Chronic liver damage caused by viral infection, alcohol, or obesity can result in increased risk for hepatocellular carcinoma (HCC). Ample epidemiological evidence suggests that there is a strong synergism between hepatitis C virus (HCV) and alcoholic liver diseases (ALD). The Toll-like receptor (TLR) signaling pathway is upregulated in chronic liver diseases. Alcoholism is associated with endotoxemia that stimulates expression of proinflammatory cytokine expression and inflammation in the liver and fat tissues. Recent studies of HCC have centered on cancer-initiating stem cell (CSC), including detection of CSC in cancer, identification of CSC markers, and isolation of CSC from human HCC cell lines. Synergism between alcohol and HCV may lead to liver tumorigenesis through TLR signaling.
1. HCV, Alcohol, and HCC
Chronic liver damage caused by viral infection, alcohol, or metabolic syndrome can result in increased risk for HCC which is the third most common cancer in the world [1]. This virus is a major cause of HCC, which is the fifth most common cancer in the world. HCC has a low five-year survival rate due to the lack of therapeutic options and is highly prevalent in the world, especially in Africa and Asia [1]. Clearly, understanding the molecular mechanisms of HCV-induced hepatocarcinogenesis is required for the eventual development of improved therapeutic modalities for this disease [2]. In particular, chronic infection with HBV or HCV represents a major risk factor for HCC [1]. HCV affects more than 170 million people worldwide [1, 3, 4].
Ample epidemiological evidence suggests that there is a strong connection between hepatitis C virus (HCV) and ALD. First, the prevalence of HCV is significantly higher among alcoholics than in the general population; for example, while the HCV positive rate in the general population of the U.S. is roughly 1%, it is 16% for alcoholics and nearly 30% for alcoholics with liver diseases [5]. Second, the presence of HCV infection correlates with the severity of the disease in alcoholic subjects, that is, HCV-infected patients with ALD develop liver cirrhosis and HCC at a significantly younger age than uninfected ALD patients, suggesting that alcohol and HCV work synergistically to cause liver damage [6]. Many studies also support synergistic interactions between HCV and alcoholism in hepatocarcinogenesis [7–11]. Heavy alcohol consumption and viral hepatitis synergistically increase the risk for HCC among blacks and whites in the U.S. [10]. HCC odds ratio increases to 48.3-fold and 47.8 from 8.1 and 8.6 by having concomitant alcohol abuse in HBV or HCV infected patients, respectively [10]. Indeed, our recent result demonstrates that the incidence of spontaneous HCC induction in the HCV core transgenic mice is increased 2-fold by chronic alcohol intake.
Recent studies with mice expressing HCV proteins have shed pivotal insights into the mechanisms underlying this synergism. The HCV core protein causes overproduction of reactive oxygen species which appears to be responsible for mitochondrial DNA damage [3, 12, 13]. The core protein also inhibits microsomal triglyceride transfer protein activity and VLDL secretion [14], which may underlie the genesis of fatty liver. The core protein also induces insulin resistance in mice and cell lines, and this effect may be mediated by degradation of insulin receptor substrates (IRS) 1 and 2 via upregulation of SOCS3 [15] in a manner dependent on PA28γ 73, or via IRS serine phosphorylation [16]. Thus, these core-induced perturbations such as oxidant stress and insulin resistance, which are also known risk factors for ALD, may underlie the synergism reproduced in alcohol-fed core transgenic mice [17]. TLR2 and TLR4 are markedly upregulated in hepatocytes, Kupffer cells, and peripheral monocytes of patients with chronic hepatitis C. TLR2-mediated activation by hepatitis C is linked to the proinflammatory cytokine induction [18]. TLR-mediated signals result in liver disease associated with hepatitis B, hepatitis C, alcoholicnonalcoholic steatohepatitis, and hepatic fibrosis [19]. The most devastating consequence of the synergism between viral hepatitis and alcohol is HCC [7–11]. The risk of developing HCC as assessed by odds ratio increases from 8~12 to 48~54 by having concomitant alcohol abuse in HCV/HBV infected patients [9, 10]. The HCV core and NS3 protein activate TLR2/TLR1 and TLR2/TLR6 on monocytes to produce inflammatory cytokines [19]. The aforementioned effects of the core protein may contribute to the mechanisms of the synergism. However, the more direct mechanistic evidence has recently been attained by our research using mice expressing the HCV nonstructural protein NS5A in a hepatocyte-specific manner. These mice, when fed alcohol for 12 months, develop liver tumors in a manner dependent on TLR4 induced by NS5A [20]. This NS5A-induced TLR4 is activated by endotoxemia associated with alcohol intake, leading to accentuated TLR4 signaling which in turn upregulates the stem cell marker Nanog required for TLR4-dependent liver oncogenesis. This finding on the NS5A-TLR4-Nanog axis in synergistic oncogenesis, is beginning to shed a novel insight into molecular mechanisms for HCC in alcoholic HCV patients.
HCV contains a 9.5-kb single-stranded positive-sense RNA genome, which encodes a polyprotein that is, processed into multiple proteins by cellular and viral proteases. Nonstructural protein NS5A may interact with an interferon-induced, double-stranded RNA-activated protein kinase PKR [21], thus accounting for the resistance of most HCV strains to interferon treatment. NS5A has also been shown to have a cryptic trans-acting activity for some cellular gene promoters [22]. The core protein deserves special mention because, in addition to being a viral structural protein, it serves multiple regulatory functions, including the activation or suppression of various cellular and viral gene promoters. Furthermore, it binds to LTβR, TNF receptor and several other cellular proteins, including apolipoprotein AII [23]. The immune- and cytokine-mediating roles of the HCV core protein may play a key role in the synergistic effects of alcohol liver disease (ALD) on HCV-associated liver damage.
