Guanine nucleotide exchange factor DOCK11-binding peptide fused with a single chain antibody inhibits hepatitis B virus infection and replication.

Hepatitis B virus (HBV) infection is a major global health problem with no established cure. Dedicator of cytokinesis 11 (DOCK11), known as a guanine nucleotide exchange factor (GEF) for Cdc42, is reported to be essential for the maintenance of HBV. However, potential therapeutic strategies targeting DOCK11 have not yet been explored. We have previously developed an in vitro virus method as a more efficient tool for the analysis of proteomics and evolutionary protein engineering. In this study, using the in vitro virus method, we screened and identified a novel antiasialoglycoprotein receptor (ASGR) antibody, ASGR3-10M, and a DOCK11-binding peptide, DCS8-42A, for potential use in HBV infection. We further constructed a fusion protein (10M-D42AN) consisting of ASGR3-10M, DCS8-42A, a fusogenic peptide, and a nuclear localization signal to deliver the peptide inside hepatocytes. We show using immunofluorescence staining that 10M-D42AN was endocytosed into early endosomes and released into the cytoplasm and nucleus. Since DCS8-42A shares homology with activated cdc42-associated kinase 1 (Ack1), which promotes EGFR endocytosis required for HBV infection, we also found that 10M-D42AN inhibited endocytosis of EGFR and Ack1. Furthermore, we show 10M-D42AN suppressed the function of DOCK11 in the host DNA repair system required for covalently closed circular DNA synthesis and suppressed HBV proliferation in mice. In conclusion, this study realizes a novel hepatocyte-specific drug delivery system using an anti-ASGR antibody, a fusogenic peptide, and DOCK11-binding peptide to provide a novel treatment for HBV.

Beta-Hydroxyisovaleryl-Shikonin Eradicates Epithelial Cell Adhesion Molecule-Positive Liver Cancer Stem Cells by Suppressing dUTP Pyrophosphatase Expression.

Cancer stem cells (CSCs) play an essential role in tumorigenesis, chemoresistance, and metastasis. Previously, we demonstrated that the development of hepatocellular carcinoma (HCC) is dictated by a subset of epithelial cell adhesion molecule-positive (EpCAM+) liver CSCs with the activation of Wnt signaling. In this study, we evaluated the expression of dUTP pyrophosphatase (dUTPase), which plays a central role in the development of chemoresistance to 5-fluorouracil, in EpCAM+ HCC cells. We further evaluated the effect of beta-hydroxyisovaleryl-shikonin (ß-HIVS), an ATP-noncompetitive inhibitor of protein tyrosine kinases, on HCC CSCs. EpCAM and dUTPase were expressed in hepatoblasts in human fetal liver, hepatic progenitors in adult cirrhotic liver, and a subset of HCC cells. Sorted EpCAM+ CSCs from HCC cell lines showed abundant nuclear accumulation of dUTPase compared with EpCAM-negative cells. Furthermore, treatment with the Wnt signaling activator BIO increased EpCAM and dUTPase expression. In contrast, ß-HIVS treatment decreased dUTPase expression. ß-HIVS treatment decreased the population of EpCAM+ liver CSCs in a dose-dependent manner in vitro and suppressed tumor growth in vivo compared with the control vehicle. Taken together, our data suggest that dUTPase could be a good target to eradicate liver CSCs resistant to 5-fluorouracil. ß-HIVS is a small molecule that could decrease dUTPase expression and target EpCAM+ liver CSCs.

Dickkopf-1 Promotes Angiogenesis and is a Biomarker for Hepatic Stem Cell-like Hepatocellular Carcinoma.

Cancer stemness evinces interest owing to the resulting malignancy and poor prognosis. We previously demonstrated that hepatic stem cell-like hepatocellular carcinoma (HpSC-HCC) is associated with high vascular invasion and poor prognosis. Dickkopf-1 (DKK-1), a Wnt signaling regulator, is highly expressed in HpSC-HCC. Here, we assessed the diagnostic and prognostic potential of serum DKK-1. Its levels were significantly higher in 391 patients with HCC compared with 205 patients with chronic liver disease. Receiver operating characteristic curve analysis revealed the optimal cutoff value of DKK-1 to diagnose HCC and predict the 3-year survival as 262.2 and 365.9 pg/mL, respectively. HCC patients with high-serum DKK-1 levels showed poor prognosis. We evaluated the effects of anti-DKK-1 antibody treatment on tumor growth in vivo and of recombinant DKK-1 on cell proliferation, invasion, and angiogenesis in vitro. DKK-1 knockdown decreased cancer cell proliferation, migration, and invasion. DKK-1 supplementation promoted angiogenesis in vitro; this effect was abolished by an anti-DKK-1 antibody. Co-injection of the anti-DKK-1 antibody with Huh7 cells inhibited their growth in NOD/SCID mice. Thus, DKK-1 promotes proliferation, migration, and invasion of HCC cells and activates angiogenesis in vascular endothelial cells. DKK-1 is a prognostic biomarker for HCC and a functional molecule for targeted therapy.

