Centromere protein U (CENPU), a centromere-binding protein required for cellular mitosis, has been reported to be closely associated with carcinogenesis in multiple malignancies; however, the role of CENPU in hepatocellular carcinoma (HCC) is still unclear. Herein, we investigated its biological role and molecular mechanism in the development of HCC. High CENPU expression in HCC tissue was observed and correlated positively with a poor prognosis in HCC patients. CENPU knockdown inhibited the proliferation, metastasis, and G1/S transition of HCC cells in vivo and in vitro, while ectopic expression of CENPU exerted the opposite effects. Mechanistically, CENPU physically interacted with E2F6 and promoted its ubiquitin-mediated degradation, thus affecting the transcription level of E2F1 and further accelerating the G1/S transition to promote HCC cell proliferation. E2F1 directly binds to the CENPU promoter and increases the transcription of CENPU, thereby forming a positive regulatory loop. Collectively, our findings indicate a crucial role for CENPU in E2F1-mediated signalling for cell cycle progression and reveal a role for CENPU as a predictive biomarker and therapeutic target for HCC patients.

• CENPU
• E2F1
• E2F6
• G1/S transition
• Hepatocellular carcinoma

• HBx protein
• cancer hallmarks
• hepatitis B virus
• hepatocellular carcinoma
• therapeutics

Hepatitis B virus (HBV) is a hepatotropic virus and an important human pathogen. There are an estimated 296 million people in the world that are chronically infected by this virus, and many of them will develop severe liver diseases including hepatitis, cirrhosis and hepatocellular carcinoma (HCC). HBV is a small DNA virus that replicates via the reverse transcription pathway. In this review, we summarize the molecular pathways that govern the replication of HBV and its interactions with host cells. We also discuss viral and non-viral factors that are associated with HBV-induced carcinogenesis and pathogenesis, as well as the role of host immune responses in HBV persistence and liver pathogenesis.

• autophagy
• immunity
• oncogenic virus
• oncolytic virus
• tumor development and progression
• tumor resistance
• tumorigenesis
• viral infection

Backgrounds and Aims: Hepatocellular Carcinoma (HCC) is the leading cause of cancer-related mortality across the world. Non-viral etiological factors including obesity and metabolic syndrome have now become prevalent cause of hepatocellular carcinoma. Sonic Hedgehog (SHH) pathway is activated in hepatocellular carcinoma but its role in regulation of lipogenic molecules during the hepatocarcinogenesis is not known. The aim of present study is to explore the role of SHH pathway in fatty changes associated with hepatocarcinogenesis at different stages and to further correlate the expression of SHH with lipogenic pathways.

Results: Our results demonstrated significant increase in lipidosis and fibrosis in DEN+CCl4 treated animals. It was simultaneously associated with the enhanced expression level of SHH, E2F1, adiponectin, and lipogenic molecules in DEN+CCl4 treated animals. These results were also corroborated with the similar findings in higher stage patients’ biospecimens.

Methods: N-Nitrosodiethylamine (DEN) and Carbon TetraChloride (CCl4) induced hepatocellular acrcinoma model in male Wistar rats were established to study the expression level of SHH pathway and associated fatty changes during different stages of hepatocarcinogenesis. The expression levels of SHH, E2F1, and lipogenic molecules were checked at different stages of hepatocellular carcinoma. These results were further compared with biospecimens of hepatocellular carcinoma patients of different stages.

Conclusions: Our results revealed an unknown aspect of SHH pathway in hepatocarcinogenesis via its control over lipogenesis. It gives insight into the lipogenic properties of DEN+CCl4 induced rodent hepatocarcinogenesis model and how SHH pathway operate to arbitrate this response.

