Development Of HBV-associated Hepatocellular Carcinoma Research Articles

Identification of key genes and carcinogenic pathways in hepatitis B virus-associated hepatocellular carcinoma through bioinformatics analysis.

Backgrounds/AimsMechanisms for the development of hepatocellular carcinoma (HCC) in hepatitis B virus (HBV)-infected patients remain unclear. The aim of the present study was to identify genes and pathways involved in the development of HBV-associated HCC.MethodsThe GSE121248 gene dataset, which included 70 HCCs and 37 adjacent liver tissues, was downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) in HCCs and adjacent liver tissues were identified. Gene ontology and Kyoto Encyclopedia of Genes and Genome pathway enrichment analyses were then performed.ResultsOf 134 DEGs identified, 34 were up-regulated and 100 were down-regulated in HCCs. The 34 up-regulated DEGs were mainly involved in nuclear division, organelle fission, spindle and midbody formation, histone kinase activity, and p53 signaling pathway, whereas the 100 down-regulated DEGs were involved in steroid and hormone metabolism, collagen-coated extracellular matrix, oxidoreductase activity, and activity on paired donors, including incorporation or reduction of molecular oxygen, monooxygenase activity, and retinol metabolism. Analyses of protein-protein interaction networks with a high degree of connectivity identified significant modules containing 14 hub genes, including ANLN, ASPM, BUB1B, CCNB1, CDK1, CDKN3, ECT2, HMMR, NEK2, PBK, PRC1, RACGAP1, RRM2, and TOP2A, which were mainly associated with nuclear division, organelle fission, spindle formation, protein serine/threonine kinase activity, p53 signaling pathway, and cell cycle.ConclusionsThis study identified key genes and carcinogenic pathways that play essential roles in the development of HBV-associated HCC. This may provide important information for the development of diagnostic and therapeutic targets for HCC.

Interaction between the Hepatitis B Virus and Cellular FLIP Variants in Viral Replication and the Innate Immune System.

During viral evolution and adaptation, many viruses have utilized host cellular factors and machinery as their partners. HBx, as a multifunctional viral protein encoded by the hepatitis B virus (HBV), promotes HBV replication and greatly contributes to the development of HBV-associated hepatocellular carcinoma (HCC). HBx interacts with several host factors in order to regulate HBV replication and evolve carcinogenesis. The cellular FADD-like IL-1β-converting enzyme (FLICE)-like inhibitory protein (c-FLIP) is a major factor that functions in a variety of cellular pathways and specifically in apoptosis. It has been shown that the interaction between HBx and c-FLIP determines HBV fate. In this review, we provide a comprehensive and detailed overview of the interplay between c-FLIP and HBV in various environmental circumstances. We describe strategies adapted by HBV to establish its chronic infection. We also summarize the conventional roles of c-FLIP and highlight the functional outcome of the interaction between c-FLIP and HBV or other viruses in viral replication and the innate immune system.

Identification of Key Genes and Carcinogenic Pathways in Hepatitis B Virus-associated Hepatocellular Carcinoma through Bioinformatics Analysis

Background: The mechanisms of development of hepatocellular carcinoma (HCC) in hepatitis B virus (HBV)-infected patients remain unclear. The present study sought to identify the genes and mechanisms involved in the development of HBV-associated HCC.

The Mechanisms of HBV-Induced Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a common malignancy, and the hepatitis B virus (HBV) is its major pathogenic factor. Over the past decades, it has been confirmed that HBV infection could promote disease progression through a variety of mechanisms, ultimately leading to the malignant transformation of liver cells. Many factors have been identified in the pathogenesis of HBV-associated HCC (HBV-HCC), including HBV gene integration, genomic instability caused by mutation, and activation of cancer-promoting signaling pathways. As research in the progression of HBV-HCC progresses, the role of many new mechanisms, such as epigenetics, exosomes, autophagy, metabolic regulation, and immune suppression, is also being continuously explored. The occurrence of HBV-HCC is a complex process caused by interactions across multiple genes and multiple steps, where the synergistic effects of various cancer-promoting mechanisms accelerate the process of disease evolution from inflammation to tumorigenesis. In this review, we aim to provide a brief overview of the mechanisms involved in the occurrence and development of HBV-HCC, which may contribute to a better understanding of the role of HBV in the occurrence and development of HCC.