BMP9-ID1 Signaling Activates HIF-1α and VEGFA Expression to Promote Tumor Angiogenesis in Hepatocellular Carcinoma.

Since hepatocellular carcinoma (HCC) is a typical hypervascular malignant tumor with poor prognosis, targeting angiogenesis is an important therapeutic strategy for advanced HCC. Involvement of bone morphologic protein 9 (BMP9), a transforming growth factor-beta superfamily member, has recently been reported in the development of liver diseases and angiogenesis. Here, we aimed to elucidate the role of BMP9 signaling in promoting HCC angiogenesis and to assess the antiangiogenic effect of BMP receptor inhibitors in HCC. By analyzing HCC tissue gene expression profiles, we found that BMP9 expression was significantly correlated with angiogenesis-associated genes, including HIF-1α and VEGFR2. In vitro, BMP9 induced HCC cell HIF-1α/VEGFA expression and VEGFA secretion. Silencing of the inhibitor of DNA-binding protein 1 (ID1), a transcription factor targeted by BMP9 signaling, suppressed BMP9-induced HIF-1α/VEGFA expression and VEGFA secretion, resulting in decreased human umbilical vein endothelial cell (HUVEC) lumen formation. BMP receptor inhibitors, which inhibit BMP9-ID1 signaling, suppressed BMP9-induced HIF-1α/VEGFA expression, VEGFA secretion, and HUVEC lumen formation. In vivo, the BMP receptor inhibitor LDN-212854 successfully inhibited HCC tumor growth and angiogenesis by inhibiting BMP9-ID1 signaling. In summary, BMP9-ID1 signaling promotes HCC angiogenesis by activating HIF-1α/VEGFA expression. Thus, targeting BMP9-ID1 signaling could be a pivotal therapeutic option for advanced HCC.

FOXM1 Is a Novel Molecular Target of AFP-Positive Hepatocellular Carcinoma Abrogated by Proteasome Inhibition.

Alpha-fetoprotein (AFP) is an oncofetal protein that is elevated in a subset of hepatocellular carcinoma (HCC) with poor prognosis, but the molecular target activated in AFP-positive HCC remains elusive. Here, we demonstrated that the transcription factor forkhead box M1 (FOXM1) is upregulated in AFP-positive HCC. We found that FOXM1 expression was highly elevated in approximately 40% of HCC cases, and FOXM1-high HCC was associated with high serum AFP levels, a high frequency of microscopic portal vein invasion, and poor prognosis. A transcriptome and pathway analysis revealed the activation of the mitotic cell cycle and the inactivation of mature hepatocyte metabolism function in FOXM1-high HCC. The knockdown of FOXM1 reduced AFP expression and induced G2/M cell cycle arrest. We further identified that the proteasome inhibitor carfilzomib attenuated FOXM1 protein expression and suppressed cell proliferation in AFP-positive HCC cells. Carfilzomib in combination with vascular endothelial growth factor receptor 2 (VEGFR2) blockade significantly prolonged survival by suppressing AFP-positive HCC growth in a subcutaneous tumor xenotransplantation model. These data indicated that FOXM1 plays a pivotal role in the proliferation of AFP-positive liver cancer cells. Carfilzomib can effectively inhibit FOXM1 expression to inhibit tumor growth and could be a novel therapeutic option in patients with AFP-positive HCC who receive anti-VEGFR2 antibodies.

Prevention of hepatocellular carcinoma by targeting MYCN-positive liver cancer stem cells with acyclic retinoid.

