Hepatocellular carcinoma (HCC), a major cause of cancer-related death in Southeast Asia, is frequently associated with hepatitis B virus (HBV) infection. HBV X protein (HBx), encoded by a viral non-structural gene, is a multifunctional regulator in HBV-associated tumor development. We investigated novel signaling pathways underlying HBx-induced liver tumorigenesis and found that the signaling pathway involving IκB kinase β (IKKβ), tuberous sclerosis complex 1 (TSC1), and mammalian target of rapamycin (mTOR) downstream effector S6 kinase (S6K1), was upregulated when HBx was overexpressed in hepatoma cells. HBx-induced S6K1 activation was reversed by IKKβ inhibitor Bay 11-7082 or silencing IKKβ expression using siRNA. HBx upregulated cell proliferation and vascular endothelial growth factor (VEGF) production, and these HBx-upregulated phenotypes were abolished by treatment with IKKβ inhibitor Bay 11-7082 or mTOR inhibitor rapamycin. The association of HBx-modulated IKKβ/mTOR/S6K1 signaling with liver tumorigenesis was verified in a HBx transgenic mouse model in which pIKKβ, pS6K1, and VEGF expression was found to be higher in cancerous than non-cancerous liver tissues. Furthermore, we also found that pIKKβ levels were strongly correlated with pTSC1 and pS6K1 levels in HBV-associated hepatoma tissue specimens taken from 95 patients, and that higher pIKKβ, pTSC1, and pS6K1 levels were correlated with a poor prognosis in these patients. Taken together, our findings demonstrate that HBx deregulates TSC1/mTOR signaling through IKKβ, which is crucially linked to HBV-associated HCC development.
S-Adenosylmethionine (SAMe), the principal biological methyl donor, is synthesized from methionine and ATP in a reaction catalyzed by methionine adenosyltransferase (MAT). In mammals, two genes (MAT1A and MAT2A), encode for two homologous MAT catalytic subunits, while a third gene MAT2β, encodes for the β-subunit that regulates MAT2A-encoded isoenzyme. Normal liver expresses MAT1A, whereas extrahepatic tissues express MAT2A. MAT2A and MAT2β are induced in human hepatocellular carcinoma (HCC), which facilitate cancer cell growth. Patients with cirrhosis of various etiologies, including alcohol, have decreased hepatic MAT activity and SAMe biosynthesis. Consequences of hepatic SAMe deficiency as illustrated by the Mat1a knock-out mouse model include increased susceptibility to steatosis and oxidative liver injury, spontaneous development of steatohepatitis and HCC. Predisposition to HCC can be partly explained by the effect of SAMe on growth. Thus, SAMe inhibits the mitogenic effect of growth factors such as hepatocyte growth factor and, following partial hepatectomy, a fall in SAMe level is required for the liver to regenerate. During liver regeneration, the fall in hepatic SAMe is transient. If the fall were to persist, it would favor a proliferative phenotype and, ultimately, development of HCC. Not only does SAMe control liver growth, it also regulates apoptosis. Interestingly, SAMe is anti-apoptotic in normal hepatocytes but pro-apoptotic in liver cancer cells. In liver cancer cells but not in normal human hepatocytes, SAMe can selectively induce Bcl-xS, an alternatively spliced isoform of Bcl-xL that promotes apoptosis. This should make SAMe an attractive agent for both chemoprevention and treatment of HCC.
apoptosis; cell proliferation; hepatocellular carcinoma; methionine adenosyltransferase; S-adenosylmethionine
Hepatitis B virus (HBV) X protein (HBx) is considered to play a role in the development of hepatocellular carcinoma (HCC) during HBV infection. HCC was shown to be more prevalent in men than in women. Estrogen, which exerts its biological function through estrogen receptor (ER), can inhibit HBV replication. ERΔ5, an ERα variant lacking exon 5, was found to be preferentially expressed in patients with HCC compared with patients with normal livers. Here, we report the biological role of ERΔ5 and a novel link between HBx and ERα signaling in hepatoma cells. ERΔ5 interacts with ERα in vitro and in vivo and functions as a dominant negative receptor. Both ERα and ERΔ5 associate with HBx. HBx decreases ERα-dependent transcriptional activity, and HBx and ERΔ5 have additive effect on suppression of ERα transactivation. The HBx deletion mutant that lacks the ERα-binding site abolishes the HBx repression of ERα. HBx, ERα and histone deacetylase 1 (HDAC1) form a ternary complex. Trichostatin A, a specific inhibitor of HDAC enzyme, can restore the transcriptional activity of ERα inhibited by HBx. Our data suggest that HBx and ERΔ5 may play a negative role in ERα signaling and that ERα agonists may be developed for HCC therapy.
