TMEM43 promotes the development of hepatocellular carcinoma by activating VDAC1 through USP7 deubiquitination.

Background: Transmembrane protein 43 (TMEM43), a member of the TMEM subfamily, is encoded by a highly conserved gene and widely expressed in most species from bacteria to humans. In previous studies, TMEM43 has been found to play an important role in a variety of tumors. However, the role of TMEM43 in cancer remains unclear. Methods: We utilized the RNA sequencing (RNA-seq) and The Cancer Genome Atlas (TGCA) databases to explore and identify genes that may play an important role in the occurrence and development of hepatocellular carcinoma (HCC), such as TMEM43. The role of TMEM43 in HCC was explored through Cell Counting Kit-8 (CCK-8) cloning, flow cytometry, and Transwell experiments. The regulatory relationship between TMEM43 and voltage-dependent anion channel 1 (VDAC1) was investigated through coimmunoprecipitation (co-IP) and western blot (WB) experiments. WB was used to study the deubiquitination effect of ubiquitin-specific protease 7 (USP7) on TMEM43. Results: In this study, we utilized the RNA-seq and TGCA databases to mine data and found that TMEM43 is highly expressed in HCC. The absence of TMEM43 in cancer cells was shown to inhibit tumor development. Further research detected an important regulatory relationship between TMEM43 and VDAC1. In addition, we found that USP7 affected the progression of HCC by regulating the ubiquitination level of TMEM43 through deubiquitination. Conclusions: Our study demonstrated that USP7 participates in the growth of HCC tumors through TMEM43/VDAC1.Our results suggest that USP7/TMEM43/VDAC1 may have predictive value and represent a new treatment strategy for HCC.

Short-chain fatty acids in cancer pathogenesis.

Cancer is a multi-step process that can be viewed as a cellular and immunological shift away from homeostasis in response to selected infectious agents, mutations, diet, and environmental carcinogens. Homeostasis, which contributes importantly to the definition of "health," is maintained, in part by the production of short-chain fatty acids (SCFAs), which are metabolites of specific gut bacteria. Alteration in the composition of gut bacteria, or dysbiosis, is often a major risk factor for some two dozen tumor types. Dysbiosis is often characterized by diminished levels of SCFAs in the stool, and the presence of a "leaky gut," permitting the penetration of microbes and microbial derived molecules (e.g., lipopolysaccharides) through the gut wall, thereby triggering chronic inflammation. SCFAs attenuate inflammation by inhibiting the activation of nuclear factor kappa B, by decreasing the expression of pro-inflammatory cytokines such as tumor necrosis factor alpha, by stimulating the expression of anti-inflammatory cytokines such as interleukin-10 and transforming growth factor beta, and by promoting the differentiation of naïve T cells into T regulatory cells, which down-regulate immune responses by immunomodulation. SCFA function epigenetically by inhibiting selected histone acetyltransferases that alter the expression of multiple genes and the activity of many signaling pathways (e.g., Wnt, Hedgehog, Hippo, and Notch) that contribute to the pathogenesis of cancer. SCFAs block cancer stem cell proliferation, thereby potentially delaying or inhibiting cancer development or relapse by targeting genes and pathways that are mutated in tumors (e.g., epidermal growth factor receptor, hepatocyte growth factor, and MET) and by promoting the expression of tumor suppressors (e.g., by up-regulating PTEN and p53). When administered properly, SCFAs have many advantages compared to probiotic bacteria and fecal transplants. In carcinogenesis, SCFAs are toxic against tumor cells but not to surrounding tissue due to differences in their metabolic fate. Multiple hallmarks of cancer are also targets of SCFAs. These data suggest that SCFAs may re-establish homeostasis without overt toxicity and either delay or prevent the development of various tumor types.

Hepatitis B x (HBx) as a Component of a Functional Cure for Chronic Hepatitis B.

