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    Altered expression of E-cadherin in hepatocellular carcinoma: correlations with genetic alterations, beta-catenin expression, and clinical features. Wei Yu,Van Nhieu Jeanne Tran,Prigent Sylvie,Srivatanakul Petcharin,Tiollais Pierre,Buendia Marie-Annick Hepatology (Baltimore, Md.) E-cadherin is a key cell adhesion protein implicated as a tumor/invasion suppressor in human carcinomas and a binding partner of beta-catenin, which plays a critical role in Wnt signaling and in tumorigenesis. Here we report genetic and expression studies of E-cadherin and beta-catenin in hepatocellular carcinoma (HCC). Immunohistochemical analysis of E-cadherin expression in 37 HCCs and adjacent nontumor tissues revealed important variations among tumor samples, ranging from complete or heterogeneous down-regulation in 35% of cases to marked overexpression in 40% of tumors. Loss of E-cadherin expression was closely associated with loss of heterozygosity (LOH) at the E-cadherin locus and methylation of CpG islands in the promoter region (P <.002), predominantly in hepatitis B virus (HBV)-related tumors (P <.005). No mutation of the E-cadherin gene could be detected in the tumors examined, suggesting the requirement for reversible mechanisms of E-cadherin down-regulation. In most HCCs, including E-cadherin-positive and -negative cases, beta-catenin was strongly expressed at the cell membrane and nuclear accumulation of the protein was correlated with the presence of mutations in the beta-catenin gene itself, but not with E-cadherin loss. At difference with a number of epithelial cancers, vascular invasion was frequently noted in HCCs showing enforced expression of the membranous E-cadherin/beta-catenin complex. In conclusion, these data support the notion that E-cadherin might play diverse and seemingly paradoxic roles in HCC, reflecting specific requirements for tumor growth and spread in the liver environment. 10.1053/jhep.2002.35342
    Infection of human bone marrow stromal cells by hepatitis B virus: implications for viral persistence and the suppression of hematopoiesis. Chai T,Prior S,Cooksley W G,Sing G K The Journal of infectious diseases Suspension cultures of bone marrow cells (BMC) were challenged with hepatitis B virus (HBV) to study interactions between the virus and the nonadherent and adherent BMC populations. Virus-challenged BMC developed an adherent stromal layer that differed in cellular composition from that of mock-infected cultures, showing a threefold increase in cells of the monocyte-macrophage lineage with an accompanying decrease in cells of the granulocytic lineage. Both viral envelope hepatitis B surface and core antigen expression was detected in adherent and nonadherent cell populations up to 10 days after virus challenge, which decreased thereafter. HBV DNA was still detectable in adherent cells 3 weeks after virus challenge, as shown by polymerase chain reaction analysis. These data indicate that HBV can infect not only bone marrow colony-forming cells but also the stromal cell populations involved with the regulation of hematopoiesis in vivo. Such virus-cell interactions could contribute to the immune dysfunction and bone marrow failure occasionally reported for patients with HBV infection as well as acting as an important site for HBV latency and persistence. 10.1093/infdis/169.4.871
    Activation of hepatic stellate cells after phagocytosis of lymphocytes: A novel pathway of fibrogenesis. Muhanna Nidal,Doron Sarit,Wald Ori,Horani Amjad,Eid Ahmed,Pappo Orit,Friedman Scott L,Safadi Rifaat Hepatology (Baltimore, Md.) UNLABELLED:Increased CD8-T lymphocytes and reduced natural killer (NK) cells contribute to hepatic fibrosis. We have characterized pathways regulating the interactions of human hepatic stellate cells (HSCs) with specific lymphocyte subsets in vivo and in vitro. Fluorescence-activated cell sorting (FACS) was used to characterize human peripheral blood lymphocytes (PBLs) and intrahepatic lymphocytes (IHLs) obtained from healthy controls and from patients with either hepatitis B virus (HBV) or hepatitis C virus (HCV) with advanced fibrosis. Liver sections were analyzed by immunohistochemistry and confocal microscopy. To investigate in vitro interactions, PBLs from healthy controls or patients with HCV cirrhosis were co-cultured with an immortalized human HSC line (LX2 cells) or with primary HSCs. Significant alterations in lymphocyte distribution were identified in IHLs but not PBLs. The hepatic CD4/CD8 ratio and NK cells were significantly reduced in HBV/HCV patients. Expression of alpha-smooth muscle actin and infiltration of CD4, CD8, and NK cells were readily apparent in liver sections from patients with cirrhosis but not in healthy controls. Lymphocytes from each subset were in proximity to HSCs primarily within the periportal regions, and some were directly attached or engulfed. In culture, HSC activation was stimulated by HCV-derived CD8-subsets but attenuated by NK cells. Confocal microscopy identified lymphocyte phagocytosis within HSCs that was completely prevented by blocking intracellular adhesion molecule 1 (ICAM-1) and integrin molecules, or by irradiation of HSCs. LX2 knockdown of either Cdc42 or Rac1 [members of the Rho-guanosine triphosphatase (GTPase) family] prevented both phagocytosis and the activation of HSC by HCV-derived lymphocytes. CONCLUSION:The CD4/CD8 ratio and NK cells are significantly decreased in livers with advanced human fibrosis. Moreover, disease-associated but not healthy lymphocytes are engulfed by cultured HSCs, which is mediated by the Rac1 and Cdc42 pathways. Ingestion of lymphocytes by HSCs in hepatic fibrosis is a novel and potentially important pathway regulating the impact of lymphocytes on the course of hepatic fibrosis. 10.1002/hep.22413
    Early molecular and genetic determinants of primary liver malignancy. Feitelson Mark A,Pan Jingbo,Lian Zhaorui The Surgical clinics of North America Although the overview above provides a partial molecular picture of the early stages of stepwise hepatocarcinogenesis. it should be emphasized that tumor and nontumor liver contain multiple changes, and that there is variability in their profile among different patients even within single studies. Variability in the number and types of genetic changes has also been observed geographically, and may be dependent upon the etiology of the tumor (viral, chemical or both). Interestingly, HBxAg inactivates tumor suppressors (such as p53 [by direct binding] and Rb [by stimulating its phosphorylation]) early in carcinogenesis that are mutated later during tumor progression. HBxAg also constitutively activates signal transduction pathways, such as those involving c-jun and ras, and activates oncogenes,such as c-nloc, that are otherwise activated by 3-catenin mutations. These findings suggest common molecular targets in hepatocarcinogenesis, despite different mechanisms of activation or inactivation. These observations need to be exploited in future drug discovery and in the development of new therapeutics. Heterogeneity in the mechanisms of tumor development, evidenced by the differences in the up- and down regulated genes reported in micro array analyses, as well as in the genetic loci that undergo mutation or LOH indifferent reports, has now been well documented. This suggests that there are multiple pathways to HCC, and that there is redundancy in the pathways that regulate cell growth and survival. These findings also reflect that,although hepatocarcinogenesis is multistep, the molecular changes that underpin histopathological changes in tumor development are likely to be different or only partially overlapping in individual tumors. Overall, the consequences of these changes suggest that the pathogenesis of HCC is accompanied by a progressive loss of differentiation, loss of normal cell adhesion, loss of the ECM, and constitutive activation of selected signal transduction pathways that promote cell growth and survival. Although mechanisms are important, attention also has to be paid to the target genes whose altered expression actually mediate the neoplastic phenotype. Other key avenues of work need to be explored. For example, it will be important to try to identify germline mutations in HBV-infected patients that are passed on to their children, resulting in the development of HCC in childhood. Clinical materials will also be important for the validation of new markers with diagnostic or prognostic potential. In this context, there is an urgent need to establish simple and low-cost tests based upon molecular changes that are hallmarks of HCC development. Identification of patients with early HCC will also significantly increase survival through its impact upon treatment. The discovery and validation of HCC markers may permit accurate staging of lesions, determine the proximity of such lesions to malignancy, and determine whether lesions with a particular genetic profile are still capable of remodeling through appropriate therapeutic intervention. The efficient reintroduction of the relevant tumor suppressors, or the inhibition of oncogene expression by siRNA, provide just some of the additional opportunities that will ultimately be useful in patient treatment. Together, these approaches will go far in reducing the very high morbidity and mortality associated with HCC. 10.1016/S0039-6109(03)00226-3
    Adhesive molecules in liver disease. Immunohistochemical distribution of thrombospondin receptors in chronic HBV infection. Volpes R,van den Oord J J,Desmet V J Journal of hepatology The distribution of the thrombospondin-receptor was studied in 26 cases of inflammatory hepatitis B virus (HBV) related liver disease and twelve non-inflammatory controls using monoclonal antibody (mcab) OKM5. OKM5 reacted in all cases with sinusoidal lining cells, portal vessel endothelium, and scattered mononuclear inflammatory cells. In 16 of the 26 cases with inflammation, one or more clusters of OKM5 positive (OKM5+) hepatocytes were found in areas of periportal or intralobular inflammation, whereas the remaining ten cases did not show hepatocellular OKM5 reactivity. In all but one case, OKM5+ liver cells coexpressed HLA-DR-antigens, and in twelve cases cytotoxic/suppressor T-cells were enriched in the OKM5+/HLA-DR+ liver cell clusters, suggesting induction of OKM5 positivity by lymphokines released by nearby T-cells. In view of its role in cytoadherence, it is suggested that OKM5 expression by hepatocytes serves in the entrapment and retention of inflammatory cells thereby facilitating their activation. Furthermore, OKM5+/HLA-DR+ hepatocytes might trigger an autologous mixed lymphocyte reaction (AMLR), resulting in down-modulation of ongoing hepatic inflammatory responses.