Hepatitis B virus (HBV) is a major etiologic agent of chronic liver disease (CLD) and hepatocellular carcinoma (HCC). HBV encoded X antigen, HBx, and pathways implicated in the self-renewal of stem cells contribute to HCC, but it is not clear whether HBx expression promotes “stemness.” Thus, experiments were designed to test the hypothesis that HBx triggers malignant transformation by promoting properties that are characteristic of cancer stem cells (CSCs). To test this hypothesis, HepG2 cells were stably transduced with HBx and then assayed for phenotypic and molecular characteristics of “stemness.” The relationship between HBx and “stemness”-associated markers was also evaluated by immunohistochemical staining of liver and tumor tissue sections from HBV infected patients. The results showed that Oct-4, Nanog, Klf4, β-catenin and EpCAM were activated by HBx in vitro and in vivo. EpCAM was detected in the nuclei of human HCC cells from infected patients. HBx promotes “stemness” by activating β-catenin and epigenetic up-regulation of miR-181, both of which target EpCAM. HBx expression was also associated with depressed levels of E-cadherin. Moreover, HBx stimulated cell migration, growth in soft agar, and spheroid formation. This work is the first to propose that HBV promotes “stemness” in the pathogenesis of HCC. HBx associated up-regulated expression of multiple “stemness” markers support the hypothesis that HBx contributes to hepatocarcinogenesis, at least in part, by promoting changes in gene expression that are characteristics of CSCs.
hepatocellular carcinoma; EpCAM; β-catenin; miR181; stemness
Hepatitis B virus (HBV) infection is a major risk for hepatocellular carcinoma (HCC), and it is a serious global health problem with two billion people exposed to it worldwide. HBx, an essential factor for viral replication and a putative oncoprotein encoded by the HBV genome, has been shown to promote oncogenic properties at multiple sites in HBV-infected liver cells. The expression level of HBx closely associates with the development and progression of HCC, therefore the mechanism(s) regulating the stability of HBx is important in oncogenesis of HBV-infected cells. We demonstrate that the X-linked tumor suppressor TSPX enhances the degradation of HBx through the ubiquitin-proteasome pathway. TSPX interacts with both HBx and a proteasome 19S lid subunit RPN3 via its C-terminal acidic tail. Most importantly, over-expression of RPN3 protects HBx from, and hence acts as a negative regulator for, proteasome-dependent degradation. TSPX abrogates the RPN3-depedent stabilization of HBx, suggesting that TSPX and RPN3 act competitively in regulation of HBx stability. Since mutation and/or epigenetic repression of X-located tumor suppressor gene(s) could significantly predispose males to human cancers, our data suggest that TSPX-induced HBx degradation could play key role(s) in hepatocarcinogenesis among HBV-infected HCC patients.
Hepatitis B virus X protein (HBx) plays an important role in the development of hepatocellular carcinoma (HCC). However, the mechanism remains unclear. Recently, we have reported that HBx promotes hepatoma cell migration through the upregulation of calpain small subunit 1 (Capn4). In addition, several reports have revealed that osteopontin (OPN) plays important roles in tumor cell migration. In this study, we investigated the signaling pathways involving the promotion of cell migration mediated by HBx. We report that HBx stimulates several factors in a network manner to promote hepatoma cell migration. We showed that HBx was able to upregulate the expression of osteopontin (OPN) through 5-lipoxygenase (5-LOX) in HepG2-X/H7402-X (stable HBx-transfected cells) cells. Furthermore, we identified that HBx could increase the expression of 5-LOX through nuclear factor-κB (NF-κB). We also found that OPN could upregulate Capn4 through NF-κB. Interestingly, we showed that Capn4 was able to upregulate OPN through NF-κB in a positive feedback manner, suggesting that the OPN and Capn4 proteins involving cell migration affect each other in a network through NF-κB. Importantly, NF-κB plays a crucial role in the regulation of 5-LOX, OPN and Capn4. Thus, we conclude that HBx drives multiple cross-talk cascade loops involving NF-κB, 5-LOX, OPN and Capn4 to promote cell migration. This finding provides new insight into the mechanism involving the promotion of cell migration by HBx.