• SHH-E2F1 pathway
• adiponectin
• adiposity
• hepatocellular carcinoma
• lipogenic molecules

• Atg6
• Autophagy
• Beclin 1
• Haploinsufficient tumor suppressor
• Monoallelic-deletion

• Caspase-3
• E2Fs
• hepatocellular carcinoma
• microRNA-34a
• prognosis

• HBV
• HCV
• cirrhosis
• hepatocellular carcinoma

• HBV
• HCV
• cirrhosis
• hepatocellular carcinoma

• EZH2
• HBV
• HCC
• epigenetics
• virus

Intestine-specific homeobox (ISX), a newly identified proto-oncogene, is involved in cell proliferation and progression of hepatocellular carcinoma (HCC). However, the underlying mechanisms linking gene expression and tumor formation remain unclear. In this study, we found that ISX transcriptionally activated E2F transcription factor 1 (E2F1) and associated oncogenic activity by directly binding to the E2 site of its promoter. Forced expression of ISX increased the expression of and phosphorylated the serine residue at position 332 of E2F1, which may be translocated into the nucleus to form the E2F1–DP-1 complex, suggesting that the promotion of oncogenic activities of the ISX–E2F1 axis plays a critical role in hepatoma cells. Coexpression of ISX and E2F1 significantly promoted p53 and RB-mediated cell proliferation and anti-apoptosis, and repressed apoptosis and autophagy. In contrast, short hairpin RNAi-mediated attenuation of ISX and E2F1 decreased cell proliferation and malignant transformation, respectively, in hepatoma cells in vitro and in vivo. The mRNA expression of E2F1 and ISX in 238 paired specimens from human HCC patients, and the adjacent, normal tissues exhibited a tumor-specific expression pattern which was highly correlated with disease pathogenesis, patient survival time, progression stage, and poor prognosis. Therefore, our results indicate that E2F1 is an important downstream gene of ISX in hepatoma progression.

• DP1
• E2F1
• ISX
• cyclin D1
• hepatocellular carcinoma (HCC)

Hepatitis B virus (HBV) has a worldwide distribution and is endemic in many populations. Due to its unique life cycle which requires an error-prone reverse transcriptase for replication, it constantly evolves, resulting in tremendous genetic variation in the form of genotypes, sub-genotypes, and mutations. In recent years, there has been considerable research on the relationship between HBV genetic variation and HBV-related pathogenesis, which has profound implications in the natural history of HBV infection, viral detection, immune prevention, drug treatment and prognosis. In this review, we attempted to provide a brief account of the influence of HBV genotype on the pathogenesis of HBV infection and summarize our current knowledge on the effects of HBV mutations in different regions on HBV-associated pathogenesis, with an emphasis on mutations in the preS/S proteins in immune evasion, occult HBV infection and hepatocellular carcinoma (HCC), mutations in polymerase in relation to drug resistance, mutations in HBV core and e antigen in immune evasion, chronicalization of infection and hepatitis B-related acute-on-chronic liver failure, and finally mutations in HBV x proteins in HCC.

• Hepatitis B virus
• Genotype
• Variation
• Pathogenesis

• Antineoplastic Agents/adverse effects
• Genetic Variation
• Genome, Viral
• Genotype
• Hepatitis B/drug therapy
• Hepatitis B/etiology
• Hepatitis B/virology
• Hepatitis B virus/genetics
• Hepatitis B virus/pathogenicity
• Humans
• Immunosuppression Therapy/adverse effects
• Mutation
• Recurrence
• Viral Proteins/genetics
• Virulence/genetics

The precise mechanism by which HBx protein of hepatitis B virus (HBV) impacts on hepato-carcinogenesis remain largely elusive despite strong evidences for its' involvement in the process. Here, we have investigated the role of HBx on expression of a novel gene hELG1/ATAD5, which is required for genome maintenance and its' importance in hepatocarcinogenesis. This study has for the first time showed that the expression of this gene was significantly higher in human cancer such as HBV-associated hepatocellular carcinoma (HCC) and in different HCC cell lines compared to normal liver. In addition, a significant elevation in ATAD5 expression was also found in HBx transfected HCC cell lines implicating HBx mediated transcriptional regulation on ATAD5. Using different deletion mutant constructs of putative promoter, the active promoter region was first identified here and subsequently the regulatory region of HBx was mapped by promoter-luciferase assay. But ChIP assay with anti-HBx antibody revealed that HBx was not physically present in ATAD5 transcription machinery whereas anti-E2F1 antibody showed the presence of E2F1 in the complex. Luciferase assay with E2F1 binding site mutant had further confirmed it. Moreover, both loss-and gain-of-function studies of ATAD5 showed that ATAD5 could enhance HBV production in transfected cells whereas knock down of ATAD5 increased the sensitivity of HCC cell line to chemotherapeutics 5-fluorouracil. Overall, this data suggests that a positive feedback loop regulation between ATAD5 and HBV contributed to both viral replication and chemo-resistance of HCC cells.