    Immunohistochemical study of the distribution of intercellular adhesion molecule-1 and lymphocyte function-associated antigen-1 in chronic type B hepatitis. Doi T,Yamada G,Mizuno M,Tsuji T Journal of gastroenterology The expression of intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-1 (LFA-1) in the livers of 11 patients with chronic hepatitis B was studied immunohistochemically by light and electron microscopy to clarify the role of these adhesion molecules in tissue damage in chronic hepatitis B. On hepatocytes, ICAM-1 expression was confined to the bile canalicular surface when the liver inflammation was mild. In contrast, when the liver inflammation was severe, ICAM-1 was distributed on the entire surface of the hepatocyte, including the sinusoidal and lateral membranes; lymphocytes which were mostly positive for LFA-1, were often observed invading deeply among these hepatocytes. The degree of ICAM-1 expression on the hepatocytes was also related to the expression of HLA class 1 antigen. In liver showing diffuse expression of ICAM-1 on the hepatocytes, strong expression of HLA class 1 antigen was observed, and amounts of HBV in the liver were decreased. Diffuse expression of ICAM-1 and HLA class 1 antigen was mostly observed after acute exacerbation of liver inflammation. These results suggest that the ICAM-1/LFA-1 pathway is involved in the immunological mechanism responsible for liver cell damage in chronic hepatitis B.
    Hepatitis B virus X protein represses E-cadherin expression via activation of DNA methyltransferase 1. Lee Jung-Ok,Kwun Hyun Jin,Jung Jin Kyu,Choi Kyung Hee,Min Do Sik,Jang Kyung Lib Oncogene E-cadherin is a key cell adhesion molecule implicated as a tumor suppressor, which is frequently altered in hepatocellular carcinoma, especially in hepatitis B virus (HBV)-related tumors. Here, we report that HBV X protein (HBx) represses E-cadherin expression at the transcription level. Based on the differential effects of HBx natural variants, we determined that Lys-130 in the transactivation domain of HBx is critical for the E-cadherin repression. The repression effect of HBx was abolished after treatment with DNA methyltransferase inhibitor, 5'-Aza-2'dC. In addition, methylation-specific PCR analysis revealed that the CpG island 1 of E-cadherin promoter is hypermethylated by HBx. Furthermore, HBx induces DNA methyltransferase 1 expression by stimulating its transcription. Therefore, we conclude that HBx represses E-cadherin expression by inducing methylation-mediated promoter inactivation. The reduced E-cadherin expression results in dramatic morphological changes of the HBx-expressing cells. In addition, HBx-expressing cells aggregate poorly in suspension culture, reflecting their altered intercellular interactions. The biological significance was further demonstrated by the increased collagen invasion ability of HBx-expressing cells. Therefore, the present study suggests that HBx plays a role during hepatocellular carcinogenesis by favoring cell detachment from the surrounding cells and migration outside of the primary tumor site. 10.1038/sj.onc.1208827
    The regulation of proteins associated with the cytoskeleton by hepatitis B virus X protein during hepatocarcinogenesis. Kong Fanyun,You Hongjuan,Tang Renxian,Zheng Kuiyang Oncology letters Hepatocellular carcinoma (HCC) is a major malignant disease worldwide, and chronic hepatitis B virus (HBV) infection is one of the primary causes for this type of cancer. Hepatitis B virus X protein (HBx) is a non-structural protein encoded by the viral genome that has significant effects on the pathogenesis of HCC. With the development of high-throughput assays and technologies, the abnormal HBx-induced expression of certain cellular proteins with assorted biological functions has been investigated. These target proteins identified by various methods include specific proteins associated with the cellular cytoskeleton, which contribute to HBx-induced hepatocarcinogenesis. In addition, the cytoskeletal proteins deregulated by HBx are involved in cell morphogenesis, adhesion, migration and proliferation. This review aims to summarize the current understanding of the expression profiles of HBx-associated cytoskeletal proteins, as well as their complex functions and underlying mechanisms in hepatocarcinogenesis. Considering that the potential therapeutics for various types of tumors may function through the stabilization of cytoskeletal proteins in order to restrict cellular movement and limit intracellular processes, clarifying the mechanisms underlying protein-associated cytoskeleton dysregulation by HBx may provide novel possibilities and potent therapeutic targets for HBV-associated HCC. 10.3892/ol.2017.5757
    Downregulation of E-cadherin by hepatitis B virus X antigen in hepatocellullar carcinoma. Liu J,Lian Z,Han S,Waye M M Y,Wang H,Wu M-C,Wu K,Ding J,Arbuthnot P,Kew M,Fan D,Feitelson M A Oncogene Hepatitis B virus (HBV)-encoded X antigen (HBxAg) contributes to the development of hepatocellular carcinoma (HCC). A frequent characteristic of HCC is reduced or absent expression of the cell adhesion protein, E-cadherin, although it is not known whether HBxAg plays a role. To address this, the levels of E-cadherin were determined in HBxAg-positive and -negative HepG2 cells in culture, and in tumor and surrounding nontumor liver from a panel of HBV carriers. The results showed an inverse relationship between HBxAg and E-cadherin expression both in tissue culture and in vivo. In HBxAg-positive cells, E-cadherin was suppressed at both the mRNA and protein levels. This was associated with hypermethylation of the E-cadherin promoter. Depressed E-cadherin correlated with HBxAg trans-activation function, as did the migration of HepG2 cells in vitro. Decreased expression of E-cadherin was also associated with the accumulation of beta-catenin in the cytoplasm and/or nuclei in tissues and cell lines, which is characteristic of activated beta-catenin. Additional work showed that HBxAg-activated beta-catenin. Together, these results suggest that the HBxAg is associated with decreased expression of E-cadherin, accumulation of beta-catenin in the cytoplasm and nucleus, and increased cell migration, which may contribute importantly to hepatocarcinogenesis. 10.1038/sj.onc.1209138
    Up-regulation of intercellular adhesion molecule 1 transcription by hepatitis B virus X protein. Hu K Q,Yu C H,Vierling J M Proceedings of the National Academy of Sciences of the United States of America Intercellular adhesion molecule 1 (ICAM-1), a counter-receptor for lymphocyte function-associated antigen 1 on T cells, is critically important to a wide variety of adhesion-dependent leukocyte functions, including antigen presentation and target cell lysis. ICAM-1 expression by hepatocytes is increased in areas of inflammation and necrosis during chronic hepatitis B. Whether induction of ICAM-1 is due to the effect of inflammatory cytokines or involves a direct effect of the hepatitis B virus (HBV) remains unknown. In the present study, transfection of the HBV genome into human hepatoma cell lines resulted in enhanced expression of ICAM-1 protein and RNA in the absence of inflammation. Results of subgenomic transfections indicated that the HBV X protein (pX) induced ICAM-1 expression. Nuclear run-on assays showed that pX induced the ICAM-1 gene by increasing its rate of transcription. Although both pX and interferon gamma induced transcription of ICAM-1, addition of interferon gamma to cells expressing pX did not show an additive or synergistic effect. These results indicate that pX can directly regulate expression of ICAM-1 and may participate in the immunopathogenesis of HBV infection. 10.1073/pnas.89.23.11441