Over 350 million people are chronically infected with hepatitis B virus (HBV), and a significant number of chronically infected individuals develop primary liver cancer. HBV encodes seven viral proteins, including the nonstructural X (HBx) protein. The results of studies with immortalized or transformed cells and with HBx-transgenic mice demonstrated that HBx can interact with mitochondria. However, no studies with normal hepatocytes have characterized the precise mitochondrial localization of HBx or the effect of HBx on mitochondrial physiology. We have used cultured primary rat hepatocytes as a model system to characterize the mitochondrial localization of HBx and the effect of HBx expression on mitochondrial physiology. We now show that a fraction of HBx colocalizes with density-gradient-purified mitochondria and associates with the outer mitochondrial membrane. We also demonstrate that HBx regulates mitochondrial membrane potential in hepatocytes and that this function of HBx varies depending on the status of NF-κB activity. In primary rat hepatocytes, HBx activation of NF-κB prevented mitochondrial membrane depolarization; however, when NF-κB activity was inhibited, HBx induced membrane depolarization through modulation of the mitochondrial permeability transition pore. Collectively, these results define potential pathways through which HBx may act in order to modulate mitochondrial physiology, thereby altering many cellular activities and ultimately contributing to the development of HBV-associated liver cancer.
Hepatitis B virus X protein (HBx) plays important roles in the development of hepatocellular carcinoma (HCC). MicroRNAs (miRNAs) contribute to cancer development by acting as oncogenes or tumor suppressors. Previously, we reported that HBx was able to promote the migration of hepatoma HepG2 cells. However, the regulation of miRNAs in the development of HBV-related HCC is poorly understood. In the present study, we reported that miR-29a was a novel regulator of migration of hepatoma cells mediated by HBx. Our data showed that the expression of miR-29a was dramatically increased in p21-HBx transgenic mice, HBx-transfected hepatoma HepG2-X (or H7402-X) cells and HepG2.2.15 cells that constitutively replicate HBV. However, our data showed that miR-29a was upregulated in 4 of the 11 clinical HCC samples. We found that the overexpression of miR-29a promoted the migration of HepG2 cells, while a specific miR-29a inhibitor could partially abolish the enhanced migration of HepG2-X cells. Moreover, we identified PTEN was one of the target genes of miR-29a in HepG2 cells. The deletion of the miR-29a-binding site was able to abolish the role of miR-29a in suppression of luciferase activity of the PTEN 3′UTR reporter. Meanwhile, the overexpression of PTEN was able to reverse the promoted migration of HepG2 cells mediated by miR-29a. Moreover, our data showed that the modulation of Akt phosphorylation, a downstream factor of PTEN, was involved in the cell migration enhanced by miR-29a, suggesting that miR-29a is responsible for the cell migration through its target gene PTEN. Thus, we conclude that miR-29a is involved in the regulation of migration of hepatoma cells mediated by HBx through PTEN in cell culture model.
Transcriptional activation of diverse cellular genes by the X protein (HBx) of hepatitis B virus (HBV) has been suggested as one of the mechanisms for HBV-associated hepatocellular carcinoma. However, such functions of HBx have been studied using transformed cells in culture and have not been examined in the normal adult hepatocytes, a natural host of HBV. Using an efficient hepatocyte-specific virus-based gene delivery system developed in our laboratory earlier, we studied the HBx action in vivo. We demonstrate that following virosome-mediated delivery of HBx DNA, a large population (>50%) of hepatocytes express the HBx protein in a dose-dependent manner, which induces a significant increase in the activity of extracellular signal-regulated kinases (ERKs) in the livers of HBx-transfected mice. Inhibition of HBx-induced ERK activation following intravenous administration of PD98059, a mitogen-activated protein kinase kinase kinase (MEK) inhibitor, confirmed the requirement for MEK in the activation of ERKs by HBx. Induction of ERK activity by HBx was sustained for up to 30 days. Interestingly, sustained activation of c-Jun N-terminal kinases (JNKs) for up to 30 days was also noted. Such constitutive ERK and JNK activation as a consequence of continued HBx expression also led to sustained stimulation of further downstream events, such as increased levels of c-Jun and c-Fos proteins along with the persistent induction of activator protein 1 binding activity. Taken together, our data suggest a critical role of these molecules in HBx-mediated cell transformation.
Hepatitis B virus (HBV) X protein (HBx) plays an essential role in viral replication and in the development of hepatocellular carcinoma. HBx has the ability to transactivate the expression of all HBV proteins, including the viral core protein HBc. Consistent with its regulatory role, HBx is relatively unstable and is present at low levels in the cell. We report here that the level of HBx was significantly reduced by the coexpression of HBc in cultured human hepatoma cells, whereas the level of HBx mRNA was unaffected. The repression of HBx by HBc was relieved by treating cells with the proteasome inhibitor MG132, indicating that HBc acts by stimulating the proteasome-mediated degradation of HBx. Moreover, the inhibitory effect of HBc was specific to HBx and did not affect other proteins, including p53, a known target of the proteasome. Although no direct physical interaction between HBc and HBx could be demonstrated, mutational analysis indicated that the C-terminal half of HBc is responsible for its inhibitory effect. These results suggest that HBc functions as a novel regulator of the HBV life cycle and of hepatocellular carcinogenesis through control of the HBx level via an inhibitory feedback type of mechanism.