2. TLRs Signaling in HCC
The TLR signaling pathway is upregulated in chronic liver diseases. Many different cell types in the liver express TLRs [19]. Hepatocytes express TLR1 through TLR9. Stellate cells express TLR2, 3, and 4. Bile duct epithelium expresses TLR2, 3, 4 and 5. Kupffer cells express TLR2, 3, and 4. Chronic alcohol consumption activates other TLRs, such as TLR1, 2, and 6–9, which further increases the TNF-α response to LPS in mice [19]. Human monocytes exposed to ethanol for a week develop hypersensitivity to LPS through decreased IRAK-M expression, which activates mitogen-activated protein kinase (MAPK) and NF-κB through TLR4 signaling, leading to activation of NF-κB, AP-1, and ERK [24].
Hepatocyte-specific deletion of TAK1 in mice causes spontaneous hepatocyte death, inflammation, fibrosis, and carcinogenesis partially mediated by TNFR signaling, indicating that TAK1 is an essential component for cellular homeostasis in the liver. In a NASH mouse model, TLR9 signaling induces production of IL-1β by Kupffer cells, leading to steatohepatitis, inflammation, and fibrosis via induction of IL-1β [25]. Furthermore, modulation of TGF-β signaling by a TLR4-MyD88-NF-κB pathway links between profibrogenic and proinflammatory signals [26].
Several possible mechanisms may explain the high prevalence rate of HCV among alcoholics and the increased severity of liver diseases in these patients. First, alcohol may enhance the replication of HCV and thus increase the expression of viral RNA and proteins, resulting in more severe HCV-induced liver injury, independent of the damage induced by alcohol alone. Indeed, HCV titer has been shown to exhibit a correlation with the amount of alcohol consumption [27]. This enhanced effect on HCV replication could be caused directly by the metabolites of ethanol, such as acetaldehyde and free radicals, which may stimulate HCV replication and gene expression. It could also be caused indirectly through alcohol-induced inhibition of the antiviral immune response. Indeed, HCV replication is more active in immunodeficient patients, such as HIV-infected patients [28], and ethanol consumption can cause immunosuppression [29].
Another potential mechanism is the involvement of cytokines. Both ALD and HCV cause enhanced secretion of TNF and other cytokines, such as IL-1, IL-6, and IL-8, [30]. TNF is particularly interesting because there is a tight correlation between the serum TNF concentration and the severity of ALD [31, 32], and TNFR1 deficiency ameliorates experimental ALD [33]. TNF may cause cell death through the activation of the TRADD/FADD signal transduction pathway. Oxidative stress may also contribute to TNF cytotoxicity [34]. On the other hand, a variety of factors can modulate the effects of TNF; for examples, NF-κB [35–37], manganous superoxide dismutase (MnSOD) [38], and GSH inhibit TNF-induced cytotoxicity [39]. In experimental ALD, the mitochondrial pool of GSH is depleted and the hepatocytes become hypersensitive to TNF [39]. We have recently shown that HCV core protein binds to lymphotoxin-β receptor and TNF receptor [40] and that the expression of this protein in several cell lines sensitize them to TNF-induced cytolysis [41]; therefore, HCV-infected cells are particularly sensitive to TNF. It is interesting to note that HCV core protein also sensitizes cells to apoptosis mediated by Fas [42], which shares with TNF receptors signal transduction molecules such as FADD. These observations suggest that HCV-infected hepatocytes are very sensitive to TNF and possibly other cytokines as well. This enhanced sensitivity, coupled with the increased secretion of TNF in ALD, may account for the synergistic effects of ALD on HCV.
3. TLR4-Mediated AP1 Activation and HCC
Alcoholism is associated with endotoxemia that stimulates expression of proinflammatory cytokine expression and inflammation in the liver and fat tissues [43]. Development of liver cancer in the HCV core mice is associated with inflammation [44]. Expression of the proinflammatory cytokine TNF-α and IL-1β is induced by HCV-infected human B cells (16) and by its core protein in the transgenic mouse model [45, 46]. Recently, we have shown that HCV infection, through NS5A protein expression, upregulates TLR4 expression and proinflammatory cytokines [47], providing a potential explanation for increased inflammation in HCV-infected livers. Further accentuation of TLR4 signaling in HCV would be expected if combined with alcohol abuse. This may serve as part of the synergistc mechanism when superimposed by other key patholophysiological events common in these comorbidities such as CYP2E1 induction. CYP2E1 induction impairs hepatic insulin signaling [48, 49], induces oxidative DNA damage [50], primes macrophages to increase LPS-induced TNF-α production [51], sensitizes hepatocytes to TNF-α-mediated cell death via c-jun [52, 53], and more importantly, leads to marked potentiation of endotoxin-induced oxidant liver injury (35). Therefore, saturated fatty acids which are implicated in obesity and diabetes could serve as additional ligands to enhance signaling via TLR4 which is already upregulated by HCV NS5A [54]. Polyunsaturated fats actually result in greater rather than less liver injury [55]. These interactive and synergistic mechanisms involving TLR4 in HCV and alcohol most likely contribute to increased incidence of HCC via oxidant stress and inflammation and are the focal points of my proposed research.
c-Jun-deficient mice die between embryonic days E12.5 and E13.5 from massive apoptosis of hepatoblasts, erythroblasts, and other cell types [56, 57]. To overcome this problem, mice harboring a “floxed” c-jun allele that can be deleted in designated cell types upon expression of the Cre recombinase have been developed. Using this system, c-Jun expression was shown to be essential for proper proliferation in postnatal hepatocytes [58]. Moreover, the deletion of c-Jun in hepatocytes compromises the ability of these cells to enter the cell cycle and undergo rapid proliferation after partial hepatectomy [58]. A well-accepted model of HCC utilizes a chemical carcinogen DEN (diethylnitrosamine) as a tumor initiator and phenobarbital as a promoter. By using this model and tissue-specific knockout mice, the loss of JNK1 in the liver was shown to reduce DEN-induced HCC development [59]. The requirement for c-jun was restricted to an early stage of tumor development in chemically induced HCC in mice [60]. In our study, c-jun knockout dramatically reduced the incidence of spontaneous and DEN-induced HCC in HCV core transgenic mice, supporting the role of c-jun in this model of hepatocarcinogenesis (Hepatology in press). Alcoholic liver disease patients have increased levels of hepatic RANTES/CCL5. Ethanol augments RANTES/CCL5 expression in rat liver sinusoidal endothelial cells and human endothelial cells via activation of NF-κB, HIF-1α, and AP-1 [61]. In vitro studies using liver-derived cell lines have demonstrated rapid activation of AP-1 by HBV or HCV proteins [62], and this mitogenic effect is implicated in hepatocytes’ susceptibility to liver cell transformation via fixation of genetic mutations caused by oxidant stress. Indeed, Further c-Jun prevents apoptosis by antagonizing p53 activity as another contributing factor for HCC development [60, 63]. Ectopic expression of HCV core protein constitutively activates AP-1 via JNK [64]. Activation of JNK is also implicated in ASH and NASH [49, 52]. Therefore, activation of JNK and c-jun most likely plays pivotal roles in synergistic induction of liver cancer by HCV and alcohol.