Hepatitis B virus X protein (HBx) enhances centrosomal P4.1-associated protein (CPAP) expression to promote hepatocarcinogenesis

BackgroundOur previous report suggested that centrosomal P4.1-associated protein (CPAP) is required for Hepatitis B virus (HBV) encoded non-structure protein X (HBx)-mediated nuclear factor kappa light chain enhancer of activated B cells (NF-κB) activation. CPAP is overexpressed in HBV-associated hepatocellular carcinoma (HCC); however, the interaction between CPAP and HBx in HBV-HCC remains unclear.MethodsThe mRNA expression of CPAP and HBx was analyzed by quantitative-PCR (Q-PCR). NF-κB transcriptional activity and CPAP promoter activity were determined using a reporter assay in Huh7 and Hep3B cells. Immunoprecipitation (IP) and in situ proximal ligation assay (PLA) were performed to detect the interaction between CPAP and HBx. Chromatin-IP was used to detect the association of cAMP response element binding protein (CREB) and HBx with the CPAP promoter. Cell proliferation was measured using cell counting kit CCK-8, Bromodeoxyuridine (5-bromo-2′-deoxyuridine, BrdU) incorporation, and clonogenic assays. The tumorigenic effects of CPAP were determined using xenograft animal models.ResultsHBx can transcriptionally up-regulate CPAP via interacting with CREB. Overexpressed CPAP directly interacted with HBx to promote HBx-mediated cell proliferation and migration; SUMO modification of CPAP was involved in interacting with HBx. Knocked-down expression of CPAP decreased the HBx-mediated tumorigenic effects, including cytokines secretion. Interestingly, overexpressed CPAP maintained the HBx protein stability in an NF-κB-dependent manner; and the expression levels of CPAP and HBx were positively correlated with the activation status of NF-κB in HCC. Increased expression of CPAP and CREB mRNAs existed in the high-risk group with a lower survival rate in HBV-HCC.ConclusionThe interaction between CPAP and HBx can provide a microenvironment to facilitate HCC development via enhancing NF-κB activation, inflammatory cytokine production, and cancer malignancies. This study not only sheds light on the role of CPAP in HBV-associated HCC, but also provides CPAP as a potential target for blocking the hyper-activated NF-κB in HCC.

Impact of Interferon-α Receptor-1 Promoter Polymorphisms on the Transcriptome of the Hepatitis B Virus-Associated Hepatocellular Carcinoma.

Genetic polymorphisms within the promoter of interferon-α receptor type-1 (IFNAR1) have been associated with the susceptibility to and the outcome of chronic hepatitis B virus (HBV) infection. However, the impact of these polymorphisms in the transcriptome of the HBV-associated hepatocellular carcinoma (HCC) remains largely unexplored. Using whole-genome and exome sequencing data from The Cancer Genome Atlas project, we characterized three single-nucleotide polymorphisms (SNPs: -568G/C, -408C/T, -3C/T) and one variable number tandem repeat [VNTR: -77(GT)n] within the IFNAR1 promoter sequence in 49 HCC patients. RNAseq data from 10 genotyped HCC samples were grouped according to their -77VNTR or -3SNP genotype to evaluate the impact of these polymorphisms on the differential expression on the HCC transcriptome. There is a fourfold higher impact of the -77VNTR on the HCC transcriptome compared to the -3SNP (q < 0.1, p < 0.001). The expression of the primary IFNAR1 transcript is not affected by these polymorphisms but a secondary, HCC-specific transcript is expressed only in homozygous -77VNTR ≤8/≤8(GT)n samples (p < 0.05). At the same time, patients carrying at least one -77VNTR >8(GT) allele, presented a strong upregulation of the fibronectin-1 (FN-1) gene, which has been associated with the development of HCC. Gene Ontology and pathway enrichment analysis of the differentially expressed genes revealed a strong disruption of the PI3K-AKT signaling pathway, which can be partially triggered by the extracellular matrix FN-1. The IFNAR-1 promoter polymorphisms are not involved in the expression levels of the main IFNAR-1 transcript. The -77VNTR has a regulatory role on the expression of a secondary, truncated, HCC-specific transcript, which in turn coincides with disruptions in cancer-associated pathways and in FN-1 expression modifications.

Modulation of Wnt signaling pathway by hepatitis B virus.