Hepatocellular carcinoma (HCC) is a highly lethal cancer that has a high rate of recurrence, in part because of cancer stem cell (CSC)-dependent field cancerization. Acyclic retinoid (ACR) is a synthetic vitamin A-like compound capable of preventing the recurrence of HCC. Here, we performed a genome-wide transcriptome screen and showed that ACR selectively suppressed the expression of MYCN, a member of the MYC family of basic helix-loop-helix-zipper transcription factors, in HCC cell cultures, animal models, and liver biopsies obtained from HCC patients. MYCN expression in human HCC was correlated positively with both CSC and Wnt/ß-catenin signaling markers but negatively with mature hepatocyte markers. Functional analysis showed repressed cell-cycle progression, proliferation, and colony formation, activated caspase-8, and induced cell death in HCC cells following silencing of MYCN expression. High-content single-cell imaging analysis and flow cytometric analysis identified a MYCN+ CSC subpopulation in the heterogeneous HCC cell cultures and showed that these cells were selectively killed by ACR. Particularly, EpCAM+ cells isolated using a cell-sorting system showed increased MYCN expression and sensitivity to ACR compared with EpCAM- cells. In a long-term (>10 y) follow-up study of 102 patients with HCC, MYCN was expressed at higher levels in the HCC tumor region than in nontumor regions, and there was a positive correlation between MYCN expression and recurrence of de novo HCC but not metastatic HCC after curative treatment. In summary, these results suggest that MYCN serves as a prognostic biomarker and therapeutic target of ACR for liver CSCs in de novo HCC.

Characteristics of Impaired Dendritic Cell Function in Patients With Hepatitis B Virus Infection.

Dendritic cells (DCs) are antigen-presenting cells with a central role in host immune response. This study analyzed gene expression and DC function in hepatitis B virus (HBV) patients, functions impaired because of HBV, and identified the genes related to these functions. Peripheral blood mononuclear cells from 64 HBV patients and 19 healthy controls were analyzed. Peripheral blood DCs were stained with antibodies against human leukocyte antigen-DR/Lin-1/CD123/CD11c and separated into plasmacytoid DCs (pDCs) and myeloid DCs by fluorescence-activated cell sorting. Using an interferon-gamma enzyme-linked immunospot assay, we analyzed antigen-specific response in HBV-infected patients. Regarding DC function, we analyzed antigen-presenting capacity, cell migration capacity, phagocytic capacity, and cytokine production capacity. DC gene expression was analyzed by microarray to identify genes related to DC function. No difference was found in the number of DCs in peripheral blood between healthy participants and HBV patients. In cell-surface marker analysis, CD80, CD83, CD86, CD40, and C-C motif chemokine receptor 7 expression levels in pDCs were related to the HBV-specific T-cell response. DCs from HBV patients exhibited decreases in antigen-presenting capacity, migration capacity, and cytokine production capacity. In gene expression analysis, immune-related genes with greatly reduced expression levels in chronic hepatitis B patients were identified. Of these genes, interleukin (IL)-6 signal transducer (IL6ST) expression level positively correlated with DC surface marker expression level. Adjustment of IL6ST expression level in DCs and treatment with oncostatin M resulted in recovery of DC function. Conclusion: IL6ST expression was identified as one cause of decline in DC function in HBV patients. Adjustment of IL6 family cytokine signaling may be useful for recovering reduced DC function in HBV infection.

Peretinoin, an Acyclic Retinoid, Inhibits Hepatitis B Virus Replication by Suppressing Sphingosine Metabolic Pathway In Vitro.

Hepatocellular carcinoma (HCC) frequently develops from hepatitis C virus (HCV) and hepatitis B virus (HBV) infection. We previously reported that peretinoin, an acyclic retinoid, inhibits HCV replication. This study aimed to examine the influence of peretinoin on the HBV lifecycle. HBV-DNA and covalently closed circular DNA (cccDNA) were evaluated by a qPCR method in HepG2.2.15 cells. Peretinoin significantly reduced the levels of intracellular HBV-DNA, nuclear cccDNA, and HBV transcript at a concentration that did not induce cytotoxicity. Conversely, other retinoids, such as 9-cis, 13-cis retinoic acid (RA), and all-trans-retinoic acid (ATRA), had no effect or rather increased HBV replication. Mechanistically, although peretinoin increased the expression of HBV-related transcription factors, as observed for other retinoids, peretinoin enhanced the binding of histone deacetylase 1 (HDAC1) to cccDNA in the nucleus and negatively regulated HBV transcription. Moreover, peretinoin significantly inhibited the expression of SPHK1, a potential inhibitor of HDAC activity, and might be involved in hepatic inflammation, fibrosis, and HCC. SPHK1 overexpression in cells cancelled the inhibition of HBV replication induced by peretinoin. This indicates that peretinoin activates HDAC1 and thereby suppresses HBV replication by inhibiting the sphingosine metabolic pathway. Therefore, peretinoin may be a novel therapeutic agent for HBV replication and chemoprevention against HCC.

Jagged1 DNA Copy Number Variation Is Associated with Poor Outcome in Liver Cancer.