    Induction of intercellular adhesion molecule-1 (CD54) on human hepatoma cell line HepG2: influence of cytokines and hepatitis B virus-DNA transfection. Volpes R,van den Oord J J,Desmet V J,Yap S H Clinical and experimental immunology Human hepatocyte expression of intercellular adhesion molecule-1 (ICAM-1) (CD54) was studied in vitro by exposing the well differentiated human hepatoblastoma cell line HepG2 to various cytokines. In addition, hepatitis B virus (HBV)-DNA transfected HepG2 cells were also analysed. Expression of ICAM-1 on HepG2 cells was then revealed with an immunohistochemical procedure. Untreated HepG2 cells were unreactive, but showed strong cytoplasmic ICAM-1 immunoreactivity after treatment with interferon-gamma (IFN-gamma). This induction was completely inhibited by addition of a neutralizing antibody directed to IFN-gamma. IL-1, IL-6, tumour necrosis factor-alpha (TNF-alpha) and IFN-alpha, used alone or in combination, did not induce ICAM-1 expression, neither did they inhibit the IFN-gamma-induced expression of this adhesion molecule on HepG2 cells. Untreated hepatitis B virus-DNA transfected HepG2 cells expressed membranous ICAM-1. These results indicate that IFN-gamma is the main cytokine trigger for ICAM-1 expression on HepG2 cells, suggesting that in areas of liver inflammation this adhesion molecule is up-regulated on hepatocytes by locally released IFN-gamma. In addition, expression of ICAM-1 by hepatitis B virus-DNA transfected HepG2 cells suggests other, still unknown, triggering mechanisms in the induction of such adhesion molecules, for instance gene activation by viral genome, or autocrine virus-induced hepatocellular cytokine production. 10.1111/j.1365-2249.1992.tb06415.x
    Hepatitis B virus X protein promotes cell migration by inducing matrix metalloproteinase-3. Yu Feng-Ling,Liu Hung-Jen,Lee Jeng-Woei,Liao Ming-Huei,Shih Wen-Ling Journal of hepatology BACKGROUND/AIMS:Hepatitis B virus (HBV), a major causative agent of hepatocellular carcinoma (HCC), encodes an oncogenic X protein (HBx) that transcriptionally activates multiple viral and cellular promoters. How this regulation influences HCC is unclear. METHODS:Global gene expression in HBx-expressing and non-expressing hepatoma cells was analyzed using cDNA microarrays. RESULTS:Genes that were markedly up- or down-regulated in the presence of HBx included those involved in signal transduction, metabolism, apoptosis, cytokine production, cell cycle, cell adhesion and oncogenesis. Other genes of ill-defined function were also affected. Trans-activation proficient HBx up-regulated the transcription, translation and secretion of matrix metalloproteinase-3 (MMP-3), manifest as a cell migratory phenotype. This HBx effect was abrogated in the presence of a MMP-3 specific peptide inhibitor. CONCLUSIONS:HBx exerts selective transcriptional control in hepatoma cells and induces cellular migration through the activation of MMP-3. 10.1016/j.jhep.2004.11.031
    Liver-intestine cadherin predicts microvascular invasion and poor prognosis of hepatitis B virus-positive hepatocellular carcinoma. Ding Zhen-Bin,Shi Ying-Hong,Zhou Jian,Shi Guo-Ming,Ke Ai-Wu,Qiu Shuang-Jian,Wang Xiao-Ying,Dai Zhi,Xu Yang,Fan Jia Cancer BACKGROUND:Liver-intestine cadherin (LI-cadherin; CDH-17) is a new member of the cadherin superfamily with distinct structural and functional features. The study was designed to investigate the role of LI-cadherin in tumor invasion and prognosis of human hepatitis B virus (HBV)-positive hepatocellular carcinoma (HCC). METHODS:LI-cadherin expression in HBV-positive hepatocellular carcinoma cell lines with low- and high-invasive potentials was evaluated by Western-blot, immunofluorescence, and real-time polymerase chain reaction (PCR) analyses. The role of LI-cadherin in tumor invasion was also evaluated in vitro by a small-interfering ribonucleic acid (siRNA)-mediated approach. The prognostic significance of LI-cadherin was validated in a cohort of HBV-positive HCC patients by immunohistochemistry and Western-blot. RESULTS:Significant high levels of LI-cadherin mRNA and protein were found in the high-invasive HCCLM3 as compared with those in low-invasive PLC/PRF/5 and Hep3B cell line. Cell migration, adhesion to extracellular matrix, and matrigel invasion were significantly reduced after LI-cadherin knockdown in HCCLM3 cells. Immunohistochemical analysis of 255 HBV-positive HCC cases showed that overexpression of LI-cadherin was well correlated with microvascular invasion, which was confirmed by Western-blot in 32 tumor tissues, and its overexpression was strongly associated with shorter overall survival as well as higher incidence of tumor recurrence. CONCLUSIONS:LI-cadherin is predictive of microvascular invasion and poor prognosis of HBV-positive HCC, and would be a potential useful intervention target for HCC. 10.1002/cncr.24513
    Diverse modes of genomic alteration in hepatocellular carcinoma. Jhunjhunwala Suchit,Jiang Zhaoshi,Stawiski Eric W,Gnad Florian,Liu Jinfeng,Mayba Oleg,Du Pan,Diao Jingyu,Johnson Stephanie,Wong Kwong-Fai,Gao Zhibo,Li Yingrui,Wu Thomas D,Kapadia Sharookh B,Modrusan Zora,French Dorothy M,Luk John M,Seshagiri Somasekar,Zhang Zemin Genome biology BACKGROUND:Hepatocellular carcinoma (HCC) is a heterogeneous disease with high mortality rate. Recent genomic studies have identified TP53, AXIN1, and CTNNB1 as the most frequently mutated genes. Lower frequency mutations have been reported in ARID1A, ARID2 and JAK1. In addition, hepatitis B virus (HBV) integrations into the human genome have been associated with HCC. RESULTS:Here, we deep-sequence 42 HCC patients with a combination of whole genome, exome and transcriptome sequencing to identify the mutational landscape of HCC using a reasonably large discovery cohort. We find frequent mutations in TP53, CTNNB1 and AXIN1, and rare but likely functional mutations in BAP1 and IDH1. Besides frequent hepatitis B virus integrations at TERT, we identify translocations at the boundaries of TERT. A novel deletion is identified in CTNNB1 in a region that is heavily mutated in multiple cancers. We also find multiple high-allelic frequency mutations in the extracellular matrix protein LAMA2. Lower expression levels of LAMA2 correlate with a proliferative signature, and predict poor survival and higher chance of cancer recurrence in HCC patients, suggesting an important role of the extracellular matrix and cell adhesion in tumor progression of a subgroup of HCC patients. CONCLUSIONS:The heterogeneous disease of HCC features diverse modes of genomic alteration. In addition to common point mutations, structural variations and methylation changes, there are several virus-associated changes, including gene disruption or activation, formation of chimeric viral-human transcripts, and DNA copy number changes. Such a multitude of genomic events likely contributes to the heterogeneous nature of HCC. 10.1186/s13059-014-0436-9