• hepatitis B virus
• hepatocellular carcinoma

• liver
• tumor suppressor
• immune function
• hcc
• gene signature

• Carcinoma, Hepatocellular/epidemiology
• Carcinoma, Hepatocellular/physiopathology
• Carcinoma, Hepatocellular/virology
• Genetic Predisposition to Disease/epidemiology
• Genetic Predisposition to Disease/genetics
• Hepatitis B/epidemiology
• Hepatitis B/physiopathology
• Hepatitis B/virology
• Hepatitis B virus/physiology
• Humans
• Liver Neoplasms/epidemiology
• Liver Neoplasms/physiopathology
• Liver Neoplasms/virology
• Prevalence
• Risk Factors

Histone lysine methyltransferase EZH2 has been reported to be frequently overexpressed in hepatocellular carcinoma (HCC) tissues and associated with hepatocarcinogenesis. However, the exact mechanism of EZH2 up-regulation in HCC has not been determined. In this study, we used murine hepatocyte AML12 cells to investigate the role of hepatitis B virus X protein (HBx) in regulating the expression of mEZH2. Western blot analysis demonstrated that the expression level of mEZH2 protein in AML12 cells was up-regulated by HBx in a dose-dependent manner. To further investigate the mechanism of mEZH2 overexpression, the 2500 bp regulatory sequence upstream from the first exon of the mEZH2 gene was amplified from AML12 genomic DNA and constructed into a luciferase reporter plasmid. The luciferase activity of the mEZH2 promoter significantly increased in AML12 cells co-transfected with HBx plasmid, and deleting the −486/−214 promoter region decreased HBx-induced mEZH2 promoter activation by nearly 50%. The −486/−214 region was then analyzed in the TRANSFAC 6.0 database and a typical E2F1-binding site was found. Mutation of this E2F1-binding site or knockdown of E2F1 expression by RNAi led to a dramatic decrease in HBx-induced activation of the mEZH2 promoter and mEZH2 overexpression in AML12 cells. These results provide evidence that HBx up-regulates mEZH2 expression by transactivating the mEZH2 promoter through E2F1 transcription factor, thereby providing new epigenetic evidence for the carcinogenic effect of HBx.

• autophagy
• cross-talking
• viral infection

• Animals
• DNA Methylation
• Epigenesis, Genetic
• Host-Pathogen Interactions/genetics
• Humans
• Infections/genetics
• Infections/microbiology

• Animals
• Apoptosis/drug effects
• Apoptosis/physiology
• Cyclin-Dependent Kinase Inhibitor p21/biosynthesis
• Cyclin-Dependent Kinase Inhibitor p21/deficiency
• Epithelial Cells/cytology
• Epithelial Cells/drug effects
• Genotype
• Hepatocytes/cytology
• Hepatocytes/drug effects
• Hepatocytes/metabolism
• Hepatocytes/physiology
• Male
• Mesoderm/cytology
• Mesoderm/drug effects
• Mice
• Mice, Transgenic
• Retinoblastoma Protein/biosynthesis
• Retinoblastoma Protein/deficiency
• Transforming Growth Factor beta/pharmacology
• Tumor Suppressor Protein p53/biosynthesis
• Tumor Suppressor Protein p53/deficiency

AIM: To evaluate correlation between histone methyltransferase SMYD3 expression in hepatoma and HBV infection.

METHODS: SMYD3 mRNA expressions and SMYD3 protein expression levels in HBV-negative HepG2 and HBV-positive hepatoma cell line HepG2.2.15 were determined using real time PCR and Western blot, respectively.

RESULTS: SMYD3 mRNA and protein levels were significantly higher in HepG2.2.15 than those in HepG2 (0.92 ± 0.12 vs 0.18 ± 0.05, 0.28 ± 0.03 vs 0.54 ± 0.05, both P < 0.01).