Patients who are carriers of the hepatitis B virus (HBV) are at high risk of chronic liver disease (CLD) which proceeds from hepatitis, to fibrosis, cirrhosis and to hepatocellular carcinoma (HCC). The hepatitis B-encoded X antigen, HBx, promotes virus gene expression and replication, protects infected hepatocytes from immunological destruction, and promotes the development of CLD and HCC. For virus replication, HBx regulates covalently closed circular (ccc) HBV DNA transcription, while for CLD, HBx triggers cellular oxidative stress, in part, by triggering mitochondrial damage that stimulates innate immunity. Constitutive activation of NF-κB by HBx transcriptionally activates pro-inflammatory genes, resulting in hepatocellular destruction, regeneration, and increased integration of the HBx gene into the host genome. NF-κB is also hepatoprotective, which sustains the survival of infected cells. Multiple therapeutic approaches include direct-acting anti-viral compounds and immune-stimulating drugs, but functional cures were not achieved, in part, because none were yet devised to target HBx. In addition, many patients with cirrhosis or HCC have little or no virus replication, but continue to express HBx from integrated templates, suggesting that HBx contributes to the pathogenesis of CLD. Blocking HBx activity will, therefore, impact multiple aspects of the host-virus relationship that are relevant to achieving a functional cure.

Hepatitis B x antigen (HBx) is an important therapeutic target in the pathogenesis of hepatocellular carcinoma.

Hepatitis B virus (HBV) is a human pathogen that has infected an estimated two billion people worldwide. Despite the availability of highly efficacious vaccines, universal screening of the blood supply for virus, and potent direct acting anti-viral drugs, there are more than 250 million carriers of HBV who are at risk for the sequential development of hepatitis, fibrosis, cirrhosis and hepatocellular carcinoma (HCC). More than 800,000 deaths per year are attributed to chronic hepatitis B. Many different therapeutic approaches have been developed to block virus replication, and although effective, none are curative. These treatments have little or no impact upon the portions of integrated HBV DNA, which often encode the virus regulatory protein, HBx. Although given little attention, HBx is an important therapeutic target because it contributes importantly to (a) HBV replication, (b) in protecting infected cells from immune mediated destruction during chronic infection, and (c) in the development of HCC. Thus, the development of therapies targeting HBx, combined with other established therapies, will provide a functional cure that will target virus replication and further reduce or eliminate both the morbidity and mortality associated with chronic liver disease and HCC. Simultaneous targeting of all these characteristics underscores the importance of developing therapies against HBx.

Studies on the Efficacy, Potential Cardiotoxicity and Monkey Pharmacokinetics of GLP-26 as a Potent Hepatitis B Virus Capsid Assembly Modulator.

While treatment options are available for hepatitis B virus (HBV), there is currently no cure. Anti-HBV nucleoside analogs and interferon-alpha 2b rarely clear HBV covalently closed circular DNA (cccDNA), requiring lifelong treatment. Recently, we identified GLP-26, a glyoxamide derivative which modulates HBV capsid assembly. The impact of GLP-26 on viral replication and integrated DNA was assessed in an HBV nude mouse model bearing HBV transfected AD38 xenografts. At day 45 post-infection, GLP-26 reduced HBV titers by 2.3-3 log10 versus infected placebo-treated mice. Combination therapy with GLP-26 and entecavir reduced HBV log10 titers by 4.6-fold versus placebo. Next, we examined the pharmacokinetics (PK) in cynomolgus monkeys administered GLP-26 via IV (1 mg/kg) or PO (5 mg/kg). GLP-26 was found to have 34% oral bioavailability, with a mean input time of 3.17 h. The oral dose produced a mean peak plasma concentration of 380.7 ng/mL, observed 0.67 h after administration (~30-fold > in vitro EC90 corrected for protein binding), with a mean terminal elimination half-life of 2.4 h and a mean area under the plasma concentration versus time curve of 1660 ng·hr/mL. GLP-26 was 86.7% bound in monkey plasma. Lastly, GLP-26 demonstrated a favorable toxicity profile confirmed in primary human cardiomyocytes. Thus, GLP-26 warrants further preclinical development as an add on to treatment for HBV infection.