S-adenosylmethionine (AdoMet, also known as SAM and SAMe) is the principal biological methyl donor synthesized in all mammalian cells but most abundantly in the liver. Biosynthesis of AdoMet requires the enzyme methionine adenosyltransferase (MAT). In mammals, two genes, MAT1A that is largely expressed by normal liver and MAT2A that is expressed by all extrahepatic tissues, encode MAT. Patients with chronic liver disease have reduced MAT activity and AdoMet levels. Mice lacking Mat1a have reduced hepatic AdoMet levels and develop oxidative stress, steatohepatitis, and hepatocellular carcinoma (HCC). In these mice, several signaling pathways are abnormal that can contribute to HCC formation. However, injury and HCC also occur if hepatic AdoMet level is excessive chronically. This can result from inactive mutation of the enzyme glycine N-methyltransferase (GNMT). Children with GNMT mutation have elevated liver transaminases, and Gnmt knockout mice develop liver injury, fibrosis, and HCC. Thus a normal hepatic AdoMet level is necessary to maintain liver health and prevent injury and HCC. AdoMet is effective in cholestasis of pregnancy, and its role in other human liver diseases remains to be better defined. In experimental models, it is effective as a chemopreventive agent in HCC and perhaps other forms of cancer as well.
Worldwide, there are over 350 million people who are chronically infected with the human hepatitis B virus (HBV); chronic HBV infections are associated with the development of hepatocellular carcinoma (HCC). The results of various studies suggest that the HBV X protein (HBx) has a role in the development of HBV-associated HCC. HBx can regulate numerous cellular signal transduction pathways, including those that modulate cell proliferation. Many previous studies that analyzed the impact of HBx on cell proliferation pathways were conducted using established or immortalized cell lines, and when HBx was expressed in the absence of HBV replication, and the precise effect of HBx on these pathways has often differed depending on experimental conditions. We have studied the effect of HBx on cell proliferation in cultured primary rat hepatocytes, a biologically relevant system. We demonstrate that HBx, both by itself and in the context of HBV replication, affected the levels and activities of various cell cycle-regulatory proteins to induce normally quiescent hepatocytes to enter the G1 phase of the cell cycle but not to proceed to S phase. We linked HBx regulation of cell proliferation to cytosolic calcium signaling and HBx stimulation of HBV replication. Cumulatively, our studies suggest that HBx induces normally quiescent hepatocytes to enter the G1 phase of the cell cycle and that this calcium-dependent HBx activity is required for HBV replication. These studies identify an essential function of HBx during HBV replication and a mechanism that may connect HBV infections to the development of HCC.
AIM: To investigate the hepatitis B virus (HBV) x gene (HBx) state in the tissues of HBV-related hepatocellular carcinoma (HCC) in Chinese patients and whether there were particular HBx mutations.
METHODS: HBx gene was amplified and direct sequencing was used in genomic DNA samples from 20 HCC and corresponding non-cancerous liver tissues from HBsAg-positive patients. HBV DNA integration and HBx deleted mutation were validated in 45 HCC patients at different stages by Southern blot analysis and polymerase chain reaction methods.
RESULTS: The frequencies of HBx point mutations were significantly lower in HCC than their corresponding non-cancerous liver tissues (11/19 vs 18/19, P = 0.019). In contrast, deletions in HBx gene were significantly higher in HCC than their non-cancerous liver tissues (16/19 vs 4/19, P < 0.001). The deletion of HBx COOH-terminal was detected in 14 HCC tissues. A specific integration of HBx at 17p13 locus was also found in 8 of 16 HCC, and all of them also exhibited full-length HBx deletions. Integrated or integrated coexistence with replicated pattern was obtained in 45.5% (20/45) - 56.8% (25/45) tumors and 40.9% (18/45) - 52.3% (23/45) non-tumor tissues.
CONCLUSION: HBx deletion, especially the COOH-terminal deletion of HBx is a frequent event in HBV-associated HCC tissues in China. HBV integration had also taken place in partial HCC tissues. This supporting the hypothesis that deletion and probably integrated forms of the HBx gene may be implicated in liver carcinogenesis.