3.1. Cancer Stem Cells and HCC
Stem cells have three major characteristics: self renewal, asymmetric and multiple cell division (clonality), and plasticity. The liver has a high regenerative potential, and hepatic small oval progenitor cells around the peripheral branches of the bile ducts, the canals of Hering, can differentiate into biliary epithelial cells and hepatocytes [65]. These oval liver progenitor cells share molecular markers with adult hepatocytes (albumin, cytokeratin 7 [CK7], CK19, oval cell markers (OV-6, A6, and OV-1), chromogranin-A, NCAM (neural cell adhesion molecule)) and fetal hepatocytes (α-fetoprotein) (Table 1) [65, 66]. They are also positive for more common stem cell markers such as CD34+, Thy-1+, c-Kit+, and Flt-3+ (FMS-like tyrosine kinase 3) [67]. Thus, it currently remains unclear whether these stem cells are derived from the bone marrow and just migrate to this periportal niche or whether they represent true resident liver stem/progenitor cells. Binding of stroma-derived factor-1α (SDF-1α) to its surface receptor CXCR4 activates oval hepatic cells [68]. Forty percent of HCC have clonality, and thus are considered to originate from progenitor/stem cells [66, 69–71]. Recent studies of HCC have centered on CSC, including detection of CSC in cancer, identification of CSC markers, and isolation of CSC from human HCC cell lines. CSC were identified as a CD117+/CD133+ hepatic precursors in regenerating liver tissue [72] and a CD45−/CD90+ subpopulation of tumor cells in HCC [73]. The CD90+ cells are not present in the normal liver and, when injected into immunodeficient mice, create tumors repeatedly. In human HCC and HCC cell lines, specifically, CD133+ cells, not CD133− cells, had the ability to self-renew, create differentiated progenies, and form tumors [74]. This coincided with the expression of genes associated with stem/progenitor status, such as β-catenin, NOTCH, BMI, and OCT3/4. When compared to CD133− cells, the CD133+ cells isolated from the HCC cell lines showed higher expression of CD44 and CD34, but both CD133 subpopulations displayed similar expression for CD29, CD49f (integrin α6), CD90 and CD117 [74]. Furthermore, CD133+/CD49f+ cells in liver tumors correlate with tumorigenecity and “stemness” genes, such as Wnt/β-catenin, Notch, Hedgehog/SMO, Bmi, and Oct3/4 [75–77]. CD133+/CD49f+ HCC cancer stem cells confer resistance to chemotherapy, and this presents a major obstacle for the treatment of HCC [78]. One potential reason for this chemoresistance may lie in the plasticity of cancer stem cells with dysregulated signaling and gene expression. Several oncogenic signaling pathways in cancer stem cells of HCC, have been described including activated PI3K/AKT [79], signal transducer and activator of transcription 3 (STAT3) [80, 81], Notch [82], Hedgehog [83, 84], and transforming growth factor-beta (TGF-β) [85, 86].
Markers for liver cancer stem cells.
Gene name
Other name
Function
Species
Organ
References
CD133
Prominin 1 (PROM1)
Glycoprotein, membrane protrusions
Human, mouse
Liver, brain
[74, 78, 79, 87–89]
CD49f
Integrinα chain α6 (ITGA6)
Cell adhesion, cell signaling
Mouse
Liver
[74, 78]
CD90
Thy-1
Glycophosphatidylinositol (GPI) anchor
Mouse
Liver
[74]
CD44
Hyaluronic acid receptor
Cell adhesion and migration, metastasis
Mouse
Liver, breast
[74, 90]
CD117
KIT
C-kit receptor cytokine receptor
Mouse
Liver
[74]
CK19
Cytokeratin 19
Biliary lineage marker
Mouse
Liver
[91, 92]
OV-6
Oval cell marker
Early progenitor cells
Human
Liver
[92]
CD34
Glycoprotein
Cell-cell adhesion factor
Mouse
Liver, leukemia
[93]
AFP
α-fetoprotein
Fetal counterpart of serum albumin
Mouse
Liver
[94]
Nanog is one of the core transcription factors found in pluripotent embryonic stem cells (ESCs) [95]. It is essential for maintaining self-renewal and pluripotency of both human and mouse embryonic stem cells [96–99]. Overexpression of Nanog induces and maintains the pluripotency and self-renewing characteristics of ESCs under what normally would be differentiation-inducing culture conditions [100]. Recently, Nanog expression has been reported in human neoplasms, including germ cell tumors [101–104], breast carcinomas [104], osteosarcoma [105], and HCC [79]. Ectopic expression of Nanog induces an oncogenic potential in NIH3T3 [106].