Designing a broad-spectrum integrative approach for cancer prevention and treatment.

Targeted therapies and the consequent adoption of "personalized" oncology have achieved notable successes in some cancers; however, significant problems remain with this approach. Many targeted therapies are highly toxic, costs are extremely high, and most patients experience relapse after a few disease-free months. Relapses arise from genetic heterogeneity in tumors, which harbor therapy-resistant immortalized cells that have adopted alternate and compensatory pathways (i.e., pathways that are not reliant upon the same mechanisms as those which have been targeted). To address these limitations, an international task force of 180 scientists was assembled to explore the concept of a low-toxicity "broad-spectrum" therapeutic approach that could simultaneously target many key pathways and mechanisms. Using cancer hallmark phenotypes and the tumor microenvironment to account for the various aspects of relevant cancer biology, interdisciplinary teams reviewed each hallmark area and nominated a wide range of high-priority targets (74 in total) that could be modified to improve patient outcomes. For these targets, corresponding low-toxicity therapeutic approaches were then suggested, many of which were phytochemicals. Proposed actions on each target and all of the approaches were further reviewed for known effects on other hallmark areas and the tumor microenvironment. Potential contrary or procarcinogenic effects were found for 3.9% of the relationships between targets and hallmarks, and mixed evidence of complementary and contrary relationships was found for 7.1%. Approximately 67% of the relationships revealed potentially complementary effects, and the remainder had no known relationship. Among the approaches, 1.1% had contrary, 2.8% had mixed and 62.1% had complementary relationships. These results suggest that a broad-spectrum approach should be feasible from a safety standpoint. This novel approach has potential to be relatively inexpensive, it should help us address stages and types of cancer that lack conventional treatment, and it may reduce relapse risks. A proposed agenda for future research is offered.

Sustained proliferation in cancer: Mechanisms and novel therapeutic targets.

Proliferation is an important part of cancer development and progression. This is manifest by altered expression and/or activity of cell cycle related proteins. Constitutive activation of many signal transduction pathways also stimulates cell growth. Early steps in tumor development are associated with a fibrogenic response and the development of a hypoxic environment which favors the survival and proliferation of cancer stem cells. Part of the survival strategy of cancer stem cells may manifested by alterations in cell metabolism. Once tumors appear, growth and metastasis may be supported by overproduction of appropriate hormones (in hormonally dependent cancers), by promoting angiogenesis, by undergoing epithelial to mesenchymal transition, by triggering autophagy, and by taking cues from surrounding stromal cells. A number of natural compounds (e.g., curcumin, resveratrol, indole-3-carbinol, brassinin, sulforaphane, epigallocatechin-3-gallate, genistein, ellagitannins, lycopene and quercetin) have been found to inhibit one or more pathways that contribute to proliferation (e.g., hypoxia inducible factor 1, nuclear factor kappa B, phosphoinositide 3 kinase/Akt, insulin-like growth factor receptor 1, Wnt, cell cycle associated proteins, as well as androgen and estrogen receptor signaling). These data, in combination with bioinformatics analyses, will be very important for identifying signaling pathways and molecular targets that may provide early diagnostic markers and/or critical targets for the development of new drugs or drug combinations that block tumor formation and progression.

Human viral oncogenesis: a cancer hallmarks analysis.

Approximately 12% of all human cancers are caused by oncoviruses. Human viral oncogenesis is complex, and only a small percentage of the infected individuals develop cancer, often many years to decades after the initial infection. This reflects the multistep nature of viral oncogenesis, host genetic variability, and the fact that viruses contribute to only a portion of the oncogenic events. In this review, the Hallmarks of Cancer framework of Hanahan and Weinberg (2000 and 2011) is used to dissect the viral, host, and environmental cofactors that contribute to the biology of multistep oncogenesis mediated by established human oncoviruses. The viruses discussed include Epstein-Barr virus (EBV), high-risk human papillomaviruses (HPVs), hepatitis B and C viruses (HBV and HCV, respectively), human T cell lymphotropic virus-1 (HTLV-1), and Kaposi's sarcoma herpesvirus (KSHV).