Hepatitis B virus; X gene; Hepatocellular carcinoma; COOH-terminal deletion mutation; Integration
The hepatitis B virus (HBV) PreS mutations C1653T, T1753V, and A1762T/G1764A were reported as a strong risk factor of hepatocellular carcinoma (HCC) in a meta-analysis. HBV core promoter overlaps partially with HBx coding sequence, so the nucleotide 1762 and 1764 mutations induce HBV X protein (HBx) 130 and 131 substitutions. We sought to elucidate the impact of HBx mutations on HCC development. Chronically HBV-infected patients were enrolled in this study: 42 chronic hepatitis B (CHB) patients, 23 liver cirrhosis (LC) patients, and 31 HCC patients. Direct sequencing showed HBx131, HBx130, HBx5, HBx94, and HBx38 amino acid mutations were common in HCC patients. Of various mutations, HBx130+HBx131 (double) mutations and HBx5+HBx130+HBx131 (triple) mutations were significantly high in HCC patients. Double and triple mutations increased the risk for HCC by 3.75-fold (95% confidence interval [CI] = 1.101 to 12.768, P = 0.033) and 5.34-fold (95% CI = 1.65 to 17.309, P = 0.005), respectively, when HCC patients were compared to CHB patients. Functionally, there were significantly higher levels of NF-κB activity in cells with the HBx5 mutant and with the double mutants than that of wild-type cells and the triple-mutant cells. The triple mutation did not increase NF-κB activity. Other regulatory pathways seem to exist for NF-κB activation. In conclusion, a specific HBx mutation may contribute to HCC development by activating NF-κB activity. The HBx5 mutation in genotype C2 HBV appears to be a risk factor for the development of HCC and may be used to predict the clinical outcomes of patients with chronic HBV infection.
Hepatitis B virus (HBV) infection accounts for over a half of cases of hepatocellular carcinoma (HCC), the most frequent malignant tumor of the liver. HBV-encoded X (HBx) plays critical roles in HBV-associated hepatocarcinogenesis. However, it is unclear whether and how HBx regulates the expression of epidermal growth factor receptor (EGFR), an important gene for cell growth. Therefore, the study aimed to investigate the association between HBx and EGFR expression. In this study, we found that HBx upregulates miR-7 expression to target 3′UTR of EGFR mRNA, which in turn results in the reduction of EGFR protein expression in HCC cells. HBx-mediated EGFR suppression renders HCC cells a slow-growth behavior. Deprivation of HBx or miR-7 expression or restoration of EGFR expression can increase the growth rate of HCC cells. Our data showed the miR-7-dependent EGFR suppression by HBx, supporting an inhibitory role of HBx in the cell growth of HCC. These findings not only identify miR-7 as a novel regulatory target of HBx, but also suggest HBx-miR-7-EGFR as a critical signaling in controlling the growth rate of HCC cells.
The hepatitis B virus (HBV) X protein (HBx) is thought to play a key role in HBV replication and the development of liver cancer. It became apparent that HBx induces mitochondrial clustering at the nuclear periphery, but the molecular basis for mitochondrial clustering is not understood. Since mitochondria move along the cytoskeleton as a cargo of motor proteins, we hypothesized that mitochondrial clustering induced by HBx occurs by an altered intracellular motility. Here, we demonstrated that the treatment of HBx-expressing cells with a microtubule-disrupting drug (nocodazole) abrogated mitochondrial clustering, while the removal of nocodazole restored clustering within 30 to 60 min, indicating that mitochondrial transport is occurring in a microtubule-dependent manner. The addition of a cytochalasin D-disrupting actin filament, however, did not measurably affect mitochondrial clustering. Mitochondrial clustering was further studied by observations of HBV-related hepatoma cells and HBV-replicating cells. Importantly, the abrogation of the dynein activity in HBx-expressing cells by microinjection of a neutralizing anti-dynein intermediate-chain antibody, dynamitin overexpression, or the addition of a dynein ATPase inhibitor significantly suppressed the mitochondrial clustering. In addition, HBx induced the activation of the p38 mitogen-activated protein kinase (MAPK) and inhibition of the p38 kinase activity by SB203580-attenuated HBx-induced mitochondrial clustering. Taken together, HBx activation of the p38 MAPK contributed to the increase in the microtubule-dependent dynein activity. The data suggest that HBx plays a novel regulatory role in subcellular transport systems, perhaps facilitating the process of maturation and/or assembly of progeny particles during HBV replication. Furthermore, mitochondrion aggregation induced by HBx may represent a cellular process that underlies disease progression during chronic viral infection.
Hepatitis B Virus (HBV) DNA integration and HBV X (HBx) deletion mutation occurs in HBV-positive liver cancer patients, and C-terminal deletion in HBx gene mutants are highly associated with hepatocarcinogenesis. Our previous study found that the HBx-d382 deletion mutant (deleted at nt 382–400) can down-regulate miR-338-3p expression in HBx-expressing cells. The aim of the present study is to examine the role of miR-338-3p in the HBx-d382-mediated liver-cell proliferation.