4. Nanog-Positive Cancer Stem Cells Induced by HCV and Alcohol
Alcohol synergistically enhances the progression of liver disease and the risk for liver cancer caused by HCV. Toll-like receptor 4 (TLR4) is induced by hepatocyte-specific transgenic (Tg) expression of the HCV nonstructural protein NS5A, and this induction mediates synergistic liver damage and tumor development by alcohol-induced endotoxemia [20]. The stem/progenitor cell marker, Nanog, is upregulated as a novel downstream gene by TLR4 activation and the presence of CD133/Nanog-positive cells in liver tumors of alcohol-fed NS5A Tg mice [20]. Transplantation of p53-deficient hepatic progenitor cells transduced with TLR4 results in liver tumor development in mice following repetitive lipopolysaccharide (LPS) injection, but concomitant transduction of Nanog short-hairpin RNA abrogates this outcome [20]. Despite the common understanding that TLR4 is one of the pattern recognition receptors expressed predominantly by innate immune cells such as macrophages and lymphocytes, our study demonstrates that hepatocytes can be the primary cellular site of both TLR4 upregulation and its pathologic consequences in the context of HCV infection. Therefore, the TLR4-dependent mechanism synergizes liver disease by HCV and alcohol and is partly dependent on Nanog, a TLR4 downstream gene.
Nanog transduction alone is not as effective as TLR4 activation in liver tumorigenesis, as shown by our cell transplantation experiment [20]. We believe that TLR4 activation induces other tumor-driver genes which cooperatively work with Nanog to cause liver oncogenesis. Thus, Nanog is still essential for TLR4-dependent oncogenesis, but it alone is poorly oncogenic. In our previous work using a cell line, we demonstrated that TLR4 promoter up-regulation by NS5A is mediated by PU.1, Oct-1, and AP-1 elements [47]. The similar transcriptional mechanism may underlie TLR4 induction in primary hepatocytes.
In summary, alcohol and HCV NS5A synergistically induce liver tumor development via induction and activation of TLR4 in mice. The importance of Nanog as a direct downstream gene of TLR4 in this oncogenesis has also been identified. Pharmacologic inhibition of TLR4 signaling may become a novel therapeutic strategy for HCV-associated liver tumors.
OkudaK.Hepatocellular carcinoma20003212252372-s2.0-0034090264CrippinJ. S.McCashlandT.TerraultN.SheinerP.CharltonM. R.A pilot study of the tolerability and efficacy of antiviral therapy in hepatitis C virus-infected patients awaiting liver transplantation2002843503552-s2.0-003624549310.1053/jlts.2002.31748OkudaM.LiK.BeardM. R.ShowalterL. A.ScholleF.LemonS. M.WeinmanS. A.Mitochondrial injury, oxidative stress, and antioxidant gene expression are induced by hepatitis C virus core protein200212223663752-s2.0-0036165333YaoF.TerraultN.Hepatitis C and hepatocellular carcinoma2001264734832-s2.0-0035748137HeintgesT.WandsJ. R.Hepatitis C virus: epidemiology and transmission19972635215262-s2.0-0030752667BrechotC.NalpasB.FeitelsonM. A.Interactions between alcohol and hepatitis viruses in the liver19961622732872-s2.0-0029889544PetersM. G.TerraultN. A.Alcohol use and hepatitis C2002365S220S2252-s2.0-003682948710.1053/jhep.2002.36811DonatoF.GelattiU.LiminaR. M.FattovichG.Southern Europe as an example of interaction between various environmental factors: a systematic review of the epidemiologic evidence20062527375637702-s2.0-3374557082210.1038/sj.onc.1209557HassanM. M.HwangL. Y.HattenC. J.SwaimM.LiD.AbbruzzeseJ. L.BeasleyP.PattY. Z.Risk factors for hepatocellular carcinoma: synergism of alcohol with viral hepatitis and diabetes mellitus2002365120612132-s2.0-003682955610.1053/jhep.2002.36780YuanJ. M.GovindarajanS.ArakawaK.YuM. C.Synergism of alcohol, diabetes, and viral hepatitis on the risk of hepatocellular carcinoma in blacks and whites in the U.S20041015100910172-s2.0-414306590710.1002/cncr.20427LaiM. S.HsiehM. S.ChiuY. H.ChenT. H. H.Type 2 diabetes and hepatocellular carcinoma: a cohort study in high prevalence area of hepatitis virus infection2006436129513022-s2.0-3374555652010.1002/hep.21208MoriyaK.NakagawaK.SantaT.ShintaniY.FujieH.MiyoshiH.TsutsumiT.MiyazawaT.IshibashiK.HorieT.ImaiK.TodorokiT.KimuraS.KoikeK.Oxidative stress in the absence of inflammation in a mouse model for hepatitis C virus-associated hepatocarcinogenesis20016111436543702-s2.0-0035360859KorenagaM.WangT.LiY.ShowalterL. A.ChanT.SunJ.WeinmanS. A.Hepatitis C virus core protein inhibits mitochondrial electron transport and increases reactive oxygen species (ROS) production20052804537481374882-s2.0-2784460426910.1074/jbc.M506412200PerlemuterG.SabileA.LetteronP.VonaG.TopilcoA.ChrétienY.KoikeK.PessayreD.ChapmanJ.BarbaG.BréchotC.Hepatitis C virus core protein inhibits microsomal triglyceride transfer protein activity and very low