The roles of hepatitis B virus-encoded X protein in virus replication and the pathogenesis of chronic liver disease.

INTRODUCTION: Hepatitis B virus (HBV) is a major cause of chronic liver disease (CLD) and hepatocellular carcinoma (HCC) worldwide. More than 350 million people are at risk for HCC, and with few treatment options available, therapeutic approaches to targets other than the virus polymerase will be needed. This review suggests that the HBV-encoded X protein, HBx, would be an outstanding target because it contributes to the biology and pathogenesis of HBV in three fundamental ways. AREAS COVERED: First, HBx is a trans-activating protein that stimulates virus gene expression and replication, thereby promoting the development and persistence of the carrier state. Second, HBx partially blocks the development of immune responses that would otherwise clear the virus, and protects infected hepatocytes from immune-mediated destruction. Thus, HBx contributes to the development of CLD without virus clearance. Third, HBx alters patterns of host gene expression that make possible the emergence of HCC. The selected literature cited is from the National Library of Medicine (Pubmed and Medline). EXPERT OPINION: Understanding the mechanisms, whereby HBx supports virus replication and promotes pathogenesis, suggests that HBx will be an important therapeutic target against both virus replication and CLD aimed at the chemoprevention of HCC.

Preclinical characterization of GLS4, an inhibitor of hepatitis B virus core particle assembly.

Hepatitis B virus (HBV)-associated chronic liver diseases are treated with nucleoside analogs that target the virus polymerase. While these analogs are potent, drugs are needed to target other virus-encoded gene products to better block the virus replication cycle and chronic liver disease. This work further characterized GLS4 and compared it to the related BAY 41-4109, both of which trigger aberrant HBV core particle assembly, where the virus replication cycle occurs. This was done in HepAD38 cells, which replicate HBV to high levels. In vitro, GLS4 was significantly less toxic for primary human hepatocytes (P < 0.01 up to 100 µM), inhibited virus accumulation in the supernantant of HepAD38 cells (P < 0.02 up to 100 nM), inhibited HBV replicative forms in the liver with a significantly lower 50% effective concentration (EC50) (P < 0.02), and more strongly inhibited core gene expression (P < 0.001 at 100 to 200 nM) compared to BAY 41-4109. In vivo characterization was performed in nude mice inoculated with HepAD38 cells, which grew out as tumors, resulting in viremia. Treatment of mice with GLS4 and BAY 41-4109 showed strong and sustained suppression of virus DNA to about the same extents both during and after treatment. Both drugs reduced the levels of intracellular core antigen in the tumors. Alanine aminotransferase levels were normal. Tumor and total body weights were not affected by treatment. Thus, GLS4 was as potent as the prototype, BAY 41-4109, and was superior to lamivudine, in that there was little virus relapse after the end of treatment and no indication of toxicity.

UCN-01 induces S and G2/M cell cycle arrest through the p53/p21(waf1) or CHK2/CDC25C pathways and can suppress invasion in human hepatoma cell lines.