CONCLUSION: HBV may promote hepatoma cell malignancy through its SMYD3 up-regulating pathways.

• SMYD3
• Epigenetics
• Hepatoma
• Hepatitis B virus
• Real time RT-PCR

TGFβ is critical to control hepatocyte proliferation by inducing G1-growth arrest through multiple pathways leading to inhibition of E2F transcription activity. The retinoblastoma protein pRb is a key controller of E2F activity and G1/S transition which can be inhibited in viral hepatitis. It is not known whether the impairment of pRb would alter the growth inhibitory potential of TGFβ in disease. We asked how Rb-deficiency would affect responses to TGFβ-induced cell cycle arrest.

Primary hepatocytes isolated from Rb-floxed mice were infected with an adenovirus expressing CRE-recombinase to delete the Rb gene. In control cells treatment with TGFβ prevented cells to enter S phase via decreased cMYC activity, activation of P16^INK4A and P21^Cip and reduction of E2F activity. In Rb-null hepatocytes, cMYC activity decreased slightly but P16^INK4A was not activated and the great majority of cells continued cycling. Rb is therefore central to TGFβ-induced cell cycle arrest in hepatocytes. However some Rb-null hepatocytes remained sensitive to TGFβ-induced cell cycle arrest. As these hepatocytes expressed very high levels of P21^Cip1 and P53 we investigated whether these proteins regulate pRb-independent signaling to cell cycle arrest by evaluating the consequences of disruption of p53 and p21^Cip1. Hepatocytes deficient in p53 or p21^Cip1 showed diminished growth inhibition by TGFβ. Double deficiency had a similar impact showing that in cells containing functional pRb; P21^Cip and P53 work through the same pathway to regulate G1/S in response to TGFβ. In Rb-deficient cells however, p53 but not p21^Cip deficiency had an additive effect highlighting a pRb-independent-P53-dependent effector pathway of inhibition of E2F activity.

The present results show that otherwise genetically normal hepatocytes with disabled p53, p21^Cip1 or Rb genes respond less well to the antiproliferative effects of TGFβ. As the function of these critical cellular proteins can be impaired by common causes of chronic liver disease and HCC, including viral hepatitis B and C proteins, we suggest that disruption of pRb function, and to a lesser extend P21^Cip1 and P53 in hepatocytes may represent an additional new mechanism of escape from TGFβ-growth-inhibition in the inflammatory milieu of chronic liver disease and contribute to cancer development.

• Blotting, Western
• Cadherins/genetics
• Cadherins/metabolism
• Cell Line, Tumor
• Cyclin D1/metabolism
• Cyclin-Dependent Kinase 4/metabolism
• Cyclin-Dependent Kinase Inhibitor p16/metabolism
• DNA Methylation
• DNA Modification Methylases/biosynthesis
• E2F1 Transcription Factor/metabolism
• Humans
• RNA Interference
• Retinoblastoma Protein/metabolism
• Reverse Transcriptase Polymerase Chain Reaction
• Signal Transduction/physiology
• Trans-Activators/metabolism
• Transfection
• Viral Regulatory and Accessory Proteins

N/A

• N/A

• Adenovirus E4 Proteins/metabolism
• Cysteine
• DNA-Binding Proteins/metabolism
• Hepatitis B virus
• Humans
• In Vitro Techniques
• Protein Binding
• Repressor Proteins/metabolism
• Retinoid X Receptors/metabolism
• Trans-Activators/chemistry
• Trans-Activators/metabolism
• Two-Hybrid System Techniques
• Viral Regulatory and Accessory Proteins

• Antigens, Viral
• Apoptosis
• Carcinoma, Hepatocellular/etiology
• Carcinoma, Hepatocellular/virology
• Cell Transformation, Neoplastic
• DNA Damage
• DNA Repair
• Hepatitis B/complications
• Hepatitis B virus/immunology
• Hepatitis B virus/pathogenicity
• Humans
• Liver Neoplasms/etiology
• Liver Neoplasms/virology
• Neoplasm Metastasis
• Risk Factors
• Signal Transduction
• Viral Proteins