Notch signaling abnormalities are reported to be involved in the acceleration of malignancy in solid tumors and stem cell formation or regeneration in various organs. We analyzed specific genes for DNA copy number variations in liver cancer cells and investigated whether these factors relate to clinical outcome. Chromosome 20p, which includes the ligand for Notch pathways, Jagged1, was found to be amplified in several types of hepatoma cells, and its mRNA was up-regulated according to α-fetoprotein gene expression levels. Notch inhibition using Jagged1 shRNA and γ-secretase inhibitors produced significant suppression of cell growth in α-fetoprotein-producing cells with suppression of downstream genes. Using in vivo hepatoma models, the administration of γ-secretase inhibitors resulted in reduced tumor sizes and effective Notch inhibition with widespread apoptosis and necrosis of viable tumor cells. The γ-secretase inhibitors suppressed cell growth of the epithelial cell adhesion molecule-positive fraction in hepatoma cells, indicating that Notch inhibitors could suppress the stem cell features of liver cancer cells. Even in clinical liver cancer samples, the expression of α-fetoprotein and Jagged1 showed significant correlation, and amplification of the copy number of Jagged1 was associated with Jagged1 mRNA expression and poor survival after liver cancer surgical resection. In conclusion, amplification of Jagged1 contributed to mRNA expression that activates the Jagged1-Notch signaling pathway in liver cancer and led to poor outcome.

Sporadic PCDH18 somatic mutations in EpCAM-positive hepatocellular carcinoma.

BACKGROUND: The relationship between specific genome alterations and hepatocellular carcinoma (HCC) cancer stem cells (CSCs) remains unclear. In this study, we evaluated the relationship between somatic mutations and epithelial cell adhesion molecule positive (EpCAM+) CSCs. METHODS: Two patient-derived HCC samples (HCC1 and HCC2) were sorted by EpCAM expression and analyzed by whole exome sequence. We measured PCDH18 expression level in eight HCC cell lines as well as HCC1 and HCC2 by real-time quantitative RT-PCR. We validated the identified gene mutations in 57 paired of HCC and matched non-cancerous liver tissues by Sanger sequence. RESULTS: Whole exome sequencing on the sorted EpCAM+ and EpCAM- HCC1 and HCC2 cells revealed 19,263 nonsynonymous mutations in the cording region. We selected mutations that potentially impair the function of the encoded protein. Ultimately, 60 mutations including 13 novel nonsense and frameshift mutations were identified. Among them, PCDH18 mutation was more frequently detected in sorted EpCAM+ cells than in EpCAM- cells in HCC1 by whole exome sequences. However, we could not confirm the difference of PCDH18 mutation frequency between sorted EpCAM+ and EpCAM- cells by Sanger sequencing, indicating that PCDH18 mutation could not explain intracellular heterogeneity. In contrast, we found novel PCDH18 mutations, including c.2556_2557delTG, c.1474C>G, c.2337A>G, and c.2976G>T, were detected in HCC1 and 3/57 (5.3%) additional HCC surgical specimens. All four HCCs with PCDH18 mutations were EpCAM-positive, suggesting that PCDH18 somatic mutations might explain the intertumor heterogeneity of HCCs in terms of the expression status of EpCAM. Furthermore, EpCAM-positive cell lines (Huh1, Huh7, HepG2, and Hep3B) had lower PCDH18 expression than EpCAM-negative cell lines (PLC/PRL/5, HLE, HLF, and SK-Hep-1), and PCDH18 knockdown in HCC2 cells slightly enhanced cell proliferation. CONCLUSIONS: Our data suggest that PCDH18 is functionally suppressed in a subset of EpCAM-positive HCCs through somatic mutations, and may play a role in the development of EpCAM-positive HCCs.

Serum cytokine profiles predict survival benefits in patients with advanced hepatocellular carcinoma treated with sorafenib: a retrospective cohort study.