BACKGROUND: UCN-01 (7-hydroxystaurosporine), a protein kinase inhibitor, has attracted a great deal of attention as a potent antitumour agent. Several clinical trials of UCN-01 alone or in combination with other agents for different tumour types are currently underway, and some of these trials have had positive results. Hepatocellular carcinoma has high incidence rates and is associated with poor prognosis and high mortality rates. METHODS: Three different hepatoma cell lines (Huh7, HepG2, and Hep3B) were treated with different concentrations of UCN-01, and the anti-tumour effects of UCN-01 were evaluated. Following UCN-01 treatment, cell growth was measured using an MTT assay, cell cycle arrest was assayed using flow cytometry, and the mechanisms of cell cycle arrest and invasion inhibition were investigated through western blotting and a Matrigel invasion assay. RESULTS: After a 72-h UCN-01 treatment, the growth of different hepatoma cell lines was significantly inhibited in a dose-dependent manner, with IC50 values ranging from 69.76 to 222.74 nM. Flow cytometry results suggested that UCN-01 inhibits proliferation in the hepatoma cells by inducing S and G2/M phase arrest, but not G1/S arrest, which differs from previous reports that used other tumour cell lines. Western blot results illustrated that UCN-01 induces a G2/M phase arrest, regardless of the status of the p53/P21(waf1) pathway, whereas the CHK2/CDC25C pathway and the p53/p21(waf1)pathway were involved in the UCN-01-induced S phase arrest. UCN-01 remarkably inhibited Huh7 cell invasion in a time-dependent manner. Suppression of Huh7 cell invasion may be due to the down-regulation of phosphorylated ß-catenin by UCN-01. CONCLUSIONS: These findings suggest that UCN-01 induces hepatoma cell growth inhibition by regulating the p53/p21(waf1) and CHK2/CDC25 pathways. Suppression of Huh7 cell invasion by UCN-01 may be due to the down-regulation of phosphorylated ß-catenin. These data lend support for further studies on UCN-01 as a promising anti-HCC candidate.

Pathogenic mechanisms in HBV- and HCV-associated hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a highly lethal cancer, with increasing worldwide incidence, that is mainly associated with chronic hepatitis B virus (HBV) and/or hepatitis C virus (HCV) infections. There are few effective treatments partly because the cell- and molecular-based mechanisms that contribute to the pathogenesis of this tumour type are poorly understood. This Review outlines pathogenic mechanisms that seem to be common to both viruses and which suggest innovative approaches to the prevention and treatment of HCC.

A spiroligomer α-helix mimic that binds HDM2, penetrates human cells and stabilizes HDM2 in cell culture.

We demonstrate functionalized spiroligomers that mimic the HDM2-bound conformation of the p53 activation domain. Spiroligomers are stereochemically defined, functionalized, spirocyclic monomers coupled through pairs of amide bonds to create spiro-ladder oligomers. Two series of spiroligomers were synthesized, one of structural analogs and one of stereochemical analogs, from which we identified compound 1, that binds HDM2 with a Kd value of 400 nM. The spiroligomer 1 penetrates human liver cancer cells through passive diffusion and in a dose-dependent and time-dependent manner increases the levels of HDM2 more than 30-fold in Huh7 cells in which the p53/HDM2 negative feed-back loop is inoperative. This is a biological effect that is not seen with the HDM2 ligand nutlin-3a. We propose that compound 1 modulates the levels of HDM2 by stabilizing it to proteolysis, allowing it to accumulate in the absence of a p53/HDM2 feedback loop.

Hedgehog signaling blockade delays hepatocarcinogenesis induced by hepatitis B virus X protein.

The hepatitis B virus (HBV) encoded X protein (HBx) contributes centrally to the pathogenesis of hepatocellular carcinoma (HCC). Aberrant activation of the Hedgehog (Hh) pathway has been linked to many tumor types including HCC. Thus, experiments were designed to test the hypothesis that HBx promotes HCC via activation of Hh signaling. HBx expression correlated with an upregulation of Hh markers in human liver cancer cell lines, in liver samples from HBV infected patients with HCC, and in the livers of HBx transgenic mice (HBxTg) that develop hepatitis, steatosis, and dysplasia, culminating in the appearance of HCC. The findings in human samples provide clinical validation for the in vitro results and those in the HBxTg. Blockade of Hh signaling inhibited HBx stimulation of cell migration, anchorage-independent growth, tumor development in HBxTg, and xenograft growth in nude mice. Results suggest that the ability of HBx to promote cancer is at least partially dependent upon the activation of the Hh pathway. This study provides biologic evidence for the role of Hh signaling in the pathogenesis of HBV-mediated HCC and suggests cause and effect for the first time. The observation that inhibition of Hh signaling partially blocked the ability of HBx to promote growth and migration in vitro and tumorigenesis in two animal models implies that Hh signaling may represent an "oncogene addiction" pathway for HBV-associated HCC. This work could be central to designing specific treatments that target early development and progression of HBx-mediated HCC.