Hepatitis B virus (HBV) has a global distribution and is one of the leading causes of hepatocellular carcinoma. The precise mechanism of pathogenicity of HBV-associated hepatocellular carcinoma (HCC) is not yet fully understood. Viral-related proteins are known to take control of several cellular pathways like Wnt/β-catenin, TGF-β, Raf/MAPK and ROS for the virus's own replication. This affects cellular persistence, multiplication, migration, alteration and genomic instability. The Wnt/FZD/β-catenin signaling pathway plays a significant role in the pathology and physiology of the liver and has been identified as a main factor in HCC development. The role of β-catenin is linked mainly to the canonical pathway of the signaling system. Progression of liver diseases is known to be accompanied by disturbances in β-catenin expression (mainly overexpression), with its cytoplasmic or nuclear translocation. In recent years, studies have documented that the HBV X protein and hepatitis B surface antigen (HBsAg) can act as pathogenic factors that are involved in the modulation and induction of canonical Wnt signaling pathway. In the present review we explore the interaction of HBV genome products with components of the Wnt/β-catenin signaling pathway that results in the enhancement of the pathway and leads to hepatocarcinogenesis.

Hepatitis B virus (HBV) X protein-mediated regulation of hepatocyte metabolic pathways affects viral replication

Chronic HBV infection is a risk factor for hepatocellular carcinoma (HCC). The HBV HBx protein stimulates HBV replication and likely influences the development of HBV-associated HCC. Whether HBx affects regulators of metabolism in normal hepatocytes has not been addressed. We used an ex vivo, cultured primary rat hepatocyte system to assess the interplay between HBV replication and mechanistic target of rapamycin complex 1 (mTORC1) signaling. HBx activated mTORC1 signaling; however, inhibition of mTORC1 enhanced HBV replication. HBx also decreased ATP levels and activated the energy-sensing factor AMP-activated protein kinase (AMPK). Inhibition of AMPK decreased HBV replication. Inhibition of AMPK activates mTORC1, and we showed that activated mTORC1 is one factor that reduces HBV replication when AMPK is inhibited. HBx activation of both AMPK and mTORC1 suggests that these activities could provide a balancing mechanism to facilitate persistent HBV replication. HBx activation of mTORC1 and AMPK could also influence HCC development.

Hepatitis B virus molecular biology and pathogenesis.

As obligate intracellular parasites, viruses need a host cell to provide a milieu favorable to viral replication. Consequently, viruses often adopt mechanisms to subvert host cellular signaling processes. While beneficial for the viral replication cycle, virus-induced deregulation of host cellular signaling processes can be detrimental to host cell physiology and can lead to virus-associated pathogenesis, including, for oncogenic viruses, cell transformation and cancer progression. Included among these oncogenic viruses is the hepatitis B virus (HBV). Despite the availability of an HBV vaccine, 350-500 million people worldwide are chronically infected with HBV, and a significant number of these chronically infected individuals will develop hepatocellular carcinoma (HCC). Epidemiological studies indicate that chronic infection with HBV is the leading risk factor for the development of HCC. Globally, HCC is the second highest cause of cancer-associated deaths, underscoring the need for understanding mechanisms that regulate HBV replication and the development of HBV-associated HCC. HBV is the prototype member of the Hepadnaviridae family; members of this family of viruses have a narrow host range and predominately infect hepatocytes in their respective hosts. The extremely small and compact hepadnaviral genome, the unique arrangement of open reading frames, and a replication strategy utilizing reverse transcription of an RNA intermediate to generate the DNA genome are distinguishing features of the Hepadnaviridae. In this review, we provide a comprehensive description of HBV biology, summarize the model systems used for studying HBV infections, and highlight potential mechanisms that link a chronic HBV-infection to the development of HCC. For example, the HBV X protein (HBx), a key regulatory HBV protein that is important for HBV replication, is thought to play a cofactor role in the development of HBV-induced HCC, and we highlight the functions of HBx that may contribute to the development of HBV-associated HCC.