BACKGROUND: Sorafenib is a multiple receptor tyrosine kinase inhibitor known to prolong overall survival in patients with advanced hepatocellular carcinoma (HCC). Predicting this drug's survival benefits is challenging because clinical responses are rarely measurable during treatment. In this study, we hypothesized that serum cytokines levels could predict the survival of advanced HCC patients, as sorafenib targets signaling pathways activated in the tumor stromal microenvironment and potentially affects serum cytokine profiles. METHODS: Of 143 patients with advanced-stage HCC, 104 who were recruited between 2003 and 2007 received hepatic arterial infusion chemotherapy (HAIC) that mainly targets tumor epithelial cells at S-phase (cohort 1); additionally, 39 recruited between 2010 and 2012 received sorafenib, which primarily targets the stromal vascular endothelial cells. Serum samples were collected and aliquoted prior to the treatment. Serum EGF, bFGF, HGF, IFN-γ, IL-10, IL-12, IL-2, IL-4, IL-5, IL-6, IL-8, IP-10, MIG, PDGF-BB, SCF, SDF1, TGF-ß, TGF-α, TNF-α, and VEGF-A were measured via enzyme-linked immunosorbent assays. The Modified Response Evaluation Criteria in Solid Tumors were used to assess tumor responses. RESULTS: The median survival time of HCC patients in cohorts 1 (HAIC-treated) and 2 (sorafenib-treated) were 12.0 and 12.4 months, respectively. Kaplan-Meier analysis revealed no significant survival differences between the 2 groups. Patients who survived more than 2 years after sorafenib treatment exhibited higher serum levels of IL-10, IL-12, TNF-a, IL-8, SDF-1, EGF, PDGF-BB, SCF, and TGF-α. Furthermore, cohort 2 patients with higher serum IL-5 (>12 pg/mL), IL-8 (>10 pg/mL), PDGF-BB (>300 pg/mL), and VEGF-A (>50 pg/mL) levels achieved longer survival; cohort 1 patients did not. Hierarchical cluster analysis of 6 cytokines robustly enriched for comparison analysis between cohorts 1 and 2 (IL-5, IL-8, TGF-α, PDGF-BB, CXCL9, and VEGF-A) revealed that elevation of these cytokines correlated with better survival when treated with sorafenib but not with HAIC. CONCLUSIONS: Patients who exhibited survival benefits owing to sorafenib treatment tended to present higher serum cytokines levels, potentially reflecting the activation of stromal signaling in the tumor microenvironment. Our study thus introduces novel biomarkers that may identify advanced HCC patients who may experience survival benefits with sorafenib treatment.

Hepatitis B Virus (HBV) Core-Related Antigen During Nucleos(t)ide Analog Therapy Is Related to Intra-hepatic HBV Replication and Development of Hepatocellular Carcinoma.

BACKGROUND: Although nucleos(t)ide analog (NA) therapy effectively reduces the hepatitis B virus (HBV) DNA load in the serum of patients with chronic hepatitis B, it does not completely reduce the incidence of hepatocellular carcinoma (HCC). METHODS AND RESULTS: A total of 109 patients who had chronic hepatitis B and were receiving NA therapy were analyzed. Multivariate Cox regression analysis showed that age (>60 years had a hazard ratio [HR] of 2.66), FIB-4 index (an index of >2.1 had a HR of 2.57), and the presence of HBV core-related antigen (HBcrAg; HR, 3.53) during treatment were significantly associated with the development of HCC. The amount of HBV DNA and pregenomic RNA in liver were significantly higher in 16 HBcrAg-positive patients, compared with 12 HBcrAg-negative patients, suggesting active HBV replication in HBcrAg-positive livers. Hepatic gene expression profiling showed that HBV-promoting transcriptional factors, including HNF4α, PPARα, and LRH1, were upregulated in HBcrAg-positive livers. HepAD38 cells overexpressing LRH1 increased HBV replication, characterized by higher HBV DNA and pregenomic RNA levels, during long-term exposure to entecavir. Conversely, overexpression of precore/core in HepG2 cells increased levels of these transcriptional factors. Metformin efficiently repressed HBV replication in primary human hepatocytes. CONCLUSIONS: Modulating HBV transcriptional factors by metformin in combination with NA therapy would potentiate anti-HBV activity and reduce the incidence of HCC in HBcrAg-positive patients.

Inhibition of microRNA-214 ameliorates hepatic fibrosis and tumor incidence in platelet-derived growth factor C transgenic mice.

Differentially regulated microRNA (miRNA) are associated with hepatic fibrosis; however, their potential usefulness for blocking hepatic fibrosis has not been exploited fully. We examined the expression of miRNA in the liver of a transgenic mouse model in which platelet-derived growth factor C (PDGF-C) is overexpressed (Pdgf-c Tg), resulting in hepatic fibrosis and steatosis and the eventual development of hepatocellular carcinoma (HCC). Robust induction of miR-214 correlated with fibrogenesis in the liver of Pdgf-c Tg mice, atherogenic high-fat diet-induced NASH mice, and patients with chronic hepatitis B or C. Pdgf-c Tg mice were injected with locked nucleic acid (LNA)-antimiR-214 via the tail vein using Invivofectamine 2.0 and the degree of hepatic fibrosis and tumor incidence were evaluated. Pdgf-c Tg mice treated with LNA-antimiR-214 showed a marked reduction in fibrosis and tumor incidence compared with saline or LNA-miR-control-injected control mice. In vitro, LNA-antimiR-214 significantly ameliorated TGF-ß1-induced pro-fibrotic gene expression in Lx-2 cells. MiR-214 targets a negative regulator of EGFR signaling, Mig-6. Mimic-miR-214 decreased the expression of Mig-6 and increased the levels of EGF-mediated p-EGFR (Y1173 and Y845) and p-Met (Tyr1234/1235) in Huh-7 cells. Conversely, LNA-antimiR-214 repressed the expression of these genes. In conclusion, miR-214 appears to participate in the development of hepatic fibrosis by modulating the EGFR and TGF-ß signaling pathways. LNA-antimiR-214 is a potential therapy for the prevention of hepatic fibrosis.