Evaluation of single and combination therapies with tenofovir disoproxil fumarate and emtricitabine in vitro and in a robust mouse model supporting high levels of hepatitis B virus replication.

Next-generation therapies for chronic hepatitis B virus (HBV) infection will involve combinations of established and/or experimental drugs. The current study investigated the in vitro and in vivo efficacy of tenofovir disoproxil fumarate (TDF) and/or emtricitabine [(-)-FTC] alone and in combination therapy for HBV infection utilizing the HepAD38 system (human hepatoblastoma cells transfected with HBV). Cellular pharmacology studies demonstrated increased levels of (-)-FTC triphosphate with coincubation of increasing concentrations of TDF, while (-)-FTC had no effect on intracellular tenofovir (TFV) diphosphate levels. Quantification of extracellular HBV by real-time PCR from hepatocytes demonstrated the anti-HBV activity with TDF, (-)-FTC, and their combination. Combination of (-)-FTC with TDF or TFV (ratio, 1:1) had a weighted average combination index of 0.7 for both combination sets, indicating synergistic antiviral effects. No cytotoxic effects were observed with any regimens. Using an in vivo murine model which develops robust HBV viremia in nude mice subcutaneously injected with HepAD38 cells, TDF (33 to 300 mg/kg of body weight/day) suppressed virus replication for up to 10 days posttreatment. At 300 mg/kg/day, (-)-FTC strongly suppressed virus titers to up to 14 days posttreatment. Combination therapy (33 mg/kg/day each drug) sustained suppression of virus titer/ml serum (<1 log(10) unit from pretreatment levels) at 14 days posttreatment, while single-drug treatments yielded virus titers 1.5 to 2 log units above the initial virus titers. There was no difference in mean alanine aminotransferase values or mean wet tumor weights for any of the groups, suggesting a lack of drug toxicity. TDF-(-)-FTC combination therapy provides more effective HBV suppression than therapy with each drug alone.

Role of miR-148a in hepatitis B associated hepatocellular carcinoma.

Hepatitis B virus encoded X antigen (HBx) is a trans-regulatory protein that alters the activity of selected transcription factors and cytoplasmic signal transduction pathways. HBx transcriptionally up-regulates the expression of a unique gene, URG11, which in turn transcriptionally up-regulates ß-catenin, thereby contributing importantly to hepatocarcinogenesis. HBx and URG11 also alter the expression of multiple microRNAs, and by miRNA array analysis, both were shown to promote the expression of miR-148a. Elevated miR-148a was also seen in HBx positive liver samples from infected patients. To study the function of miR-148a, anti-148a was introduced into HepG2 and Hep3B cells stably expressing HBx or stably over-expressing URG11. Anti-miR-148a suppressed cell proliferation, cell cycle progression, cell migration, anchorage independent growth in soft agar and subcutaneous tumor formation in SCID mice. Introduction of anti-miR-148a increased PTEN protein and mRNA expression, suggesting that PTEN was targeted by miR-148a. Anti-miR-148a failed to suppress PTEN expression when co-transfected with reporter gene mutants in the 3'UTR of PTEN mRNA. Introduction of anti-miR-148a also resulted in depressed Akt signaling by HBx and URG11, resulting in decreased expression of ß-catenin. Thus, miR-148a may play a central role in HBx/URG11 mediated HCC, and may be an early diagnostic marker and/or therapeutic target associated with this tumor type.