We established HBx-expressing LO2 cells by Lipofectamine 2000 transfection. A miR-338-3p mimics or inhibitor was transfected into LO2/HBx-d382 and LO2/HBx cells using miR-NC as a control miRNA. In silico analysis of potential miR-338-3p targets revealed that miR-338-3p could target the cell cycle regulatory protein CyclinD1. To confirm that CyclinD1 is negatively regulated by miR-338-3p, we constructed luciferase reporters with wild-type and mutated CyclinD1-3′UTR target sites for miR-338-3p binding. We examined the CyclinD1 expression by real-time PCR and western blot, and proliferation activity by flow cytometric cell cycle analysis, Edu incorporation, and soft agar colony.
HBx-d382 exhibited enhanced proliferation and CyclinD1 expression in LO2 cells. miR-338-3p expression inhibited cell proliferation in LO2/HBx-d382 cells (and LO2/HBx cells), and also negatively regulated CyclinD1 protein expression. Of the two putative miR-338-3p binding sites in the CyclinD1-3′UTR region, the effect of miR-338-3p on the second binding site (nt 2397–2403) was required for the inhibition.
miR-338-3p can directly regulate CyclinD1 expression through binding to the CyclinD1-3′UTR region, mainly at nt 2397–2403. Down-regulation of miR-338-3p expression is required for liver cell proliferation in both LO2/HBx and LO2/HBx-d382 mutant cells, although the effect is more pronounced in LO2/HBx-d382 cells. Our study elucidated a novel mechanism, from a new miRNA-regulation perspective, underlying the propensity of HBx deletion mutants to induce hepatocarcinogenesis at a faster rate than HBx.
Methionine adenosyltransferase (MAT) is an essential enzyme that catalyzes the biosynthesis of S-adenosylmethionine. Hepatic MAT activity falls in chronic liver diseases, and mice lacking Mat1a are predisposed to liver injury and develop hepatocellular carcinoma (HCC) spontaneously by 18 months. The current work examined the hypothesis that liver cancer stem cells contribute to HCC in this model. Livers from 6- and 18-month-old Mat1a-knockout (KO) mice and their wild-type (WT) littermates were fractionated and isolated by flow cytometry. CD45− nonparenchymal (NP) cells were cultured using liver stem cell conditions. Cells were analyzed by real-time PCR and fluorescent immunohistochemistry (FIHC). Tumor formation was assessed by injecting 1 × 106 CD133+CD49f+ cells intraperitoneally into immune-deficient mice. The proportion of CD49f+ and CD133+ cells in the CD45-NP fraction increased 4.5- to 5.5-fold from 6 to 18 months in KO mice but not in their WT littermates. Compared to CD49f− cells from old KO mice, CD49f+ cells from the same animals had a markedly increased expression of several oncogenes. CD133+ cells with CD49f coexpression were selected in vitro and exhibited rapid growth, with the expression of biliary cytokeratins, α-fetoprotein, and c-Met by FIHC. Clonal expansion of single CD133+CD49f+ cells revealed maintenance of bipotency. After CD133+CD49f+ cells were injected into immune-deficient mice, 3 of the 8 mice developed tumors of liver epithelial cells after 6–8 weeks.
Matla−/− mice have expansion of liver stem cells as they age. These cells have increased expression of several oncogenes and are tumorigenic in vivo. This is the first demonstration of adult liver stem cells possessing tumorigenic potential without the use of a carcinogen or manipulation of tumor-suppressor or oncogene expression.
Methionine adenosyltransferase (MAT) is an essential enzyme required for S-adenosylmethionine biosynthesis. Hepatic MAT activity falls during chronic liver injury, and mice lacking Mat1a develop spontaneous hepatocellular carcinoma by 18 months. We have previously demonstrated that CD133+CD45− oval cells isolated from 16-month-old Mat1a−/− mice represent a liver cancer stem cell population. The transforming growth factor β (TGF-β) pathway constitutes a central signaling network in proliferation, apoptosis, and tumorigenesis. In this study, we tested the response of tumorigenic liver stem cells to TGF-β. CD133+CD45− oval cells were isolated from premalignant 16-month-old Mat1a−/− mice by flow cytometry and expanded as five clone lines derived from a single cell. All clone lines demonstrated expression of both hepatocyte and cholangiocyte markers and maintained a small population (0.5% to 2%) of CD133+ cells in vitro, and three of five clone lines produced tumors. Although TGF-β1 inhibited cell growth equally in CD133− and CD133+ cells from each clone line, the CD133+ population demonstrated significant resistance to TGF-β–induced apoptosis compared with CD133+ cells. Furthermore, CD133+ cells demonstrated a substantial increase in mitogen-activated protein kinase (MAPK) pathway activation, as demonstrated by phosphorylated extra-cellular signal-regulated kinase levels before and after TGF-β stimulation. MAPK inhibition using mitogen-activated protein kinase kinase 1 (MEK1) inhibitor PD98059 led to a significant increase in TGF-β–induced apoptosis in CD133+ cells. Conversely, a constitutively active form of MEK1 blocked the apoptotic effects of TGF-β in CD133− cells.