Defeating EpCAM(+) liver cancer stem cells by targeting chromatin remodeling enzyme CHD4 in human hepatocellular carcinoma.

BACKGROUND & AIMS: Hepatocellular carcinoma is composed of a subset of cells with enhanced tumorigenicity and chemoresistance that are called cancer stem (or stem-like) cells. We explored the role of chromodomain-helicase-DNA-binding protein 4, which is encoded by the CHD4 gene and is known to epigenetically control gene regulation and DNA damage responses in EpCAM(+) liver cancer stem cells. METHODS: Gene and protein expression profiles were determined by microarray and immunohistochemistry in 245 and 144 hepatocellular carcinoma patients, respectively. The relationship between gene/protein expression and prognosis was examined. The functional role of CHD4 was evaluated in primary hepatocellular carcinoma cells and in cell lines in vitro and in vivo. RESULTS: CHD4 was abundantly expressed in EpCAM(+) hepatocellular carcinoma with expression of hepatic stem cell markers and poor prognosis in two independent cohorts. In cell lines, CHD4 knockdown increased chemosensitivity and CHD4 overexpression induced epirubicin chemoresistance. To inhibit the functions of CHD4 that are mediated through histone deacetylase and poly (ADP-ribose) polymerase, we evaluated the effect of the histone deacetylase inhibitor suberohydroxamic acid and the poly (ADP-ribose) polymerase inhibitor AG-014699. Treatment with either suberohydroxamic acid or AG-014699 reduced the number of EpCAM(+) liver cancer stem cells in vitro, and suberohydroxamic acid and AG-014699 in combination successfully inhibited tumor growth in a mouse xenograft model. CONCLUSIONS: CHD4 plays a pivotal role in chemoresistance and the maintenance of stemness in liver cancer stem cells and is therefore a good target for the eradication of hepatocellular carcinoma.

Hepatic interferon-stimulated genes are differentially regulated in the liver of chronic hepatitis C patients with different interleukin-28B genotypes.

UNLABELLED: Pretreatment up-regulation of hepatic interferon (IFN)-stimulated genes (ISGs) has a stronger association with the treatment-resistant interleukin (IL)28B minor genotype (MI; TG/GG at rs8099917) than with the treatment-sensitive IL28B major genotype (MA; TT at rs8099917). We compared the expression of ISGs in the liver and blood of 146 patients with chronic hepatitis C who received pegylated IFN and ribavirin combination therapy. Gene expression profiles in the liver and blood of 85 patients were analyzed using an Affymetrix GeneChip (Affymetrix, Santa Clara, CA). ISG expression was correlated between the liver and blood of the MA patients, whereas no correlation was observed in the MI patients. This loss of correlation was the result of the impaired infiltration of immune cells into the liver lobules of MI patients, as demonstrated by regional gene expression analysis in liver lobules and portal areas using laser capture microdissection and immunohistochemical staining. Despite having lower levels of immune cells, hepatic ISGs were up-regulated in the liver of MI patients and they were found to be regulated by multiple factors, namely, IL28A/B, IFN-λ4, and wingless-related MMTV integration site 5A (WNT5A). Interestingly, WNT5A induced the expression of ISGs, but also increased hepatitis C virus replication by inducing the expression of the stress granule protein, GTPase-activating protein (SH3 domain)-binding protein 1 (G3BP1), in the Huh-7 cell line. In the liver, the expression of WNT5A and its receptor, frizzled family receptor 5, was significantly correlated with G3BP1. CONCLUSIONS: Immune cells were lost and induced the expression of other inflammatory mediators, such as WNT5A, in the liver of IL28B minor genotype patients. This might be related to the high level of hepatic ISG expression in these patients and the treatment-resistant phenotype of the IL28B minor genotype.

Impaired interferon signaling in chronic hepatitis C patients with advanced fibrosis via the transforming growth factor beta signaling pathway.