Novel pre-mRNA splicing of intronically integrated HBV generates oncogenic chimera in hepatocellular carcinoma

Hepatitis B virus (HBV) integration is common in HBV-associated hepatocellular carcinoma (HCC) and may play an important pathogenic role through the production of chimeric HBV-human transcripts. We aimed to screen the transcriptome for HBV integrations in HCCs. Transcriptome sequencing was performed on paired HBV-associated HCCs and corresponding non-tumorous liver tissues to identify viral-human chimeric sites. Validation was further performed in an expanded cohort of human HCCs. Here we report the discovery of a novel pre-mRNA splicing mechanism in generating HBV-human chimeric protein. This mechanism was exemplified by the formation of a recurrent HBV-cyclin A2 (CCNA2) chimeric transcript (A2S), as detected in 12.5% (6 of 48) of HCC patients, but in none of the 22 non-HCC HBV-associated cirrhotic liver samples examined. Upon the integration of HBV into the intron of the CCNA2 gene, the mammalian splicing machinery utilized the foreign splice sites at 282nt. and 458nt. of the HBV genome to generate a pseudo-exon, forming an in-frame chimeric fusion with CCNA2. The A2S chimeric protein gained a non-degradable property and promoted cell cycle progression, demonstrating its potential oncogenic functions. A pre-mRNA splicing mechanism is involved in the formation of HBV-human chimeric proteins. This represents a novel and possibly common mechanism underlying the formation of HBV-human chimeric transcripts from intronically integrated HBV genome with functional impact. HBV is involved in the mammalian pre-mRNA splicing machinery in the generation of potential tumorigenic HBV-human chimeras. This study also provided insight on the impact of intronic HBV integration with the gain of splice sites in the development of HBV-associated HCC.

E3 Ubiquitin Ligase Siah-1 is Down-regulated and Fails to Target Natural HBx Truncates for Degradation in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a common worldwide malignancy with high morbidity and mortality. Hepatitis B viral (HBV)-encoded X protein (HBx) and natural HBx variants play important roles in HBV-associated HCC development. HBx is an unstable protein that can be degraded in vivo. Our previous study found that the E3 ubiquitin ligase Siah-1 could target HBx for poly-ubiquitylation and proteasomal degradation and attenuate the transcriptional activity of HBx. However, in HCC patients, high expression levels of HBx and HBx variants are frequently observed and are associated with HCC progression. The mechanism underlying their up-regulation is largely unknown. In this study, we screened for Siah-1 mutations in 270 HCC samples and 9 HCC cell lines, and examined Siah-1 mRNA and protein expression in a subset of paired HCC specimens. Our results demonstrate that Siah-1 is highly conserved, as no somatic mutation was identified, with the exception of one synonymous transition from G to A at codon 67. Both the mRNA and protein levels of Siah-1 were significantly down-regulated in HCC tissues compared with their adjacent normal counterparts. We constructed three natural HBx truncates that were identified in our HCC cases. We found that Siah-1 failed to decrease the stability of these HBx variants and was unable to inhibit the transcriptional activity of these HBx truncates at heat shock elements (HSEs). Moreover, Siah-1 had weaker association with three HBx mutants than full length HBx. Therefore, our findings suggest that down-regulation of Siah-1, but not its mutations, and natural HBx variants resistant to Siah-1-induced degradation may be a novel mechanism for HCC development.

HBx co-localizes with COXIII in HL-7702 cells to upregulate mitochondrial function and ROS generation.

Hepatocellular carcinoma (HCC) is one of the most common malignant diseases, and HBx leads to the development of HBV-associated HCC. Mitochondria are key organelles that regulate apoptosis, cellular energetics and signal transduction pathways, and are the source of HBx-induced reactive oxygen species (ROS). Recent findings have shown that HBx interacts with the inner mitochondrial membrane protein, COXIII, via the yeast two-hybrid system, mating experiment and coimmunoprecipitation. The aim of the present study was to examine the co-localizaiton of HBx and COXIII in HL-7702 cells and to investigate ensuing alterations of mitochondrial function. An HL-7702 cell line stably expressing the HBx gene by lentivirus vectors was constructed. Confocal microscopy was utilized to assess the interaction between HBx protein and COXIII. Expression of COXIII, activities of cytochrome c oxidase (COX) and the mitochondrial membrane potential, which were functionally relevant to the HBx protein-COXIII interaction, were investigated in cell cultures. Moreover, the intracellular ROS levels were detected by flow cytometry. The results demonstrated that HBx co-localized with the inner mitochondrial protein, COXIII, in HL-7702 cells, causing the upregulation of COXIII protein expression as well as COX activity. However, HBx did not alter the mitochondrial membrane potential and mitochondria exhibited only slight swelling in HL-7702-HBx cells. Moreover, HBx elevated the generation of mitochondrial ROS in HL-7702-HBx cells. The main finding of the present study was that the co-localization of HBx and COXIII leads to upregulation of the mitochondrial function and ROS generation, which are associated with the oncogenesis of HBV-associated HCC.