CD133+ liver cancer stem cells exhibit relative resistance to TGF-β–induced apoptosis. One mechanism of resistance to TGF-β–induced apoptosis in CD133+ cancer stem cells is an activated mitogen-activated protein kinase/extracellular signal-regulated kinase pathway.
Hepatitis B virus X protein (HBx) is recognized as an oncogene in hepatocellular carcinoma (HCC). HBx regulates microRNA expression, including down-regulating miR-338-3p in LO2 cells. Here, we investigated miR-338-3p function in HBx-mediated hepatocarcinogenesis. In 23 HBV-infected HCC clinical patient tumor and adjacent non-tumor control tissues, 17 and 19 tumors expressed HBx mRNA and protein, respectively. When considered as a group, HBV-infected HCC tumors had lower miR-338-3p expression than controls; however, miR-338-3p was only significantly down-regulated in HBx-positive tumors, indicating that HBx inversely correlated with miR-338-3p. Functional characterization of miR-338-3p indicated that miR-338-3p mimics inhibited cell proliferation by inducing cell cycle arrest at the G1/S phase as assessed by EdU and cell cycle assays in HBx-expressing LO2 cells. CyclinD1, containing two putative miR-338-3p targets, was confirmed as a direct target using 3′-UTR luciferase reporter assays from cells transfected with mutated binding sites. Mutating the 2397–2403 nt binding site conferred the greatest resistance to miR-338-3p suppression of CyclinD1, indicating that miR-338-3p suppresses CyclinD1 at this site. Overall, this study demonstrates that miR-338-3p inhibits proliferation by regulating CyclinD1, and HBx down-regulates miR-338-3p in HCC. This newly identified miR-338-3p/CyclinD1 interaction provides novel insights into HBx-mediated hepatocarcinogenesis and may facilitate therapeutic development against HCC.
hepatocellular carcinoma; hepatitis B virus X protein; miR-338-3p; CyclinD1; cell proliferation
Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide. Hepatitis B virus X protein (HBx) contributes to the development of HCC, whereas HBx with COOH-terminal deletion is a frequent event in the HCC tissues. Previously, we identified a natural mutant of HBx-truncated 27 amino acids at the COOH-terminal (termed HBxΔ127), which strongly enhanced cell growth. In the present study, we focused on investigating the mechanism. Accordingly, fatty acid synthase (FAS) plays a crucial role in cancer cell survival and proliferation; thus, we examined the signaling pathways involving FAS. Our data showed that HBxΔ127 strongly increased the transcriptional activities of FAS in human hepatoma HepG2 and H7402 cells. Moreover, we found that 5-lipoxygenase (5-LOX) was responsible for the up-regulation of FAS by using MK886 (an inhibitor of 5-LOX) and 5-LOX small interfering RNA. We observed that HBxΔ127 could upregulate 5-LOX through phosphorylated extracellular signal-regulated protein kinases 1/2 and thus resulted in the increase of released leukotriene B4 (LTB4, a metabolite of 5-LOX) by ELISA. The additional LTB4 could upregulate the expression of FAS in the cells as well. Interestingly, we found that FAS was able to upregulate the expression of 5-LOX in a feedback manner by using cerulenin (an inhibitor of FAS). Collectively, HBxΔ127 promotes cell growth through a positive feedback loop involving 5-LOX and FAS, in which released LTB4 is involved in the up-regulation of FAS. Thus, our finding provides a new insight into the mechanism involving the promotion of cell growth mediated by HBxΔ127.
Loss of E-cadherin is associated with acquisition of metastatic capacity. Numerous studies suggest histone deacetylation and/or hypermethylation of CpG islands in E-cadherin gene (CDH1) are major mechanisms responsible for E-cadherin silencing in different tumors and cancer cell lines. The Hepatitis B virus (HBV) encoded X antigen, HBx, contributes importantly to the development of hepatocellular carcinoma (HCC) using multiple mechanisms. Experiments were designed to test if in addition to CDH1 hypermethylation HBx promotes epigenetic modulation of E-cadherin transcriptional activity through histone deacetylation and miR-373. The relationships between HBx, E-cadherin, mSin3A, Snail-1 and miR-373 were evaluated in HBx expressing (HepG2X) and control (HepG2CAT) cells by western blotting, immunoprecipitation, chromatin immunoprecipitation as well as by immunohistochemical staining of liver and tumor tissue sections from HBV infected patients. In HepG2X cells, decreased levels of E-cadherin and elevated levels of mSin3A and Snail-1 were detected. Reciprocal immunoprecipitation with anti-HBx and anti-mSin3A demonstrated mutual binding. Further, HBx-mSin3A co-localization was detected by immunofluorescent staining. HBx down-regulated E-cadherin expression by the recruitment of the mSin3A/HDAC complex to the Snail-binding sites in human CDH1. Histone deacetylation inhibition by Trichostatin A treatment restored E-cadherin expression. Mir-373, a positive regulator of E-cadherin expression, was down-regulated by HBx in HepG2X cells and tissue sections from HBV infected patients. Thus, histone deacetylation of CDH1 and down-regulation of miR-373, together with the previously demonstrated hyper-methylation of CDH1 by HBx, may be important for the understanding of HBV-related carcinogenesis.