density lipoprotein secretion: a model of viral-related steatosis20021621851942-s2.0-1824436586610.1096/fj.01-0396comKawaguchiT.YoshidaT.HaradaM.HisamotoT.NagaoY.IdeT.TaniguchiE.KumemuraH.HanadaS.MaeyamaM.BabaS.KogaH.KumashiroR.UenoT.OgataH.YoshimuraA.SataM.Hepatitis C virus down-regulates insulin receptor substrates 1 and 2 through up-regulation of suppressor of cytokine signaling 320041655149915082-s2.0-7244243810BanerjeeS.SaitoK.Ait-GoughoulteM.MeyerK.RayR. B.RayR.Hepatitis C virus core protein upregulates serine phosphorylation of insulin receptor substrate-1 and impairs the downstream Akt/protein kinase B signaling pathway for insulin resistance2008826260626122-s2.0-4014908926210.1128/JVI.01672-07KoikeK.TsutsumiT.MiyoshiH.ShinzawaS.ShintaniY.FujieH.YotsuyanagiH.MoriyaK.Molecular basis for the synergy between alcohol and hepatitis C virus in hepatocarcinogenesis200823supplement 1S87S912-s2.0-4074915832610.1111/j.1440-1746.2007.05292.xSzaboG.DolganiucA.MandrekarP.Pattern recognition receptors: a contemporary view on liver diseases20064422872982-s2.0-3374704082510.1002/hep.21308TestroA. G.VisvanathanK.Toll-like receptors and their role in gastrointestinal disease20092469439542-s2.0-6654909305310.1111/j.1440-1746.2009.05854.xMachidaK.TsukamotoH.MkrtchyanH.DuanL.DynnykA.LiuH. M.AsahinaK.GovindarajanS.RayR.JamesJ. H.SekiE.DeshaiesR.MiyakeK.LaiM. M. C.Toll-like receptor 4 mediates synergism between alcohol and HCV in hepatic oncogenesis involving stem cell marker Nanog20091065154815532-s2.0-6084910214910.1073/pnas.0807390106GaleM. J.Jr.KorthM. J.TangN. M.TanS. L.HopkinsD. A.DeverT. E.PolyakS. J.GretchD. R.KatzeM. G.Evidence that hepatitis C virus resistance to interferon is mediated through repression of the PKR protein kinase by the nonstructural 5A protein199723022172272-s2.0-034392435710.1006/viro.1997.8493KatoN.LanK. H.Ono-NitaS. K.ShiratoriY.OmataM.Hepatitis C virus nonstructural region 5A protein is a potent transcriptional activator19977111885688592-s2.0-0030879817BarbaG.HarperF.HaradaT.KoharaM.GoulinetS.MatsuuraY.EderG.SchaffZS.ChapmanM. J.MiyamuraT.BréchotC.Hepatitis C virus core protein shows a cytoplasmic localization and associates to cellular lipid storage droplets1997944120012052-s2.0-1114435727410.1073/pnas.94.4.1200MandrekarP.BalaS.CatalanoD.KodysK.SzaboG.The opposite effects of acute and chronic alcohol on lipopolysaccharide- induced inflammation are linked to IRAK-M in human monocytes20091832132013272-s2.0-7024909117610.4049/jimmunol.0803206MiuraK.KodamaY.InokuchiS.SchnablB.AoyamaT.OhnishiH.OlefskyJ. M.BrennerD. A.SekiE.Toll-like receptor 9 promotes steatohepatitis by induction of interleukin-1β in mice201013913233342-s2.0-7795389162710.1053/j.gastro.2010.03.052SekiE.De MinicisS.ÖsterreicherC. H.KluweJ.OsawaY.BrennerD. A.SchwabeR. F.TLR4 enhances TGF-β signaling and hepatic fibrosis20071311132413322-s2.0-3594895895510.1038/nm1663OshitaM.HayashiN.KasaharaA.HagiwaraH.MitaE.NaitoM.KatayamaK.FusamotoH.KamadaT.Increased serum hepatitis C virus RNA levels among alcoholic patients with chronic hepatitis C1994205111511202-s2.0-002803000810.1016/0270-9139(94)90745-5ShermanK. E.O'BrienJ.GutierrezA. G.HarrisonS.UrdeaM.NeuwaldP.WilberJ.Quantitative evaluation of hepatitis C virus RNA in patients with concurrent human immunodeficiency virus infections19933110267926822-s2.0-0027359543ParonettoF.Immunologic reactions in alcoholic liver disease19931321831952-s2.0-0027164362McClainC. J.CohenD. A.Increased tumor necrosis factor production by monocytes in alcoholic hepatitis1989933493512-s2.0-0024587645KhorutsA.StahnkeL.McClainC. J.LoganG.AllenJ. I.Circulating tumor necrosis factor, interleukin-1 and interleukin-6 concentrations in chronic alcoholic patients19911322672762-s2.0-002602663910.1016/0270-9139(91)92439-FBirdG. L. A.SheronN.GokaA. K. J.AlexanderG. J.WilliamsR. S.Increased plasma tumor necrosis factor in severe alcoholic hepatitis1990112129179202-s2.0-0025363385JiC.DengQ.KaplowitzN.Role of TNF-α in ethanol-induced hyperhomocysteinemia and murine alcoholic liver injury20044024424512-s2.0-404306439110.1002/hep.20309AdamsonG. M.BillingsR. E.Tumor necrosis factor induced oxidative stress in isolated mouse hepatocytes199229412232292-s2.0-002660271910.1016/0003-9861(92)90161-OBegA. A.BaltimoreD.An essential role for NF-κB in preventing TNF-α-induced cell death199627452887827842-s2.0-002997681710.1126/science.274.5288.782Van AntwerpD. J.MartinS. J.KafriT.GreenD. R.VermaI. M.Suppression of TNF-α-induced apoptosis by NF-κB199627452887877892-s2.0-002999260910.1126/science.274.5288.787WangC. Y.MayoM. W.BaldwinA. S.TNF- and cancer therapy-induced apoptosis: potentiation by inhibition of NF-κB199627452887847872-s2.0-002985838710.1126/science.274.5288.784WongG. H. W.ElwellJ. H.OberlyL. W.GoeddelD. V.Manganous superoxide dismutase is essential for