    High expression of beta2-glycoprotein I is associated significantly with the earliest stages of hepatitis B virus infection. Liu Ya-Ming,Zhang Wen-Yan,Wang Zhong-Feng,Yan Chao-Ying,Gao Pu-Jun Journal of medical virology Human beta2-glycoprotein I (beta2-GPI) binds to recombinant hepatitis B surface antigen (rHBsAg) and can bind specifically to annexin II, which is located on the cell membrane of human hepatoma SMMC-7721 cells. Viral envelope proteins are essential for mediating cellular entry. The aim of this study was to investigate the role of beta2-GPI in the early stages of hepatitis B virus (HBV) infection. Western blot and qRT-PCR analyses revealed that beta2-GPI expression was upregulated in HepG2.2.15 cells at both the mRNA and protein level and was almost non-existent in 293T and CHO cells. Furthermore, annexin II was expressed at lower levels in HepG2.2.15 cells compared to L02, HepG2, and SMMC-7721 cells. Additionally, ELISA analyses demonstrated that beta2-GPI enhanced the ability of HBsAg to bind to cell surfaces, and there was differential adhesion to L02, HepG2, HepG2.2.15, and 293T cells. Western blot and ELISA were then performed to assess the effects of HBV and the HBsAg domain on beta2-GPI expression in co-transfected 293T cells. This study revealed that HBV and the large HBV envelope protein increased beta2-GPI expression. Further investigation indicated that beta2-GPI colocalized with HBsAg in the cytosol of HepG2.2.15 cells, with sodium taurocholate co-transporting polypeptide (NTCP) on the cell membrane in NTCP-complemented HepG2 cells, and with annexin II in the cytosol of HepG2 and HepG2.2.15 cells. These data suggest that high expression of beta2-GPI enhances HBsAg binding to cell surfaces, thus contributing to virus particle transfer to the NTCP receptor and interaction with annexin II for viral membrane fusion. 10.1002/jmv.23961
    Cancer-associated circulating large extracellular vesicles in cholangiocarcinoma and hepatocellular carcinoma. Julich-Haertel Henrike,Urban Sabine K,Krawczyk Marcin,Willms Arnulf,Jankowski Krzysztof,Patkowski Waldemar,Kruk Beata,Krasnodębski Maciej,Ligocka Joanna,Schwab Robert,Richardsen Ines,Schaaf Sebastian,Klein Angelina,Gehlert Sebastian,Sänger Hanna,Casper Markus,Banales Jesus M,Schuppan Detlef,Milkiewicz Piotr,Lammert Frank,Krawczyk Marek,Lukacs-Kornek Veronika,Kornek Miroslaw Journal of hepatology BACKGROUND & AIMS:Large extracellular vesicles, specifically AnnexinV EpCAM CD147 tumour-associated microparticles (taMPs), facilitate the detection of colorectal carcinoma (CRC), non-small cell lung carcinoma (NSCLC) as well as pancreas carcinoma (PaCa). Here we assess the diagnostic value of taMPs for detection and monitoring of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). Specifically, the aim of this study was to differentiate liver taMPs from other cancer taMPs, such as CRC and NSCLC. METHODS:Fluorescence-activated cell scanning (FACS) was applied to detect various taMP populations in patients' sera that were associated with the presence of a tumour (AnnexinV EpCAM CD147 taMPs) or could discriminate between cirrhosis (due to HCV or HBV) and liver cancers (AnnexinV EpCAM ASGPR1 taMPs). In total 172 patients with liver cancer (HCC or CCA), 54 with cirrhosis and no liver neoplasia, and 202 control subjects were enrolled. RESULTS:The results indicate that AnnexinV EpCAM CD147 taMPs were elevated in HCC and CCA. Furthermore, AnnexinV EpCAM ASGPR1 CD133 taMPs allowed the distinction of liver malignancies (HCC or CCA) and cirrhosis from tumour-free individuals and, more importantly, from patients carrying other non-liver cancers. In addition, AnnexinV EpCAM ASGPR1 taMPs were increased in liver cancer-bearing patients compared to patients with cirrhosis that lacked any detectable liver malignancy. The smallest sizes of successfully detected cancers were ranging between 11-15mm. AnnexinV EpCAM ASGPR1 taMPs decreased at 7days after curative R0 tumour resection suggesting close correlations with tumour presence. ROC values, sensitivity/specificity scores and positive/negative predictive values (>78%) indicated a potent diagnostic accuracy of AnnexinV EpCAM ASGPR1 taMPs. CONCLUSION:These data provide strong evidence that AnnexinV EpCAM ASGPR1 taMPs are a novel biomarker of HCC and CCA liquid biopsy that permit a non-invasive assessment of the presence and possible extent of these cancers in patients with advanced liver diseases. LAY SUMMARY:Microparticles (MPs) are small vesicles that bleb from the membrane of every cell, including cancer cells, and are released to circulate in the bloodstream. Since their surface composition is similar to the surface of their underlying parental cell, MPs from the bloodstream can be isolated and by screening their surface components, the presence of their parental cells can be identified. This way, it was possible to detect and discriminate between patients bearing liver cancer and chronic liver cirrhosis. 10.1016/j.jhep.2017.02.024
    Activation of focal adhesion kinase by hepatitis B virus HBx protein: multiple functions in viral replication. Bouchard Michael J,Wang Lihua,Schneider Robert J Journal of virology The hepatitis B virus (HBV) X protein (HBx) is a multifunctional regulator of cellular signal transduction and transcription pathways and has a critical role in HBV replication. Much of the cytoplasmic signal transduction activity associated with HBx expression and its stimulation of viral replication is attributable to HBx-induced activation of calcium signaling pathways involving Pyk2 and Src tyrosine kinases. To further characterize upstream signal transduction pathways that are required for HBx activity, including activation of Src and mitogen-activated protein kinase (MAPK) cascades, we determined whether focal adhesion kinase (FAK), a known regulator of Src family kinases and the other member of the Pyk2/FAK kinase family, is activated by HBx. We report that HBx activates FAK and that FAK activation is important for multiple HBx functions. Dominant inhibiting forms of FAK blocked HBx activation of Src kinases and downstream signal transduction, HBx stimulation of NF-kappaB and AP-1-dependent transcription, and HBV DNA replication. We also demonstrate that HBx-induced activation of FAK is dependent on cellular calcium signaling, which is modulated by HBx. Moreover, prolonged expression of HBx increases both FAK activity and its level of expression. FAK activation may play a role in cellular transformation and cancer progression. HBx stimulation of FAK activity and abundance may also be relevant as a potential cofactor in HBV-associated hepatocellular carcinoma. 10.1128/JVI.80.9.4406-4414.2006
    Expression of epithelial cellular adhesion molecule (Ep-CAM) in chronic (necro-)inflammatory liver diseases and hepatocellular carcinoma. Breuhahn Kai,Baeuerle Patrick A,Peters Malte,Prang Nadja,Töx Ulrich,Köhne-Volland Rudolph,Dries Volker,Schirmacher Peter,Leo Eugen Hepatology research : the official journal of the Japan Society of Hepatology Epithelial cell adhesion molecule (Ep-CAM) is expressed in a several epithelial tissues and carcinomas, but not on mature hepatocytes. Here, we analysed the expression of Ep-CAM in 230 patients suffering from various liver diseases like chronic hepatitis B and C (HBV and HCV infection), chronic autoimmune hepatitis (AIH), chronic alcoholic liver disease (ALD), primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), hereditary hemochromatosis and dysplastic nodules (DNs) as well as hepatocellular carcinomas (HCCs) and cholangiocellular carcinomas (CCCs) by immunohistochemistry. De novo hepatocellular Ep-CAM expression was found in 75.9% of ALD (22/29), 63.6% of HCV (21/33) and 55.6% of each AIH and HBV cases (5/9 and 15/27, respectively). Lower Ep-CAM expression levels were observed for primary sclerosing liver diseases (PBC and PSC) with 25% (3/12) and 7.7% (1/13) of cases. Moreover, only 14.3% of HCCs (9/63) manifested expression, while all CCCs showed strong Ep-CAM expression (5/5). For DNs and hereditary hemochromatosis, Ep-CAM expression was found in 10 and 50% (3/30 and 2/4), respectively. In HBV and HCV, Ep-CAM expression correlated significantly with inflammatory activity as assessed by histological parameters and to the extent of fibrosis. In addition, for HCV also transaminase levels correlated significantly with Ep-CAM expression. Our results indicate that de novo Ep-CAM expression in hepatocytes is frequent in inflammatory liver diseases and is potentially linked to regenerative activity. CCCs and Ep-CAM positive HCCs may represent an attractive target group for Ep-CAM-directed immunotherapies, yet unwanted toxicity may limit the use of such strategies due to Ep-CAM expression in biliary epithelium and several chronic liver diseases such as HBV-and HCV-hepatitis. 10.1016/j.hepres.2005.10.006
