervention for HBV-related HCC.


Subject(s)
Carcinoma, Hepatocellular/metabolism , Liver Neoplasms/metabolism , Nanog Homeobox Protein/metabolism , Neoplastic Stem Cells/metabolism , STAT3 Transcription Factor/metabolism , Trans-Activators/metabolism , Adult , Aged , Animals , Antineoplastic Agents/pharmacology , Blotting, Western , Carcinogenesis/genetics , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Cell Self Renewal/genetics , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Female , Humans , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Male , Mice, Inbred NOD , Mice, SCID , Middle Aged , Mutation , Nanog Homeobox Protein/genetics , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/genetics , Trans-Activators/genetics , Transplantation, Heterologous , Viral Regulatory and Accessory Proteins
2.
Liver Int ; 36(5): 737-45, 2016 05.
Article in English | MEDLINE | ID: mdl-26351778

ABSTRACT

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is often associated with metastasis and recurrence leading to a poor prognosis. Therefore, development of novel treatment regimens is urgently needed to improve the survival of HCC patients. In this study, we aimed to investigate the in vitro and in vivo effects of anti-CD47 antibody alone and in combination with chemotherapy in HCC. METHODS: In this study, we examined the functional effects of anti-CD47 antibody (B6H12) on cell proliferation, sphere formation, migration and invasion, chemosensitivity, macrophage-mediated phagocytosis and tumourigenicity both in vitro and in vivo. The therapeutic efficacy of anti-CD47 antibody alone or in combination with doxorubicin was examined in patient-derived HCC xenograft. RESULTS: Blocking CD47 with anti-CD47 monoclonal antibody (B6H12) at 10 µg/ml could suppress self-renewal, tumourigenicity and migration and invasion abilities of MHCC-97L and Huh-7 cells. Interestingly, anti-CD47 antibody synergized the effect of HCC cells to chemotherapeutic drugs including doxorubicin and cisplatin. Blockade of CD47 by anti-CD47 antibody induced macrophage-mediated phagocytosis. Using a patient-derived HCC xenograft mouse model, we found that anti-CD47 antibody (400 µg/mouse) in combination with doxorubicin (2 mg/kg) exerted maximal effects on tumour suppression, as compared with doxorubicin and anti-CD47 antibody alone. CONCLUSIONS: Anti-CD47 antibody treatment could complement chemotherapy which may be a promising therapeutic strategy for the treatment of HCC patients.


Subject(s)
Antibodies, Monoclonal/pharmacology , CD47 Antigen/immunology , Carcinoma, Hepatocellular/drug therapy , Liver Neoplasms/drug therapy , Macrophages/drug effects , Animals , Apoptosis/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cisplatin/pharmacology , Doxorubicin/pharmacology , Humans , Mice , Xenograft Model Antitumor Assays
3.
Oncotarget ; 6(41): 43483-95, 2015 Dec 22.
Article in English | MEDLINE | ID: mdl-26536659

ABSTRACT

Hepatocellular carcinoma (HCC) is frequently complicated by the occurrence of intrahepatic and extrahepatic metastases, leading to poor prognosis. To improve the prognosis for HCC patients, there is an urgent need to understand the molecular mechanisms of metastasis in HCC. Since protein Serine/Threonine phosphorylation emerges to be an important posttranslational modification critical in signaling process associated with cell proliferation, survival and metastasis, we employed a pair of primary tumor-derived and corresponding lung-metastatic counterparts (PLC/PRF/5-PT and PLC/PRF/5-LM) and aimed to identify these changes using CelluSpot Serine/Threonine kinase peptide array. Upon analysis, we found phosphorylated level of nucleophosmin (NPM) at Threonine 234/237 (p-NPM-Thr234/237) had remarkably high level in metastatic HCC cells (PLC-LM) than the corresponding primary HCC cell line (PLC-PT). Similar observation was observed in another match primary and their metastatic counterparts (MHCC-97L and MHCC-97H). By immunohistochemical staining, p-NPM-Thr234/237 was consistently found to be preferentially expressed in metastatic HCCs when compared with primary HCC in 28 HCC cases (p < 0.0001). By overexpressing Flag-tagged NPM and its phosphorylation site mutant (Thr234/237A) into low p-NPM-Thr234/237 expressing cells (Hep3B and Huh7) using a lentiviral based approach, we demonstrated that p-NPM-Thr234/237 is critical in invasion and migration of HCC cells, and this effect was mediated by cyclin-dependent kinase 1 (CDK1). Wild-type NPM was found to physically interact with a metastatic gene, ROCK2, and defective in Thr234/237 phosphorylation decreased its binding affinity, resulting in decrease in ROCK2 mediated signaling pathway. Identification of CDK1/p-NPM/ROCK2 signaling pathway provides a novel target for molecular therapy against HCC metastasis.