cellular resistance to cytotoxicity of tumor necrosis factor19895859239312-s2.0-0024428198Fernandez-ChecaJ. C.Garcia-RuizC.OokhtensM.KaplowitzN.Impaired uptake of glutathione by hepatic mitochondria from chronic ethanol-fed rats. Tracer kinetic studies in vitro and in vivo and susceptibility to oxidant stress19918723974052-s2.0-0025979356MatsumotoM.HsiehT. Y.ZhuN.Van ArsdaleT.HwangS. B.JengK. S.GorbalenyaA. E.LoS. Y.OuJ. H.WareC. F.LaiM. M. C.Hepatitis C virus core protein interacts with the cytoplasmic tail of lymphotoxin-β receptor1997712130113092-s2.0-18844480671ZhuN.KhoshnanA.SchneiderR.MatsumotoM.DennertG.WareC.LaiM. M. C.Hepatitis C virus core protein binds to the cytoplasmic domain of tumor necrosis factor (TNF) receptor 1 and enhances TNF-induced apoptosis1998725369136972-s2.0-0031957325RuggieriA.HaradaT.MatsuuraY.MiyamuraT.Sensitization to Fas-mediated apoptosis by hepatitis C virus core protein1997229168762-s2.0-003155080610.1006/viro.1996.8420RibeiroP. S.Cortez-PintoH.SoláS.CastroR. E.RamalhoR. M.BaptistaA.MouraM. C.CamiloM. E.RodriguesC. M. P.Hepatocyte apoptosis, expression of death receptors, and activation of NF-κB in the liver of nonalcoholic and alcoholic steatohepatitis patients2004999170817172-s2.0-454433515510.1111/j.1572-0241.2004.40009.xMoriyaK.FujieH.ShintaniY.YotsuyanagiH.TsutsumiT.IshibashiK.MatsuurasY.KimuraS.MiyamuraT.KoikeK.The core protein of hepatitis C virus induces hepatocellular carcinoma in transgenic mice199849106510672-s2.0-003171281310.1038/2053MachidaK.ChengK. T. H.PavioN.SungV. M. H.LaiM. M. C.Hepatitis C virus E2-CD81 interaction induces hypermutation of the immunoglobulin gene in B cells20057913807980892-s2.0-2074444850010.1128/JVI.79.13.8079-8089.2005TsutsumiT.SuzukiT.MoriyaK.YotsuyanagiH.ShintaniY.FujieH.MatsuuraY.KimuraS.KoikeK.MiyamuraT.Alteration of intrahepatic cytokine expression and AP-1 activation in transgenic mice expressing hepatitis C virus core protein200230424154242-s2.0-1224427679610.1006/viro.2002.1702MachidaK.ChengK. T. H.SungV. M. H.LevineA. M.FoungS.LaiM. M. C.Hepatitis C virus induces toll-like receptor 4 expression, leading to enhanced production of beta interferon and interleukin-620068028668742-s2.0-3034444447510.1128/JVI.80.2.866-874.2006SongM. J.KimK. H.YoonJ. M.KimJ. B.Activation of Toll-like receptor 4 is associated with insulin resistance in adipocytes200634637397452-s2.0-3374518711210.1016/j.bbrc.2006.05.170SchattenbergJ. M.WangY.SinghR.RigoliR. M.CzajaM. J.Hepatocyte CYP2E1 overexpression and steatohepatitis lead to impaired hepatic insulin signaling200528011988798942-s2.0-1544437534110.1074/jbc.M410310200BradfordB. U.KonoH.IsayamaF.KosykO.WheelerM. D.AkiyamaT. E.BleyeL.KrauszK. W.GonzalezF. J.KoopD. R.RusynI.Cytochrome P450 CYP2E1, but not nicotinamide adenine dinucleotide phosphate oxidase, is required for ethanol-induced oxidative DNA damage in rodent liver20054123363442-s2.0-1994443298710.1002/hep.20532CaoQ.MakK. M.LieberC. S.Cytochrome P4502E1 primes macrophages to increase TNF-α production in response to lipopolysaccharide20052891G95G1072-s2.0-2174443554910.1152/ajpgi.00383.2004LiuH.JonesB. E.BradhamC.CzajaM. J.Increased cytochrome P-450 2E1 expression sensitizes hepatocytes to c-Jun-mediated cell death from TNF-α20022822G257G2662-s2.0-0036086898LuY.CederbaumA. I.Enhancement by pyrazole of lipopolysaccharide-induced liver injury in mice: role of cytochrome P450 2E1 and 2A520064412632742-s2.0-3374592772410.1002/hep.21241LeeJ. Y.YeJ.GaoZ.YounH. S.LeeW. H.ZhaoL.SizemoreN.HwangD. H.Reciprocal modulation of toll-like receptor-4 signaling pathways involving MyD88 and phosphatidylinositol 3-kinase/AKT by saturated and polyunsaturated fatty acids20032783937041370512-s2.0-014184465310.1074/jbc.M305213200NanjiA. A.Role of different dietary fatty acids in the pathogenesis of experimental alcoholic liver disease200434121252-s2.0-1274426654310.1016/j.alcohol.2004.08.005HilbergF.AguzziA.HowellsN.WagnerE. F.c-Jun is essential for normal mouse development and hepatogenesis199336564421791812-s2.0-002724218010.1038/365179a0JohnsonR. S.Van LingenB.PapaioannouV. E.SpiegelmanB. M.A null mutation at the c-jun locus causes embryonic lethality and retarded cell growth in culture199377130913172-s2.0-0027219303BehrensA.SibiliaM.DavidJ. P.Möhle-SteinleinU.TroncheF.SchützG.WagnerE. F.Impaired postnatal hepatocyte proliferation and liver regeneration in mice lacking c-jun in the liver2002217178217902-s2.0-003700720810.1093/emboj/21.7.1782SakuraiT.MaedaS.ChangL.KarinM.Loss of hepatic NF-κB activity enhances chemical hepatocarcinogenesis through sustained c-Jun N-terminal kinase 1 activation20061032810544105512-s2.0-3374603257110.1073/pnas.0603499103EferlR.RicciR.KennerL.ZenzR.DavidJ. P.RathM.WagnerE. F.Liver tumor development: c-Jun antagonizes the proapoptotic activity of