    Circulating Tumor Cells Undergoing EMT Provide a Metric for Diagnosis and Prognosis of Patients with Hepatocellular Carcinoma. Qi Lu-Nan,Xiang Bang-De,Wu Fei-Xiang,Ye Jia-Zhou,Zhong Jian-Hong,Wang Yan-Yan,Chen Yuan-Yuan,Chen Zu-Shun,Ma Liang,Chen Jie,Gong Wen-Feng,Han Ze-Guang,Lu Yan,Shang Jin-Jie,Li Le-Qun Cancer research To clarify the significance of circulating tumor cells (CTC) undergoing epithelial-mesenchymal transition (EMT) in patients with hepatocellular carcinoma (HCC), we used an advanced CanPatrol CTC-enrichment technique and hybridization to enrich and classify CTC from blood samples. One hundred and one of 112 (90.18%) patients with HCC were CTC positive, even with early-stage disease. CTCs were also detected in 2 of 12 patients with hepatitis B virus (HBV), both of whom had small HCC tumors detected within 5 months. CTC count ≥16 and mesenchymal-CTC (M-CTC) percentage ≥2% prior to resection were significantly associated with early recurrence, multi-intrahepatic recurrence, and lung metastasis. Postoperative CTC monitoring in 10 patients found that most had an increased CTC count and M-CTC percentage before clinically detectable recurrence nodules appeared. Analysis of HCC with high CTC count and high M-CTC percentage identified 67 differentially expressed cancer-related genes involved in cancer-related biological pathways (e.g., cell adhesion and migration, tumor angiogenesis, and apoptosis). One of the identified genes, BCAT1, was significantly upregulated, and knockdown in Hepg2, Hep3B, and Huh7 cells reduced cell proliferation, migration, and invasion while promoting apoptosis. A concomitant increase in epithelial marker expression (EpCAM and E-cadherin) and reduced mesenchymal marker expression (vimentin and Twist) suggest that BCAT1 may trigger the EMT process. Overall, CTCs were highly correlated with HCC characteristics, representing a novel marker for early diagnosis and a prognostic factor for early recurrence. BCAT1 overexpression may induce CTC release by triggering EMT and may be an important biomarker of HCC metastasis. In liver cancer, CTC examination may represent an important "liquid biopsy" tool to detect both early disease and recurrent or metastatic disease, providing cues for early intervention or adjuvant therapy. . 10.1158/0008-5472.CAN-17-2459
    Expression of EpCAM increases in the hepatitis B related and the treatment-resistant hepatocellular carcinoma. Kimura Osamu,Kondo Yasuteru,Kogure Takayuki,Kakazu Eiji,Ninomiya Masashi,Iwata Tomoaki,Morosawa Tatsuki,Shimosegawa Tooru BioMed research international Increasing evidence supports the important role of cancer stem cells (CSCs). Many reports suggest that epithelial cell adhesion molecule (EpCAM) is a useful marker for cancer stem cells in hepatocellular carcinoma (HCC). To elucidate the mechanisms of cancer stem cells, the development of specific molecular targeted drugs has become very important. In the present study, we examined the EpCAM expression pattern and its characteristic expression in resected HCC. We studied the drug resistance of EpCAM expression cells. EpCAM expression was detected significantly more frequently with hepatitis B virus (HBV) than with other etiologies. In HCC resection patients who had received prior treatment (transcatheter arterial embolization or hepatic arterial infusion chemotherapy), EpCAM was strongly expressed. In particular, very strong expression was observed after hepatic arterial infusion chemotherapy. The PLC/PRF/5 human HCC cell line expressed bimodal EpCAM, and EpCAM-positive cells had CSC cell potency. The EpCAM expression in EpCAM-positive cells increased significantly by treatment with cisplatin. EpCAM-positive cells showed better viability than EpCAM-negative cells when treated with ciplatin. Collectively, our results suggest that cancer stem cells are highly expressed in hepatitis B and have potential anticancer drug resistance. 10.1155/2014/172913
    Hepatitis B Virus Stimulated Fibronectin Facilitates Viral Maintenance and Replication through Two Distinct Mechanisms. Ren Sheng,Wang Jun,Chen Tie-Long,Li Hao-Yu,Wan Yu-Shun,Peng Nan-Fang,Gui Xi-En,Zhu Ying PloS one Fibronectin (FN) is a high molecular weight extracellular matrix protein that functions in cell adhesion, growth, migration, and embryonic development. However, little is known about the role of FN during viral infection. In the present study, we found significantly higher levels of FN in sera, and liver tissues from hepatitis B virus (HBV) patients relative to healthy individuals. HBV expression enhanced FN mRNA and protein levels in the hepatic cell lines Huh7 and HepG2. HBV infection of susceptible HepG2-sodium taurocholate co-transporting polypeptide cells also increased FN expression. We also found that transcriptional factor specificity protein 1 was involved in the induction of FN by HBV. Knockdown of FN expression significantly inhibited HBV DNA replication and protein synthesis through activating endogenous IFN-α production. In addition, FN interacted with the transforming growth factor β-activated protein kinase 1 (TAK1) and TAK1-binding protein complex and attenuated interferon signaling by inhibiting TAK1 phosphorylation. Furthermore, the nuclear translocation of NF-κB/p65 was found to be inhibited by FN. We also observed that FN promoted HBV enhancers to support HBV expression. These results suggest novel functions of endogenous FN involved in immune evasion and maintenance of HBV replication. 10.1371/journal.pone.0152721
    Recurrence and poor prognosis following resection of small hepatitis B-related hepatocellular carcinoma lesions are associated with aberrant tumor expression profiles of glypican 3 and osteopontin. Yu Ming-Chin,Lee Yun-Shien,Lin Sey-En,Wu Hsiang-Yao,Chen Tse-Ching,Lee Wei-Chen,Chen Miin-Fu,Tsai Chi-Neu Annals of surgical oncology BACKGROUND:Early detection and following appropriate treatments of hepatocellular carcinoma (HCC) is still the gold standard for favored outcome of HCC patients; nevertheless, a small portion of hepatitis B virus (HBV)-related small HCC (<5 cm) patients got poor prognosis. Furthermore, the study for small HBV-HCC was limited. Therefore, the aim of this study was to explore the potential genetic signature for HBV-related small HCC as novel prognostic factors. METHODS:We examined expression profiles of HBV-related small HCC using an Affymetrix U133A GeneChip, evaluated differential gene expression by quantitative real-time polymerase chain reaction (qRT-PCR), and finally validated these expression patterns by immunohistochemistry (IHC). RESULTS:A total of 57 genes were differentially expressed between tumor and normal parts (n = 20 pairs) using Affymetrix U133A chip, and 16 genes were further evaluated by qRT-PCR. The result was compatible with the finding of oligonucleotide microarray (Pearson's correlation, r = 0.87). Furthermore, the expression pattern in HCC tissue by IHC in another group of small HBV-HCC (n = 100) showed overexpression of either osteopontin (OPN) or glypican 3 (GPC3) is an independent prognostic factor for disease-free survival (DFS) in HBV-positive small HCC (P < 0.01 and 0.03, respectively). Long-term DFS and overall survival (OS) for small HBV-HCC patients with high risk (both elevated GPC3(+)/OPN(+)) were DFS 0%, OS 0%, respectively; on the other hand, DFS and OS in patients with moderate (only 1 gene elevated) or low (OPN(-)/GPC3(-)) risk were 35.0 and 46.5%, respectively. CONCLUSIONS:Elevation of both OPN and GPC3 may act as an adverse indicator for HBV-related small HCC patients after curative resection. 10.1245/s10434-011-1946-2
