Hikari Okada

MicroRNA-27a Regulates Lipid Metabolism and Inhibits Hepatitis C Virus Replication in Human Hepatoma Cells

ABSTRACT 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/cm3, 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.

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

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. (Hepatology 2014;60:1674–1685)

Acyclic retinoid targets platelet-derived growth factor signaling in the prevention of hepatic fibrosis and hepatocellular carcinoma development

Hepatocellular carcinoma (HCC) often develops in association with liver cirrhosis, and its high recurrence rate leads to poor patient prognosis. Although recent evidence suggests that peretinoin, a member of the acyclic retinoid family, may be an effective chemopreventive drug for HCC, published data about its effects on hepatic mesenchymal cells, such as stellate cells and endothelial cells, remain limited. Using a mouse model in which platelet-derived growth factor (PDGF)-C is overexpressed (Pdgf-c Tg), resulting in hepatic fibrosis, steatosis, and eventually, HCC development, we show that peretinoin significantly represses the development of hepatic fibrosis and tumors. Peretinoin inhibited the signaling pathways of fibrogenesis, angiogenesis, and Wnt/β-catenin in Pdgf-c transgenic mice. In vitro, peretinoin repressed the expression of PDGF receptors α/β in primary mouse hepatic stellate cells (HSC), hepatoma cells, fibroblasts, and endothelial cells. Peretinoin also inhibited PDGF-C-activated transformation of HSCs into myofibroblasts. Together, our findings show that PDGF signaling is a target of peretinoin in preventing the development of hepatic fibrosis and HCC.

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

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. (Hepatology 2014;59:828–838)

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.

La protein required for internal ribosome entry site-directed translation is a potential therapeutic target for hepatitis C virus replication.

BACKGROUND Translation of the hepatitis C virus (HCV) is mediated by an internal ribosome entry site (IRES). Here, we analyzed the functional relevance of La protein for replication of HCV using an infectious HCV clone, JFH-1. METHODS A single-nucleotide mutation from A to U was introduced at the 338th nucleotide in the stem-loop domain IV structure of HCV IRES, which stabilized stem-loop IV and abolished translation and replication of JFH-1 almost completely. RESULTS During JFH-1 replication, translation initiation factors required for HCV IRES activity, including La protein, polypyrimidine tract binding protein (PTB), PSMA7, and PCBP2, were significantly induced in Huh-7.5 cells. Interestingly, JFH-1 infection increased telomerase activity and induced the expression of human telomerase RNA (hTR) in Huh-7.5 cells. In 37 tissue specimens from patients with chronic hepatitis C, La protein significantly correlated with the representative essential telomerase components hTR, p23, and HSP90 (P<.001). Recombinant adenovirus that expressed short-hairpin RNA against La protein successfully suppressed the levels of La protein and core protein of JFH-1 to 30% of that in the control cells. CONCLUSIONS HCV infection might be strongly related to telomerase activity in the liver through La protein induction. Inhibition of La protein substantially repressed JFH-1 replication; therefore, La protein is a potential therapeutic target for HCV.

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.

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

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)‐β 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‐β signaling was significantly up‐regulated in the advanced fibrosis stage of CH‐C. A significant positive correlation was observed between the expression of TGF‐β2 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‐β1 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‐β1 also inhibited mTORC1 and IFN signaling. Interestingly, c‐Jun and TGF‐β 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‐β receptor inhibitor canceled these effects and showed an additive effect on the anti‐HCV activity of direct‐acting antiviral drugs (DAAs). Conclusion: Blocking TGF‐β 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. (Hepatology 2014;60:1519–1530)

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‐β1‐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‐β signaling pathways. LNA‐antimiR‐214 is a potential therapy for the prevention of hepatic fibrosis.

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

Significance Hepatocellular carcinoma (HCC) is a highly lethal cancer, partly because of its high rate of recurrence, which is caused by the presence of liver cancer stem cells (CSCs). Here, using a selective chemopreventive agent, acyclic retinoid (ACR), as a bioprobe, we identified MYCN, which is mostly recognized as an oncogene in neuroblastoma, as a therapeutic target of ACR for HCC through a selective deletion of MYCN+ liver CSCs. We also demonstrated that the expression of MYCN in HCC served as a prognostic biomarker and positively correlated with recurrence of de novo HCC after curative treatment. Our study highlighted MYCN as a biomarker and therapeutic target in drug discovery for screening chemopreventive agents against the recurrence of HCC. 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/β-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.