p53200311221811922-s2.0-003746245410.1016/S0092-8674(03)00042-4YeligarS. M.MachidaK.TsukamotoH.KalraV. K.Ethanol augments RANTES/CCL5 expression in rat liver sinusoidal endothelial cells and human endothelial cells via activation of NF-κB, HIF-1α, and AP-120091839596459762-s2.0-7795415728010.4049/jimmunol.0901564KatoN.YoshidaH.Kioko Ono-NitaS.KatoJ.GotoT.OtsukaM.LanK. H.MatsushimaK.ShiratoriY.OmataM.Activation of intracellular signaling by hepatitis B and C viruses: C- viral core is the most potent signal inducer20003224054122-s2.0-0033863120MaedaS.KarinM.Oncogene at last—c-Jun promotes liver cancer in mice2003321021042-s2.0-014224412110.1016/S1535-6108(03)00025-4ShrivastavaA.MannaS. K.RayR.AggarwalB. B.Ectopic expression of hepatitis C virus core protein differentially regulates nuclear transcription factors19987212972297282-s2.0-0031797725RoskamsT. A.TheiseN. D.BalabaudC.BhagatG.BhathalP. S.Bioulac-SageP.BruntE. M.CrawfordJ. M.CrosbyH. A.DesmetV.FinegoldM. J.GellerS. A.GouwA. S. H.HytiroglouP.KniselyA. S.KojiroM.LefkowitchJ. H.NakanumaY.OlynykJ. K.ParkY. N.PortmannB.SaxenaR.ScheuerP. J.StrainA. J.ThungS. N.WanlessI. R.WestA. B.Nomenclature of the finer branches of the biliary tree: canals, ductules, and ductular reactions in human livers2004396173917452-s2.0-254259553710.1002/hep.20130RoskamsT.Liver stem cells and their implication in hepatocellular and cholangiocarcinoma20062527381838222-s2.0-3374553355910.1038/sj.onc.1209558BurkeZ. D.ThowfeequS.PeranM.ToshD.Stem cells in the adult pancreas and liver200740421691782-s2.0-3424979959110.1042/BJ20070167HatchH. M.ZhengD.JorgensenM. L.PetersenB. E.SDF-1α/CXCR4: a mechanism for hepatic oval cell activation and bone marrow stem cell recruitment to the injured liver of rats2002443393512-s2.0-0036961904AlisonM. R.Liver stem cells: implications for hepatocarcinogenesis2005132532602-s2.0-2844444906510.1385/SCR:1:3:253ZenderL.SpectorM. S.XueW.FlemmingP.Cordon-CardoC.SilkeJ.FanS. T.LukJ. M.WiglerM.HannonG. J.MuD.LucitoR.PowersS.LoweS. W.Identification and validation of oncogenes in liver cancer using an integrative oncogenomic approach20061257125312672-s2.0-3374525310910.1016/j.cell.2006.05.030TangY.KitisinK.JogunooriW.LiC.DengC. X.MuellerS. C.RessomH. W.RashidA.HeA. R.MendelsonJ. S.JessupJ. M.ShettyK.ZasloffM.MishraB.ReddyE. P.JohnsonL.MishraL.Progenitor/stem cells give rise to liver cancer due to aberrant TGF-β and IL-6 signaling20081057244524502-s2.0-4064911694310.1073/pnas.0705395105CraigC. E. H.QuagliaA.SeldenC.LowdellM.HodgsonH.DhillonA. P.The histopathology of regeneration in massive hepatic necrosis200424149642-s2.0-194251927710.1055/s-2004-823101YangZ. F.HoD. W.NgM. N.LauC. K.YuW. C.NgaiP.ChuP. W. K.LamC. T.PoonR. T. P.FanS. T.Significance of CD90+ cancer stem cells in human liver cancer20081321531662-s2.0-3854913914010.1016/j.ccr.2008.01.013MaS.ChanK. W.HuL.LeeT. K.WoJ. Y.NgI. O.ZhengBO. J.GuanX. Y.Identification and characterization of tumorigenic liver cancer stem/progenitor cells20071327254225562-s2.0-3424998034310.1053/j.gastro.2007.04.025Valk-LingbeekM. E.BruggemanS. W. M.Van LohuizenM.Stem cells and cancer: the polycomb connection200411844094182-s2.0-464431920410.1016/j.cell.2004.08.005ChambersI.SmithA.Self-renewal of teratocarcinoma and embryonic stem cells20042343715071602-s2.0-604426915710.1038/sj.onc.1207930BeachyP. A.KarhadkarS. S.BermanD. M.Tissue repair and stem cell renewal in carcinogenesis200443270153243312-s2.0-924422117210.1038/nature03100RountreeC. B.SenadheeraS.MatoJ. M.CrooksG. M.LuS. C.Expansion of liver cancer stem cells during aging in methionine adenosyltransferase 1A-deficient mice2008474128812972-s2.0-4224909435010.1002/hep.22141MaS.LeeT. K.ZhengB. J.ChanK. W.GuanX. Y.CD133+ HCC cancer stem cells confer chemoresistance by preferential expression of the Akt/PKB survival pathway20082712174917582-s2.0-4074915074010.1038/sj.onc.1210811WurmbachE.ChenY. B.KhitrovG.ZhangW.RoayaieS.SchwartzM.FielI.ThungS.MazzaferroV.BruixJ.BottingerE.FriedmanS.WaxmanS.LlovetJ. M.Genome-wide molecular profiles of HCV-induced dysplasia and hepatocellular carcinoma20074549389472-s2.0-3424754934710.1002/hep.21622YeohG. C. T.ErnstM.Rose-JohnS.AkhurstB.PayneC.LongS.AlexanderW.CrokerB.GrailD.MatthewsV. B.Opposing roles of gp130-mediated STAT-3 and ERK-1/2 signaling in liver progenitor cell migration and proliferation20074524864942-s2.0-3384773862910.1002/hep.21535DandoJ. S.TavianM.CatelainC.PoiraultS.Bennaceur-GriscelliA.SaintenyF.VainchenkerW.PéaultB.LauretE.Notch/Delta4 interaction in human embryonic liver CD34CD38 cells: positive influence on BFU-E production and LTC-IC potential maintenance20052345505602-s2.0-1644437593110.1634/stemcells.2004-0205SicklickJ. K.LiY. X.JayaramanA.KannangaiR.QiYI.VivekanandanP.LudlowJ. W.OwzarK.ChenW.TorbensonM. S.DiehlA. M.Dysregulation of the Hedgehog pathway in human