    Hepatitis B virus induced coupling of deadhesion and migration of HepG2 cells on thermo-responsive polymer. Li Xi,Feng Huixing,Chen Wei Ning,Chan Vincent Biomaterials The unique physical property of thermo-responsive polymer (TRP) has recently prompted its increasing applications in tissue engineering. On the other hand, TRP has not been exploited for potential applications in quantitative cell screening against external stimulations. In this study, TRP is applied as a model system for elucidating the effect of HBV replication on the biophysical responses of HepG2 cells transfected by wild type HBV genome. Moreover, mutant HBV genome is designed to assess the specific activity of the SH3-binding domain of HBx during HBV replication. The adhesion contact recession and geometry transformation of HepG2 cells transfected with empty vector (pcDNA3.1 cells), wild type HBV (wtHBV cells) and mutant HBV genome (mHBV cells) are probed during the thermal transformation across lower solution critical temperature of TRP. In comparison with pcDNA3.1 cells and mHBV cells, the initial rate of reduction in degree of deformation and average adhesion energy for wtHBV cells is significantly increased. Interestingly, migration speed and persistence time of cells are found to be correlated with the cell deadhesion kinetics. Immuno-fluorescence microscopy demonstrates that HBV replication reduces the actin concentration and focal adhesions at cell periphery during the initial 30 min cell deadhesion. The results strongly suggested that HBV infection triggers the dynamic responses of HepG2 cells through the cytoskeleton remodeling and subsequent mechanochemical transduction. Overall, it is shown that TRP provides a convenient platform for quantifying biological stimulations on adherent cells. 10.1016/j.biomaterials.2009.11.031
    Dysregulation of β-catenin by hepatitis B virus X protein in HBV-infected human hepatocellular carcinomas. Chen Lei,Hu Liang,Li Liang,Liu Yuan,Tu Qian-Qian,Chang Yan-Xin,Yan He-Xin,Wu Meng-Chao,Wang Hong-Yang Frontiers of medicine in China β-catenin is a key molecule involved in both cell-cell adhesion and Wnt signaling pathway. In our study, we found that, in the development of hepatocellular carcinoma (HCC), β-catenin was correlated with hepatitis B virus (HBV) X gene encoded protein, which is essential for HBV infectivity and is a potential cofactor in viral carcinogenesis. The expression levels of wild-type β-catenin and E-cadherin were decreased in HepG2 cells expressing hepatitis B virus X protein (HBx), accompanied by destabilization of adherens junction. Reverse transcriptase PCR (RT-PCR), Northern and Western blot showed that reduction of wild-type β-catenin expression involved degradation of the protein. However, RNA interference (RNAi) and luciferase assay indicated that HBx enhanced β-catenin mediated signaling in HepG2 cells. In addition, immunohistochemical and Western blot analysis of β-catenin revealed that a decrease in the β-catenin protein level was found in 58.3% of HBV-related HCCs versus 19.2% of non-HBV-related tumors. Our data suggest that the expression of HBx contributed to the development of HCC, in part, by repressing the wild-type β-catenin expression and enforcing β-catenin-dependent signaling pathway, thus inducing cellular changes leading to acquisition of metastatic and/or proliferation properties. 10.1007/s11684-010-0170-y
    EGF and HGF levels are increased during active HBV infection and enhance survival signaling through extracellular matrix interactions in primary human hepatocytes. Barreiros Ana Paula,Sprinzl Martin,Rosset Sylvia,Höhler Thomas,Otto Gerd,Theobald Matthias,Galle Peter R,Strand Dennis,Strand Susanne International journal of cancer The hepatitis B virus (HBV) is a major causative agent of chronic liver disease and subsequent liver cirrhosis worldwide. The reduced sensitivity of virus-infected liver cells to apoptosis may play a role in the failure to remove virus-infected cells and eventually promote viral chronicity. The purpose of our study was to investigate whether survival factors induced during compensatory liver regeneration may protect hepatocytes against apoptosis. We evaluated the serum levels of hepatocyte growth factor (HGF) and epidermal growth factor (EGF) in HBV-infected patients and found significant increases in HGF and EGF in patients with active virus infection. In primary human hepatocytes we show that HGF and EGF have a protective effect against CD95-mediated apoptosis and cytotoxic T-cell killing. Simultaneous treatment with both regeneration factors enhanced the cytoprotective effect. The PI 3-K/Akt kinase inhibitor, wortmannin, and the STAT3 pathway inhibitor, Tyrphostin AG490, both effectively attenuated the cytoprotective effect of HGF and EGF. Furthermore, we show an EGF/HGF-dependent upregulation of beta(1)-integrin chains, increased adhesion to extracellular matrix and an increase in focal adhesions, suggesting outside-in signaling from the extracellular matrix as an additional cytoprotective mechanism. Our study demonstrates that HGF and EGF can interfere with CD95-mediated apoptosis and the action of cytotoxic T-cells through multiple mechanisms in human hepatocytes. Together our results argue that a survival mileau generated by activation of liver regeneration factors may be a risk factor for establishing viral persistence. 10.1002/ijc.23921
    E-cadherin Plays a Role in Hepatitis B Virus Entry Through Affecting Glycosylated Sodium-Taurocholate Cotransporting Polypeptide Distribution. Hu Qin,Zhang Feifei,Duan Liang,Wang Bo,Ye Yuanyuan,Li Pu,Li Dandan,Yang Shengjun,Zhou Lan,Chen Weixian Frontiers in cellular and infection microbiology Hepatitis B virus (HBV) infection is a major cause of chronic liver disease and hepatocellular carcinoma. Current antiviral therapy does not effectively eradicate HBV and further investigations into the mechanisms of viral infection are needed to enable the development of new therapeutic agents. The sodium-taurocholate cotransporting polypeptide (NTCP) has been identified as a functional receptor for HBV entry in liver cells. However, the NTCP receptor is not sufficient for entry and other membrane proteins contribute to modulate HBV entry. This study seeks to understand how the NTCP functions in HBV entry. Herein we show that knockdown of the cell-cell adhesion molecule, E-cadherin significantly reduced infection by HBV particles and entry by HBV pseudoparticles in infected liver cells and cell lines. The glycosylated NTCP localizes to the plasma membrane through interaction with E- cadherin, which increases interaction with the preS1 portion of the Large HBV surface antigen. Our study contributes novel insights that advance knowledge of HBV infection at the level of host cell binding and viral entry. 10.3389/fcimb.2020.00074