Hepatitis B virus and microRNAs: Complex interactions affecting hepatitis B virus replication and hepatitis B virus-associated diseases.

Chronic infection with the hepatitis B virus (HBV) is the leading risk factor for the development of hepatocellular carcinoma (HCC). With nearly 750000 deaths yearly, hepatocellular carcinoma is the second highest cause of cancer-related death in the world. Unfortunately, the molecular mechanisms that contribute to the development of HBV-associated HCC remain incompletely understood. Recently, microRNAs (miRNAs), a family of small non-coding RNAs that play a role primarily in post-transcriptional gene regulation, have been recognized as important regulators of cellular homeostasis, and altered regulation of miRNA expression has been suggested to play a significant role in virus-associated diseases and the development of many cancers. With this in mind, many groups have begun to investigate the relationship between miRNAs and HBV replication and HBV-associated disease. Multiple findings suggest that some miRNAs, such as miR-122, and miR-125 and miR-199 family members, are playing a role in HBV replication and HBV-associated disease, including the development of HBV-associated HCC. In this review, we discuss the current state of our understanding of the relationship between HBV and miRNAs, including how HBV affects cellular miRNAs, how these miRNAs impact HBV replication, and the relationship between HBV-mediated miRNA regulation and HCC development. We also address the impact of challenges in studying HBV, such as the lack of an effective model system for infectivity and a reliance on transformed cell lines, on our understanding of the relationship between HBV and miRNAs, and propose potential applications of miRNA-related techniques that could enhance our understanding of the role miRNAs play in HBV replication and HBV-associated disease, ultimately leading to new therapeutic options and improved patient outcomes.

The hepatitis B virus (HBV) HBx protein activates AKT to simultaneously regulate HBV replication and hepatocyte survival.

Chronic infection with hepatitis B virus (HBV) is a risk factor for developing liver diseases such as hepatocellular carcinoma (HCC). HBx is a multifunctional protein encoded by the HBV genome; HBx stimulates HBV replication and is thought to play an important role in the development of HBV-associated HCC. HBx can activate the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway in some cell lines; however, whether HBx regulates PI3K/AKT signaling in normal hepatocytes has not been evaluated. In studies described here, we assessed HBx activation of PI3K/AKT signaling in an ex vivo model of cultured primary hepatocytes and determined how this HBx activity affects HBV replication. We report that HBx activates AKT in primary hepatocytes and that the activation of AKT decreases HBV replication and HBV mRNA and core protein levels. We show that the transcription factor hepatocyte nuclear factor 4α (HNF4α) is a target of HBx-regulated AKT, and we link HNF4α to HBx-regulated AKT modulation of HBV transcription and replication. Although we and others have shown that HBx stimulates and is likely required for HBV replication, we now report that HBx also activates signals that can diminish the overall level of HBV replication. While this may seem counterintuitive, we show that an important effect of HBx activation of AKT is inhibition of apoptosis. Consequently, our studies suggest that HBx balances HBV replication and cell survival by stimulating signaling pathways that enhance hepatocyte survival at the expense of higher levels of HBV replication. Chronic hepatitis B virus (HBV) infection is a common cause of the development of liver cancer. Regulation of cell signaling pathways by the HBV HBx protein is thought to influence the development of HBV-associated liver cancer. HBx stimulates, and may be essential for, HBV replication. We show that HBx activates AKT in hepatocytes to reduce HBV replication. While this seems contradictory to an essential role of HBx during HBV replication, HBx activation of AKT inhibits hepatocyte apoptosis, and this may facilitate persistent, noncytopathic HBV replication. AKT regulates HBV replication by reducing the activity of the transcription factor hepatocyte nuclear factor 4α (HNF4α). HBx activation of AKT may contribute to the development of liver cancer by facilitating persistent HBV replication, augmenting the dedifferentiation of hepatocytes by inhibiting HNF4α functions, and activating AKT-regulated oncogenic pathways. AKT-regulated factors may provide therapeutic targets for inhibiting HBV replication and the development of HBV-associated liver cancer.

Metastatic tumor antigen in hepatocellular carcinoma: golden roads toward personalized medicine.