Does the hepatitis B antigen HBx promote the appearance of liver cancer stem cells?

Hepatitis B virus (HBV) is a major etiologic agent of chronic liver disease 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 (CSC). 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, Klf-4, ß-catenin, and epithelial cell adhesion molecule (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 ß-catenin and epigenetic upregulation 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 upregulated expression of multiple "stemness" markers supports the hypothesis that HBx contributes to hepatocarcinogenesis, at least in part, by promoting changes in gene expression that are characteristics of CSCs.

Application of HBx-induced anti-URGs as early warning biomarker of cirrhosis and HCC.

BACKGROUND: Hepatitis B virus (HBV) carriers are at high risk for the development of hepatocellular carcinoma (HCC), but there are no reliable markers that will identify such high-risk patients. HBV up-regulates the expression of selected genes (URGs) in the liver during chronic infection. These aberrantly expressed proteins trigger corresponding antibodies (anti-URGs) that appear prior to the detection of HCC. This study was undertaken to see if the anti-URGs could be used as early warning biomarker of HBV-induced liver cirrhosis and HCC. METHODS: A cross sectional study using a total of 625 serum samples from HBV infected and uninfected controls were tested for the anti-URGs using specific ELISAs. RESULTS: The number and specificity of anti-URGs correlated with the severity of liver disease Anti-URGs were predominantly present among patients with HBV-associated HCC (55.2%) and cirrhosis (60.7%), and at a lower frequency among patients with chronic hepatitis (35.8%), and at still lower frequencies in most asymptomatic carriers (12.3%) with normal ALT, among patients with chronic hepatitis C (38.5%) and blood donors (0.9%). These anti-URGs were rarely detected in sera from those with tumors other than HCC, except among HBV infected patients with cholangioicarcinoma and in some patients with drug induced hepatitis. 3 or more anti-URGs could precede the diagnosis of cirrhosis or HCC 11.8 months on average, and HBV hepatitis patients with 3 or more anti-URGs have much higher risk (5/20 vs 0/30) to develop cirrhosis and HCC than those patients with less anti-URGs. As the early warning biomarker, 3 or more anti-URGs were served as the threshold to separate the cirrhosis and HCC from others with a moderate sensitivity (58.3%) and specificity (80.0%), which was better than other biomarkers (AFP, AFP-L3, GPC3 and GP73) and would improve up to 70.3% when combined with another biomarker. CONCLUSIONS: The results of this clinical validation study suggest that the anti-URGs might have diagnostic/prognostic utility among patients at high risk for the development of cirrhosis and HCC.

Adenoviral-mediated RNA interference targeting URG11 inhibits growth of human hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the second most common malignancy in Asia, with a 5-year survival rate of less than 5% due to high recurrence after surgery and resistance to chemotherapy. A variety of therapeutic interventions to treat HCC, particularly gene therapy, have recently been investigated in tumor model systems to provide a more complete understanding of hepatocarcinogenesis and effectively design therapeutic strategies to treat this disease. In our study, we constructed an adenoviral vector expressing small interfering RNA (siRNA) targeting a newly discovered gene named upregulated gene 11 (URG11). We introduced this vector into HCC cells to investigate the role of URG11 in HCC carcinogenesis. We observed that upon URG11 knockdown, HCC cell proliferation was inhibited through downregulation of several G1-S phase related molecules including cyclin D1 and apoptosis was induced as a result of Bcl-2 downregulation. Besides decreased expression of cyclin D1, CDK4, pRb and Bcl-2, URG11 also suppressed several other proteins including CAPN9, which was identified by cDNA microarray and 2D gel electrophoresis. Moreover, Ad-URG11-siRNA significantly suppressed HCC tumor growth in nude mice. In conclusion, Ad-URG11-siRNA can significantly suppress HCC tumor growth in vitro and in vivo by silencing the URG11 gene, and the use of this vector for gene therapy may represent a novel strategy to treat human HCC.