    Role of Dendritic Cell-Specific ICAM-3-Grabbing Nonintegrin on Dendritic Cells in the Recognition of Hepatitis B Virus. Wang Minxin,Zou Xiaojing,Tian Deying,Hu Song,Jiang Libin Viral immunology Dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) is an essential process for virus infection, such as HIV and hepatitis C, and plays a role in immune escape. However, the role of DC-SIGN in hepatitis B virus (HBV) infection is still unknown. The aim of this study was to investigate the role of DC-SIGN in mediating the maturation and activation of dendritic cells (DCs) when infected by HBV. Highly mannosylated HBV particles were obtained by treating HBV-producing HepG2.2.15 cells with the a-mannosidase I-inhibitor kifunensine. Highly mannosylated HBV or wild type HBV was added to infect the DCs of the DC-SIGN gene-silencing group and normal group, respectively. Then, the expression of CDla, CD80, CD83, CD86 and HLA-DR on DCs was detected by flow cytometry, the capacity of stimulating lymphocyte proliferation was tested by MTT assay, the level of IL-12p70 that was released by DCs was measured by enzyme-linked immunosorbent assay, and the expression of the proteins NF-κBp65 and p38 was detected by western blot. Both wild type and highly mannosylated HBV could promote DCs maturation and activation. However, the highly mannosylated HBV could promote DCs immune activation more strongly. The difference in the effect on DCs between the two types of HBV could be eliminated by DC-SIGN gene silencing. DC-SIGN can promote the maturation and activation of DCs when recognized HBV, but wild type HBV can escape recognition by DC-SIGN to a certain extent with the help of demannosylated modification, leading to defective DCs function and chronic HBV infection. 10.1089/vim.2014.0128
    Up-regulation of human cervical cancer proto-oncogene contributes to hepatitis B virus-induced malignant transformation of hepatocyte by down-regulating E-cadherin. Li Junfeng,Dai Xiaopeng,Zhang Hongfei,Zhang Wei,Sun Shihui,Gao Tongtong,Kou Zhihua,Yu Hong,Guo Yan,Du Lanying,Jiang Shibo,Zhang Jianying,Zhou Yusen Oncotarget Hepatocellular carcinoma (HCC) is one of the most fatal human malignancies, Human cervical cancer proto-oncogene (HCCR) aberrantly expressed in a number of malignant tumors, including HCC. HCC is associated with Hepatitis B virus (HBV) infection in a large percentage of cases. To explore the regulation and function of HCCR expression in the development of HCC, we detected HCCR expression in HBV expressing hepatocytes. Results showed that the expression of HCCR was higher in HBV-expressing hepatocytes than that in control cells. Examining different components of HBV revealed that the HBx promotes HCCR expression in hepatocytes via the T-cell factor (TCF)/β-catenin pathway. HCCR expression in HBx transgenic mice increased with as the mice aged and developed tumors. We also found that overexpression of HCCR in hepatocytes promoted cell proliferation, migration, and invasion and reduced cell adhesion. Suppressing HCCR expression abolished the effect of HBx-induced hepatocyte growth. In addition, HCCR represses the expression of E-cadherin by inhibition its promoter activity, which might correlate with the effects of HCCR in hepatocytes. Taken together, these results demonstrate that HBx-HCCR-E-cadherin regulation pathway might play an important role in HBV-induced hepatocarcinogenesis. They also imply that HCCR is a potential risk marker for HCC and/or a potential therapeutic target. 10.18632/oncotarget.5039
    Hepatitis B virus X protein induces EpCAM expression via active DNA demethylation directed by RelA in complex with EZH2 and TET2. Fan H,Zhang H,Pascuzzi P E,Andrisani O Oncogene Chronic hepatitis B virus (HBV) infection is a major risk factor for developing hepatocellular carcinoma (HCC), and HBV X protein (HBx) acts as cofactor in hepatocarcinogenesis. In liver tumors from animals modeling HBx- and HBV-mediated hepatocarcinogenesis, downregulation of chromatin regulating proteins SUZ12 and ZNF198 induces expression of several genes, including epithelial cell adhesion molecule (EpCAM). EpCAM upregulation occurs in HBV-mediated HCCs and hepatic cancer stem cells, by a mechanism not understood. Herein we demonstrate HBx induces EpCAM expression via active DNA demethylation. In hepatocytes, EpCAM is silenced by polycomb repressive complex 2 (PRC2) and ZNF198/LSD1/Co-REST/HDAC1 chromatin-modifying complexes. Cells with stable knockdown of SUZ12, an essential PRC2 subunit, upon HBx expression demethylate a CpG dinucleotide located adjacent to NF-κB/RelA half-site. This NF-κB/RelA site is in a CpG island downstream from EpCAM transcriptional start site (TSS). Chromatin immunoprecipitation (ChIP) assays demonstrate HBx-dependent RelA occupancy of NF-κB half-site, whereas RelA knockdown suppresses CpG demethylation and EpCAM expression. Tumor necrosis factor-α activates RelA, propagating demethylation to nearby CpG sites, shown by sodium bisulfite sequencing. RelA-dependent demethylation occurring upon HBx expression requires methyltrasferase EZH2, TET2 a key factor in cytosine demethylation and inactive DNMT3L, shown by knockdown assays and sodium bisulfite sequencing. Co-immunoprecipitations and sequential ChIP assays demonstrate that RelA in the presence of HBx forms a complex with EZH2, TET2 and DNMT3L, although the role of DNMT3L remains to be understood. Interestingly, the human EpCAM gene also has a CpG island downstream from its TSS, and a NF-κB-binding site flanked by CpGs. HepG2 cells derived from human HCC exhibit demethylation of these NF-κB-flanking CpG sites, and HBV replication propagates demethylation to nearby CpG sites. DLK1, another PRC2 target gene, also upregulated in HBV-mediated HCCs, is demethylated in liver tumors at CpG dinucleotides flanking the NF-κB-binding sequence, supporting that this active DNA demethylation mechanism functions during oncogenic transformation. 10.1038/onc.2015.122
    Peritumoral EpCAM Is an Independent Prognostic Marker after Curative Resection of HBV-Related Hepatocellular Carcinoma. Dai Xiao-Meng,Huang Tao,Yang Sheng-Li,Zheng Xiu-Mei,Chen George G,Zhang Tao Disease markers Accumulating evidence suggests that the tumor microenvironment has a profound influence on tumor initiation and progression, opening a new avenue for studying tumor biology. Nonetheless, the prognostic values of the peritumoral expression of EpCAM and CD13 remain to be elucidated in hepatocellular carcinoma (HCC) patients. In this study, the expression of EpCAM and CD13 was assessed by immunohistochemistry in peritumoral liver hepatocytes from 106 hepatitis B virus- (HBV-) related HCC patients who had undergone curative hepatectomy. The peritumoral EpCAM-positive group had a significantly worse overall survival (OS) ( = 0.003) and recurrence-free survival (RFS) ( = 0.022) compared to the negative group. Peritumoral CD13-positive patients were also associated with poor OS ( = 0.038), while not significantly associated with RFS. The adjusted multivariate COX proportional hazard regression analysis suggested that only the positive expression of peritumoral EpCAM precisely predicted poor OS. Being peritumoral EpCAM positive was also significantly associated with a larger tumor size, liver cirrhosis, and more frequent vascular invasion; however, no statistically significant association was observed between CD13 and any clinicopathological features. Taken together, peritumoral EpCAM and CD13 expression was associated with a poor prognosis, but EpCAM may be a better prognostic marker than CD13 in HBV-related HCC patients. In the future, peritumoral EpCAM could be a good target for adjuvant therapy after curative hepatectomy. 10.1155/2017/8495326