Hepatocellular carcinoma (HCC), a prototype of hypervascular tumors, is one of the most common malignancies in the world, especially hyperendemic in the Far East where chronic hepatitis B virus (HBV) infection is highly prevalent. It is characterized by the clinical feature of a poor prognosis or a high mortality due to its already far advanced stages at diagnosis. It is so multifactorial that hepatocarcinogenesis cannot be explained by a single molecular mechanism. To date, a number of pathways have been known to contribute to the development, growth, angiogenesis, and even metastasis of HCC. Among the various factors, metastatic tumor antigens (MTAs) or metastasis-associated proteins have been vigorously investigated as an intriguing target in the field of hepatocarcinogenesis. According to recent studies including ours, MTAs are not only involved in the HCC development and growth (molecular carcinogenesis), but also closely associated with the post-operative recurrence and a poor prognosis or a worse response to post-operative anti-cancer therapy (clinical significance). Herein, we review MTAs in light of their essential structure, functions, and molecular mechanism in hepatocarcinogenesis. We will also focus in detail on the interaction between hepatitis B x protein (HBx) of HBV and MTA in order to clarify the HBV-associated HCC development. Finally, we will discuss the prognostic significance and clinical application of MTA in HCC. We believe that this review will help clinicians to understand the meaning and use of the detection of MTA in order to more effectively manage their HCC patients.

Hepatitis B virus X protein inhibits autophagic degradation by impairing lysosomal maturation

Deficiency in autophagy, a lysosome-dependent cell degradation pathway, has been associated with a variety of diseases especially cancer. Recently, the activation of autophagy by hepatitis B virus X (HBx) protein, which is implicated in hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC), has been identified in hepatic cells. However, the underlying mechanism and the relevance of HBx-activated autophagy to the carcinogenesis caused by HBV remain elusive. Here, by transfection of HBV genomic DNA and HBx in hepatic and hepatoma cells, we showed that HBV- or HBx-induced autophagosome formation was accompanied by unchanged MTOR (mechanistic target of rapamycin) activity and decreased degradation of LC3 and SQSTM1/p62, the typical autophagic cargo proteins. Further functional and morphological analysis indicated that HBx dramatically impaired lysosomal acidification leading to a drop in lysosomal degradative capacity and the accumulation of immature lysosomes possibly through interaction with V-ATPase affecting its lysosome targeting. Moreover, clinical specimen test showed increased SQSTM1 and immature lysosomal hydrolase CTSD (cathepsin D) in human liver tissues with chronic HBV infection and HBV-associated liver cancer. These data suggest that a repressive effect of HBx on lysosomal function is responsible for the inhibition of autophagic degradation, and this may be critical to the development of HBV-associated HCC.

HBx mutants differentially affect the activation of hypoxia-inducible factor-1α in hepatocellular carcinoma

Background:Mutations in HBx gene are frequently found in HBV-associated hepatocellular carcinoma (HCC). Activation of hypoxia-inducible factor-1α (HIF-1α) contributes to HCC development and progression. Wild-type HBx has been demonstrated to activate HIF-1α, but the effect of HBx mutations on HIF-1α has not been elucidated.Methods:HBx mutations were identified by gene sequencing in 101 HCC tissues. Representative HBx mutants were cloned and transfected into HCC cells. Expression and activation of HIF-1α were analysed by western blot and luciferase assays, respectively. The relationship between HBx mutants and HIF-1α expression in HCC tissues was also evaluated.Results:The dual mutations K130M/V131I enhanced the functionality of HBx as they upregulated the expression and transcriptional activity of HIF-1α. The C-terminal truncations and deletion mutations, however, weakened the ability of HBx to upregulate HIF-1α. Meanwhile, the C-terminus was further found to be essential for the stability and transactivation of HBx. In the HCC tissues, there was a positive association between the HBx mutants and HIF-1α expression.Conclusion:Different mutations of HBx exert differentiated effects on the functionality of HIF-1α, however, the overall activity of HBx mutants appears to increase the expression and transcriptional activity of HIF-1α.

Modulation of apoptotic signaling by the hepatitis B virus X protein.