UNLABELLED: Malnutrition in the advanced fibrosis stage of chronic hepatitis C (CH-C) impairs interferon (IFN) signaling by inhibiting mammalian target of rapamycin complex 1 (mTORC1) signaling. However, the effect of profibrotic signaling on IFN signaling is not known. Here, the effect of transforming growth factor (TGF)-ß signaling on IFN signaling and hepatitis C virus (HCV) replication was examined in Huh-7.5 cells by evaluating the expression of forkhead box O3A (Foxo3a), suppressor of cytokine signaling 3 (Socs3), c-Jun, activating transcription factor 2, ras homolog enriched in brain, and mTORC1. The findings were confirmed in liver tissue samples obtained from 91 patients who received pegylated-IFN and ribavirin combination therapy. TGF-ß signaling was significantly up-regulated in the advanced fibrosis stage of CH-C. A significant positive correlation was observed between the expression of TGF-ß2 and mothers against decapentaplegic homolog 2 (Smad2), Smad2 and Foxo3a, and Foxo3a and Socs3 in the liver of CH-C patients. In Huh-7.5 cells, TGF-ß1 activated the Foxo3a promoter through an AP1 binding site; the transcription factor c-Jun was involved in this activation. Foxo3a activated the Socs3 promoter and increased HCV replication. TGF-ß1 also inhibited mTORC1 and IFN signaling. Interestingly, c-Jun and TGF-ß signaling was up-regulated in treatment-resistant IL28B minor genotype patients (TG/GG at rs8099917), especially in the early fibrosis stage. Branched chain amino acids or a TGF-ß receptor inhibitor canceled these effects and showed an additive effect on the anti-HCV activity of direct-acting antiviral drugs (DAAs). CONCLUSION: Blocking TGF-ß signaling could potentiate the antiviral efficacy of IFN- and/ or DAA-based treatment regimens and would be useful for the treatment of difficult-to-cure CH-C patients.

Gd-EOB-DTPA-enhanced magnetic resonance imaging and alpha-fetoprotein predict prognosis of early-stage hepatocellular carcinoma.

UNLABELLED: The survival of patients with hepatocellular carcinoma (HCC) is often individually different even after surgery for early-stage tumors. Gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid (Gd-EOB-DTPA)-enhanced magnetic resonance imaging (MRI) has been introduced recently to evaluate hepatic lesions with regard to vascularity and the activity of the organic anion transporter OATP1B3. Here we report that Gd-EOB-DTPA-enhanced MRI (EOB-MRI) in combination with serum alpha-fetoprotein (AFP) status reflects the stem/maturational status of HCC with distinct biology and prognostic information. Gd-EOB-DTPA uptake in the hepatobiliary phase was observed in ∼15% of HCCs. This uptake correlated with low serum AFP levels, maintenance of hepatocyte function with the up-regulation of OATP1B3 and HNF4A expression, and good prognosis. By contrast, HCC showing reduced Gd-EOB-DTPA uptake with high serum AFP levels was associated with poor prognosis and the activation of the oncogene FOXM1. Knockdown of HNF4A in HCC cells showing Gd-EOB-DTPA uptake resulted in the increased expression of AFP and FOXM1 and the loss of OATP1B3 expression accompanied by morphological changes, enhanced tumorigenesis, and loss of Gd-EOB-DTPA uptake in vivo. HCC classification based on EOB-MRI and serum AFP levels predicted overall survival in a single-institution cohort (n=70), and its prognostic utility was validated independently in a multi-institution cohort of early-stage HCCs (n=109). CONCLUSION: This noninvasive classification system is molecularly based on the stem/maturation status of HCCs and can be incorporated into current staging practices to improve management algorithms, especially in the early stage of disease.

MicroRNA-27a regulates lipid metabolism and inhibits hepatitis C virus replication in human hepatoma cells.

The replication and infectivity of the lipotropic hepatitis C virus (HCV) are regulated by cellular lipid status. Among differentially expressed microRNAs (miRNAs), we found that miR-27a was preferentially expressed in HCV-infected liver over hepatitis B virus (HBV)-infected liver. Gene expression profiling of Huh-7.5 cells showed that miR-27a regulates lipid metabolism by targeting the lipid synthetic transcription factor RXRα and the lipid transporter ATP-binding cassette subfamily A member 1. In addition, miR-27a repressed the expression of many lipid metabolism-related genes, including FASN, SREBP1, SREBP2, PPARα, and PPARγ, as well as ApoA1, ApoB100, and ApoE3, which are essential for the production of infectious viral particles. miR-27a repression increased the cellular lipid content, decreased the buoyant density of HCV particles from 1.13 to 1.08 g/cm(3), and increased viral replication and infectivity. miR-27a overexpression substantially decreased viral infectivity. Furthermore, miR-27a enhanced in vitro interferon (IFN) signaling, and patients who expressed high levels of miR-27a in the liver showed a more favorable response to pegylated IFN and ribavirin combination therapy. Interestingly, the expression of miR-27a was upregulated by HCV infection and lipid overload through the adipocyte differentiation transcription factor C/EBPα. In turn, upregulated miR-27a repressed HCV infection and lipid storage in cells. Thus, this negative feedback mechanism might contribute to the maintenance of a low viral load and would be beneficial to the virus by allowing it to escape host immune surveillance and establish a persistent chronic HCV infection.