    EpCAM-regulated intramembrane proteolysis induces a cancer stem cell-like gene signature in hepatitis B virus-infected hepatocytes. Mani Saravana Kumar Kailasam,Zhang Hao,Diab Ahmed,Pascuzzi Pete E,Lefrançois Lydie,Fares Nadim,Bancel Brigitte,Merle Philippe,Andrisani Ourania Journal of hepatology BACKGROUND & AIMS:Hepatocytes in which the hepatitis B virus (HBV) is replicating exhibit loss of the chromatin modifying polycomb repressive complex 2 (PRC2), resulting in re-expression of specific, cellular PRC2-repressed genes. Epithelial cell adhesion molecule (EpCAM) is a PRC2-repressed gene, normally expressed in hepatic progenitors, but re-expressed in hepatic cancer stem cells (hCSCs). Herein, we investigated the functional significance of EpCAM re-expression in HBV-mediated hepatocarcinogenesis. METHODS:Employing molecular approaches (transfections, fluorescence-activated cell sorting, immunoblotting, qRT-PCR), we investigated the role of EpCAM-regulated intramembrane proteolysis (RIP) in HBV replicating cells in vitro, and in liver tumors from HBV X/c-myc mice and chronically HBV infected patients. RESULTS:EpCAM undergoes RIP in HBV replicating cells, activating canonical Wnt signaling. Transfection of Wnt-responsive plasmid expressing green fluorescent protein (GFP) identified a GFP population of HBV replicating cells. These GFP/Wnt cells exhibited cisplatin- and sorafenib-resistant growth resembling hCSCs, and increased expression of pluripotency genes NANOG, OCT4, SOX2, and hCSC markers BAMBI, CD44 and CD133. These genes are referred as EpCAM RIP and Wnt-induced hCSC-like gene signature. Interestingly, this gene signature is also overexpressed in liver tumors of X/c-myc bitransgenic mice. Clinically, a group of HBV-associated hepatocellular carcinomas was identified, exhibiting elevated expression of the hCSC-like gene signature and associated with reduced overall survival post-surgical resection. CONCLUSIONS:The hCSC-like gene signature offers promise as prognostic tool for classifying subtypes of HBV-induced HCCs. Since EpCAM RIP and Wnt signaling drive expression of this hCSC-like signature, inhibition of these pathways can be explored as therapeutic strategy for this subtype of HBV-associated HCCs. LAY SUMMARY:In this study, we provide evidence for a molecular mechanism by which chronic infection by the hepatitis B virus results in the development of poor prognosis liver cancer. Based on this mechanism our results suggest possible therapeutic interventions. 10.1016/j.jhep.2016.05.022
    Serum levels of soluble vascular cell adhesion molecule are related to hyperdynamic circulation in patients with liver cirrhosis. Lo Iacono Oreste,Rincón Diego,Hernando Ana,Ripoll Cristina,Catalina Maria Vega,Salcedo Magdalena,Clemente Gerardo,Gomez Judith,Nuñez Oscar,Matilla Ana,Bañares Rafael Liver international : official journal of the International Association for the Study of the Liver BACKGROUND:In patients with liver cirrhosis, serum levels of soluble vascular cell adhesion molecule-1 (sVCAM-1) have been associated with increasing fibrosis and are related to angiogenesis. AIM:To assess the possible correlation between sVCAM-1 and splanchnic and systemic haemodynamic and clinical staging of cirrhotic patients. METHODS:We assessed, using immunoassays, the serum levels of sVCAM-1, in the peripheral and hepatic vein, in all consecutive patients with liver cirrhosis, who underwent a haemodynamic study as part of its routine clinical work-up. RESULTS:We studied 86 patients [61 M/25 F; age 51.1 (8.3) years] with alcoholic (31) or viral (HBV:6, HCV:49) cirrhosis, 10 of them with hepatocellular carcinoma (Milan criteria). The mean follow-up was 391(187) days; 29 patients died or underwent transplantion during follow-up. A strong correlation in serum levels of sVCAM-1 was observed between the peripheral and the hepatic vein (r=0.8; P=0.0001). There was no correlation between levels of sVCAM-1 and hepatic venous pressure gradient. At univariate analysis, sVCAM-1 was inversely related with mean arterial pressure (r=-0.292; P=0.007), systemic vascular resistance (SVR) (r=-0.37; P=0.005) and serum sodium levels (r=-0.326; P=0.002). In multivariate linear regression only SVR remained as an independent variable associated to sVCAM-1. A correlation of sVCAM-1 with Child-Pugh scores, model for end-stage liver disease (MELD) and the clinical stage proposed in the Baveno IV consensus conference was also observed. Finally, patients who died or underwent transplantion during follow-up had significantly greater values of sVCAM-1 at baseline than those who did not [3505(1329) vs. 2488(1208) P=0.001]. CONCLUSION:This study supports a potential role of sVCAM-1 as a marker of hyperdynamic circulation, closely related to the different stage of liver cirrhosis. 10.1111/j.1478-3231.2008.01763.x
    Natural Killer Cell-Derived Interferon-Gamma Promotes Hepatocellular Carcinoma Through the Epithelial Cell Adhesion Molecule-Epithelial-to-Mesenchymal Transition Axis in Hepatitis B Virus Transgenic Mice. Chen Yongyan,Hao Xiaolei,Sun Rui,Wei Haiming,Tian Zhigang Hepatology (Baltimore, Md.) Hepatitis B virus (HBV) is a major risk factor for development of hepatocellular carcinoma (HCC), at least partially due to dysfunctional anti-HBV adaptive immunity; however, the role of innate immune response to HBV in this process is not well understood. In this study, low-dose polyinosinic:polycytidylic acid (poly [I:C]), a natural killer (NK) cell activator (3 μg/g body weight, twice/week for 8 weeks), induced HCC in HBV transgenic (HBs-Tg) mice, with an incidence of 100% after 6 months, while HBs-Tg mice without treatment only had HCC with an incidence of 16.7%. In HBs-Tg mice, poly (I:C) induced liver inflammation with markedly increased infiltrating lymphocytes, along with the concurrently increased apoptosis and proliferation of hepatocytes, leading to the accelerated epithelial-to-mesenchymal transition (EMT) of hepatocytes shown by increased expression of the typical transcriptional factors (Slug, Twist, and mothers against decapentaplegic-interacting protein 1) and phenotypic proteins (vimentin and chemokine [C-X-C motif] receptor 4). The EMT and tumorigenesis in this model depended on the presence of NK cells because depletion of these cells significantly reduced the HCC rate to 28.6%. Further, intrahepatic NK cells highly expressed interferon-gamma (IFN-γ), anti-IFN-γ neutralizing monoclonal antibody might obviously alleviate the hepatitis, and hepatocyte-specific IFN-γ overexpression promoted HCC. Moreover, IFN-γ deficiency in HBs-Tg mice prevented HCC occurring, though hepatic NK cells existed and could be activated, suggesting the critical role of IFN-γ in NK cell-mediated tumorigenesis. In an in vitro experiment, IFN-γ up-regulated epithelial cell adhesion molecule (EpCAM) expression through phosphorylated signal transducer and activator of transcription (p-STAT1) pathway, which was followed by EMT, and p-STAT1 inhibitor might absolutely abolish the expression of EpCAM and EMT in HBV surface antigen-positive hepatocytes. Conclusion: This work demonstrates that NK cell-derived IFN-γ promotes HCC through the EpCAM-EMT axis in HBs-Tg mice, revealing the importance of innate immunity in pathogenesis of HBV-associated HCC. 10.1002/hep.30317