Worldwide, an estimated 350 million people are chronically infected with the Hepatitis B Virus (HBV); chronic infection with HBV is associated with the development of severe liver diseases including hepatitis and cirrhosis. Individuals who are chronically infected with HBV also have a significantly higher risk of developing hepatocellular carcinoma (HCC) than uninfected individuals. The HBV X protein (HBx) is a key regulatory HBV protein that is important for HBV replication, and likely plays a cofactor role in the development of HCC in chronically HBV-infected individuals. Although some of the functions of HBx that may contribute to the development of HCC have been characterized, many HBx activities, and their putative roles during the development of HBV-associated HCC, remain incompletely understood. HBx is a multifunctional protein that localizes to the cytoplasm, nucleus, and mitochondria of HBV‑infected hepatocytes. HBx regulates numerous cellular signal transduction pathways and transcription factors as well as cell cycle progression and apoptosis. In this review, we will summarize reports in which the impact of HBx expression on cellular apoptotic pathways has been analyzed. Although various effects of HBx on apoptotic pathways have been observed in different model systems, studies of HBx activities in biologically relevant hepatocyte systems have begun to clarify apoptotic effects of HBx and suggest mechanisms that could link HBx modulation of apoptotic pathways to the development of HBV-associated HCC.

Hepatitis B Virus X Protein Upregulates mTOR Signaling through IKKβ to Increase Cell Proliferation and VEGF Production in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC), a major cause of cancer-related death in Southeast Asia, is frequently associated with hepatitis B virus (HBV) infection. HBV X protein (HBx), encoded by a viral non-structural gene, is a multifunctional regulator in HBV-associated tumor development. We investigated novel signaling pathways underlying HBx-induced liver tumorigenesis and found that the signaling pathway involving IκB kinase β (IKKβ), tuberous sclerosis complex 1 (TSC1), and mammalian target of rapamycin (mTOR) downstream effector S6 kinase (S6K1), was upregulated when HBx was overexpressed in hepatoma cells. HBx-induced S6K1 activation was reversed by IKKβ inhibitor Bay 11-7082 or silencing IKKβ expression using siRNA. HBx upregulated cell proliferation and vascular endothelial growth factor (VEGF) production, and these HBx-upregulated phenotypes were abolished by treatment with IKKβ inhibitor Bay 11-7082 or mTOR inhibitor rapamycin. The association of HBx-modulated IKKβ/mTOR/S6K1 signaling with liver tumorigenesis was verified in a HBx transgenic mouse model in which pIKKβ, pS6K1, and VEGF expression was found to be higher in cancerous than non-cancerous liver tissues. Furthermore, we also found that pIKKβ levels were strongly correlated with pTSC1 and pS6K1 levels in HBV-associated hepatoma tissue specimens taken from 95 patients, and that higher pIKKβ, pTSC1, and pS6K1 levels were correlated with a poor prognosis in these patients. Taken together, our findings demonstrate that HBx deregulates TSC1/mTOR signaling through IKKβ, which is crucially linked to HBV-associated HCC development.

High HBV-DNA Titer in Surrounding Liver Rather Than in Hepatocellular Carcinoma Tissue Predisposes to Recurrence After Curative Surgical Resection

In this study the authors intended to investigate the relationship between intrahepatic hepatitis B virus (HBV)-DNA concentrations and posthepatectomy recurrence of HBV-associated hepatocellular carcinoma (HCC). High HBV-DNA level is strongly associated with HCC development in chronic HBV infection and considered to be a risk factor of HCC recurrence. A total of 109 patients with HBV-associated HCC who underwent curative surgical resection were followed up every 3 to 6 months for a median of 82 months. Intrahepatic total HBV-DNA titer was measured in HCC and surrounding liver tissues using a TaqMan probe-based real-time polymerase chain reaction method. HBV-DNA titers in HCC and surrounding liver were compared in accordance with patients' clinical, radiologic, and histopathological characteristics. The relationships between HBV-DNA titers in HCC or surrounding liver tissues and cumulative HCC recurrence rates were determined. Of the 109 patients, 67 (62%) showed posthepatectomy recurrence of HCC. In all patients, total HBV-DNA titers were significantly higher in HCCs than in surrounding liver tissues (P=0.019). HCC recurred more frequently in patients with higher than those with lower HBV-DNA titers in surrounding liver tissues (P=0.009). In contrast, the HCC recurrence rates were similar in patients with higher and those with lower HBV-DNA titers in HCC specimens (P=0.301). Multivariate analysis showed that tumor size >5 cm (P=0.008), the presence of portal vein thrombus (P=0.001), and high HBV-DNA titer in surrounding liver tissues (P=0.002) were independent risk factors for posthepatectomy HCC recurrence in patients with HBV-associated HCC. In patients with HBV-associated HCC, high HBV-DNA titer in surrounding liver rather than in the HCC itself is associated with posthepatectomy HCC recurrence after curative surgical resection.