Potential utility of l-carnitine for preventing liver tumors derived from metabolic dysfunction-associated steatohepatitis.

BACKGROUND: Recent reports have unveiled the potential utility of l-carnitine to alleviate metabolic dysfunction-associated steatohepatitis (MASH) by enhancing mitochondrial metabolic function. However, its efficacy at preventing the development of HCC has not been assessed fully. METHODS: l-carnitine (2 g/d) was administered to 11 patients with MASH for 10 weeks, and blood liver function tests were performed. Five patients received a serial liver biopsy, and liver histology and hepatic gene expression were evaluated using this tissue. An atherogenic plus high-fat diet MASH mouse model received long-term l-carnitine administration, and liver histology and liver tumor development were evaluated. RESULTS: Ten-week l-carnitine administration significantly improved serum alanine transaminase and aspartate transaminase levels along with a histological improvement in the NAFLD activity score, while steatosis and fibrosis were not improved. Gene expression profiling revealed a significant improvement in the inflammation and profibrotic gene signature as well as the recovery of lipid metabolism. Long-term l-carnitine administration to atherogenic plus high-fat diet MASH mice substantially improved liver histology (inflammation, steatosis, and fibrosis) and significantly reduced the incidence of liver tumors. l-carnitine directly reduced the expression of the MASH-associated and stress-induced transcriptional factor early growth response 1. Early growth response 1 activated the promoter activity of neural precursor cell expressed, developmentally downregulated protein 9 (NEDD9), an oncogenic protein. Thus, l-carnitine reduced the activation of the NEDD9, focal adhesion kinase 1, and AKT oncogenic signaling pathway. CONCLUSIONS: Short-term l-carnitine administration ameliorated MASH through its anti-inflammatory effects. Long-term l-carnitine administration potentially improved the steatosis and fibrosis of MASH and may eventually reduce the risk of HCC.

Identification of a Transmembrane Protein Involved in Shear Stress Signaling and Hepatocarcinogenesis After a Sustained Virological Response to Hepatitis C Virus.

BACKGROUND & AIMS: The risk of hepatocellular carcinoma (HCC) remains after achieving a sustained virological response (SVR) in patients with chronic hepatitis C (CHC). Epigenetic abnormalities might be key regulators in the development of HCC. This study aimed to identify the genes involved in hepatocarcinogenesis after an SVR. METHODS: DNA methylation in liver tissue was compared between 21 CHC patients without HCC and 28 CHC patients with HCC, all of whom had achieved an SVR. Additional comparisons with 23 CHC patients before treatment and 10 normal livers were performed. The characteristics of a newly identified gene were explored in vitro and in vivo. RESULTS: We found that the transmembrane protein no. 164 (TMEM164) gene was demethylated by hepatitis C virus infection and HCC development after achieving an SVR. TMEM164 was expressed mainly in endothelial cells, alpha smooth muscle actin-positive cells, and some capillarized liver sinusoidal endothelial cells. TMEM164 expression was significantly correlated with liver fibrosis and relapse-free survival in HCC patients. TMEM164 was induced by shear stress, interacted with GRP78/BiP, accelerated ATF6 (activating transcription factor 6)-mediated endoplasmic reticulum (ER) stress signaling, and activated interleukin-6/STAT3 (signal transducer and activator of transcription 3) signaling in the TMNK1 liver endothelial cell line. Therefore, we termed TMEM164 "shear stress-induced transmembrane protein associated with ER stress signaling" (SHERMER). SHERMER knockout mice were protected against CCL4-induced liver fibrosis. SHERMER overexpression in TMNK1 cells accelerated HCC growth in a xenograft model. CONCLUSIONS: We identified a new transmembrane protein, SHERMER, in CHC patients with HCC after achieving an SVR. SHERMER was induced by shear stress and accelerated ATF6-mediated ER stress signaling in endothelial cells. Thus, SHERMER is a novel endothelial marker associated with liver fibrosis, hepatocarcinogenesis, and progression of HCC.