Subject(s)
Carcinoma, Hepatocellular/metabolism , Liver Neoplasms/metabolism , Nuclear Proteins/metabolism , Blotting, Western , CDC2 Protein Kinase , Carcinoma, Hepatocellular/pathology , Cyclin-Dependent Kinases/metabolism , Fluorescent Antibody Technique , Gene Knockdown Techniques , Humans , Liver Neoplasms/pathology , Neoplasm Invasiveness , Nucleophosmin , Phosphorylation , Polymerase Chain Reaction , Signal Transduction/physiology , Threonine/metabolism , rho-Associated Kinases/metabolism
4.
Hepatology ; 62(2): 534-45, 2015 Aug.
Article in English | MEDLINE | ID: mdl-25902734

ABSTRACT

UNLABELLED: Sorafenib is a new standard treatment for patients with advanced hepatocellular carcinoma (HCC). However, the survival benefit of this treatment is modest, partly owing to drug resistance. Recent evidence has demonstrated the existence of tumor-initiating cells (T-ICs) as the culprit for treatment resistance. To examine whether sorafenib resistance was a result of the presence of liver T-ICs, we developed sorafenib-resistant HCC cells both in vitro and in vivo through continuous exposure to sorafenib. Using these models, we found that sorafenib-resistant clones demonstrated enhanced T-IC properties, including tumorigenicity, self-renewal, and invasiveness. In addition, several T-IC markers were found to be up-regulated, among which CD47 was found to be most significant. Using chromatin immunoprecipitation assays and expression analyses, CD47 expression was found to be regulated by nuclear factor kappa B (NF-κB) through a specific response element in the promoter of CD47, and the site occupancy and expression were increased and decreased upon stimulation and inhibition of NF-κB, respectively. Consistently, NF-κB was activated in sorafenib-resistant HCC cells, and this finding was confirmed in clinical HCC samples, which showed a positive correlation between NF-κB and CD47 expression. Functional characterization of CD47 in sorafenib-resistant HCC cells was evaluated using a lentivirus-based knockdown approach and showed increased sensitization to sorafenib upon CD47 knockdown. Furthermore, blockade of CD47 using anti-CD47 antibody (Ab) showed a similar effect. Using a patient-derived HCC xenograft mouse model, we found that anti-CD47 Ab (500 µg/mouse) in combination with sorafenib (100 mg/kg, orally) exerted synergistic effects on tumor suppression, as compared with sorafenib and anti-CD47 Ab alone. CONCLUSIONS: NF-κB-mediated CD47 up-regulation promotes sorafenib resistance, and targeting CD47 in combination with sorafenib is an attractive therapeutic regimen for the treatment of HCC patients.


Subject(s)
CD47 Antigen/genetics , Carcinoma, Hepatocellular/drug therapy , Drug Resistance, Neoplasm/genetics , Liver Neoplasms/drug therapy , Niacinamide/analogs & derivatives , Phenylurea Compounds/pharmacology , Animals , Antineoplastic Agents/pharmacology , Carcinoma, Hepatocellular/genetics , Disease Models, Animal , Humans , Liver Neoplasms/genetics , Mice , Mice, SCID , Molecular Targeted Therapy , NF-kappa B/drug effects , NF-kappa B/metabolism , Niacinamide/pharmacology , Random Allocation , Signal Transduction/drug effects , Sorafenib , Treatment Outcome , Tumor Cells, Cultured , Up-Regulation , Xenograft Model Antitumor Assays
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