Hepatocellular carcinoma; mSin3A; Snail-1; HDAC; miR-373
To understand the molecular mechanisms of caveolin-1 downregulation by hepatitis B virus X protein (HBx).
The DNA methylation status of the caveolin-1 promoter was examined by nested methylation-specific PCR of 33 hepatitis B virus (HBV)-infected hepatocellular carcinoma (HCC) samples. The SMMC-7721 hepatoma cell line was transfected with a recombinant HBx adenoviral vector, and the effects of HBx protein on caveolin-1 expression and promoter methylation were examined and confirmed by sequencing. A reporter gene containing the caveolin-1 promoter region was constructed, and the effects of HBx on the transcriptional activity of the promoter were also studied.
Methylation of the caveolin-1 promoter was detected in 84.8% (28/33) of HBV-infected HCC samples. Expression of caveolin-1 was significantly downregulated (P = 0.022), and multiple CpG sites in the promoter region of caveolin-1 were methylated in SMMC-7721 cells after HBx transfection. Transfected HBx significantly suppressed caveolin-1 promoter activity (P = 0.001).
HBx protein induces methylation of the caveolin-1 promoter region and suppresses its expression.
Hepatitis B virus X protein; Hepatocellular carcinoma; Caveolin-1; Methylation
TGF-beta 1 has been implicated in the pathogenesis of liver disease. The high frequency of detection of the hepatitis B virus X (HBx) antigen in liver cells from patients with chronic hepatitis, cirrhosis, and liver cancer suggested that expression of HBx and TGF-beta 1 may be associated. To test this possibility, we examined the expression of TGF-beta 1 in the liver of transgenic mice expressing the HBx gene. We show that the patterns of expression of TGF-beta 1 and Hbx protein are similar in these mice and that HBx activates transcription of the TGF-beta 1 gene in transfected hepatoma cells. The cis-acting element within the TGF-beta 1 gene that is responsive to regulation by Hbx is the binding site for the Egr family of transcription factors. We further show that the Egr-1 protein associates with the HBx protein, allowing HBx to participate in the transcriptional regulation of immediate-early genes. Our results suggest that expression of Hbx might induce expression of TGF-beta 1 in the early stages of infection and raise the possibility that TGF-beta 1 may play a role in hepatitis B virus pathogenesis.
The biological pathways and functional properties by which misexpressed microRNAs (miRNAs) contribute to liver carcinogenesis have been intensively investigated. However, little is known about the upstream mechanisms that deregulate miRNA expressions in this process. In hepatocellular carcinoma (HCC), hepatitis B virus (HBV) X protein (HBx), a transcriptional trans-activator, is frequently expressed in truncated form without carboxyl-terminus but its role in miRNA expression and HCC development is unclear.
Human non-tumorigenic hepatocytes were infected with lentivirus-expressing full-length and carboxyl-terminal truncated HBx (Ct-HBx) for cell growth assay and miRNA profiling. Chromatin immunoprecipitation microarray was performed to identify the miRNA promoters directly associated with HBx. Direct transcriptional control was verified by luciferase reporter assay. The differential miRNA expressions were further validated in a cohort of HBV-associated HCC tissues using real-time PCR.
Hepatocytes expressing Ct-HBx grew significantly faster than the full-length HBx counterparts. Ct-HBx decreased while full-length HBx increased the expression of a set of miRNAs with growth-suppressive functions. Interestingly, Ct-HBx bound to and inhibited the transcriptional activity of some of these miRNA promoters. Notably, some of the examined repressed-miRNAs (miR-26a, -29c, -146a and -190) were also significantly down-regulated in a subset of HCC tissues with carboxyl-terminal HBx truncation compared to their matching non-tumor tissues, highlighting the clinical relevance of our data.
Our results suggest that Ct-HBx directly regulates miRNA transcription and in turn promotes hepatocellular proliferation, thus revealing a viral contribution of miRNA deregulation during hepatocarcinogenesis.