hepatocarcinogenesis20062747487572-s2.0-3364537150910.1093/carcin/bgi292SicklickJ. K.LiY. X.MelhemA.SchmelzerE.ZdanowiczM.HuangJ.CaballeroM.FairJ. H.LudlowJ. W.McClellandR. E.ReidL. M.DiehlA. M.Hedgehog signaling maintains resident hepatic progenitors throughout life20062905G859G8702-s2.0-3364582369910.1152/ajpgi.00456.2005KitisinK.GanesanN.TangY.JogunooriW.VolpeE. A.KimS. S.KaturiV.KallakuryB.PishvaianM.AlbaneseC.MendelsonJ.ZasloffM.RashidA.FishbeinT.EvansS. R. T.SidawyA.ReddyE. P.MishraB.JohnsonL. B.ShettyK.MishraL.Disruption of transforming growth factor-β signaling through β-spectrin ELF leads to hepatocellular cancer through cyclin D1 activation20072650710371102-s2.0-3584895165210.1038/sj.onc.1210513NguyenL. N.FuruyaM. H.WolfraimL. A.NguyenA. P.HoldrenM. S.CampbellJ. S.KnightB.YeohG. C. T.FaustoN.ParksW. T.Transforming growth factor-beta differentially regulates oval cell and hepatocyte proliferation200745131412-s2.0-3384645466710.1002/hep.21466HoJ. W. Y.PangR. W. C.LauC.SunC. K.WanC. Y.SheungT. F.PoonR. T. P.Significance of circulating endothelial progenitor cells in hepatocellular carcinoma20064448368432-s2.0-3375062110310.1002/hep.21353SinghS. K.HawkinsC.ClarkeI. D.SquireJ. A.BayaniJ.HideT.HenkelmanR. M.CusimanoM. D.DirksP. B.Identification of human brain tumour initiating cells200443270153964012-s2.0-924424157610.1038/nature03128ShmelkovS. V.ClairR. ST.LydenD.RafiiS.AC133/CD133/Prominin-120053747157192-s2.0-1344427889610.1016/j.biocel.2004.08.010Al-HajjM.WichaM. S.Benito-HernandezA.MorrisonS. J.ClarkeM. F.Prospective identification of tumorigenic breast cancer cells20031007398339882-s2.0-003738820410.1073/pnas.0530291100Tirnitz-ParkerJ. E. E.TonkinJ. N.KnightB.OlynykJ. K.YeohG. C. T.Isolation, culture and immortalisation of hepatic oval cells from adult mice fed a choline-deficient, ethionine-supplemented diet20073912222622392-s2.0-3474891007710.1016/j.biocel.2007.06.008LibbrechtL.De VosR.CassimanD.DesmetV.AertsR.RoskamsT.Hepatic progenitor cells in hepatocellular adenomas20012511138813962-s2.0-003475863810.1097/00000478-200111000-00006LapidotT.SirardC.VormoorJ.MurdochB.HoangT.Caceres-CortesJ.MindenM.PatersonB.CaligiuriM. A.DickJ. E.A cell initiating human acute myeloid leukaemia after transplantation into SCID mice199436764646456482-s2.0-002809119410.1038/367645a0ChibaT.KitaK.ZhengY. W.YokosukaO.SaishoH.IwamaA.NakauchiH.TaniguchiH.Side population purified from hepatocellular carcinoma cells harbors cancer stem cell-like properties20064412402512-s2.0-3374592469610.1002/hep.21227MartinG. R.Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells19817812763476382-s2.0-0001007610LohY. H.WuQ.ChewJ. L.VegaV. B.ZhangW.ChenXI.BourqueG.GeorgeJ.LeongB.LiuJ.WongK. Y.SungK. W.LeeC. W. H.ZhaoX. D.ChiuK. P.LipovichL.KuznetsovV. A.RobsonP.StantonL. W.WeiC. L.RuanY.LimB.NgH. H.The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells20063844314402-s2.0-3364538193610.1038/ng1760WangJ.RaoS.ChuJ.ShenX.LevasseurD. N.TheunissenT. W.OrkinS. H.A protein interaction network for pluripotency of embryonic stem cells200644471173643682-s2.0-3375109224610.1038/nature05284RaoS.OrkinS. H.Unraveling the transcriptional network controlling ES cell pluripotency200678, article 2302-s2.0-3384556204310.1186/gb-2006-7-8-230PanG.ThomsonJ. A.Nanog and transcriptional networks in embryonic stem cell pluripotency200717142492-s2.0-3384618475210.1038/sj.cr.7310125ChambersI.ColbyD.RobertsonM.NicholsJ.LeeS.TweedieS.SmithA.Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells200311356436552-s2.0-003814361110.1016/S0092-8674(03)00392-1Hoei-HansenC. E.AlmstrupK.NielsenJ. E.Brask SonneS.GraemN.SkakkebaekN. E.LeffersH.Rajpert-De MeytsE.Stem cell pluripotency factor NANOG is expressed in human fetal gonocytes, testicular carcinoma in situ and germ cell tumours200547148562-s2.0-2214444640910.1111/j.1365-2559.2005.02182.xHartA. H.HartleyL.ParkerK.IbrahimM.LooijengaL. H. J.PauchnikM.ChowC. W.RobbL.The pluripotency homeobox gene NANOG is expressed in human germ cell tumors200510410209220982-s2.0-2764444881710.1002/cncr.21435SantagataS.LigonK. L.HornickJ. L.Embryonic stem cell transcription factor signatures in the diagnosis of primary and metastatic germ cell tumors20073168368452-s2.0-3444730384710.1097/PAS.0b013e31802e708aEzehU. I.TurekP. J.Reijo PeraR. A.ClarkA. T.Human embryonic stem cell genes OCT4, NANOG, STELLAR, and GDF3 are expressed in both seminoma and breast carcinoma200510410225522652-s2.0-2764447369510.1002/cncr.21432GibbsC. P.KukekovV. G.ReithJ. D.TchigrinovaO.SuslovO. N.ScottE. W.GhivizzaniS. C.IgnatovaT. N.SteindlerD. A.Stem-like cells in bone sarcomas: implications for tumorigenesis20057119679762-s2.0-3364482546510.1593/neo.05394ZhangJ.WangX.ChenB.SuoG.ZhaoY.DuanZ.DaiJ.Expression of Nanog gene promotes NIH3T3 cell proliferation20053382109811022-s2.0-2774447516810.1016/j.bbrc.2005.10.071