Teruyuki Ueda

Differential microRNA expression between hepatitis B and hepatitis C leading disease progression to hepatocellular carcinoma

MicroRNA (miRNA) plays an important role in the pathology of various diseases, including infection and cancer. Using real‐time polymerase chain reaction, we measured the expression of 188 miRNAs in liver tissues obtained from 12 patients with hepatitis B virus (HBV)‐related hepatocellular carcinoma (HCC) and 14 patients with hepatitis C virus (HCV)‐related HCC, including background liver tissues and normal liver tissues obtained from nine patients. Global gene expression in the same tissues was analyzed via complementary DNA microarray to examine whether the differentially expressed miRNAs could regulate their target genes. Detailed analysis of the differentially expressed miRNA revealed two types of miRNA, one associated with HBV and HCV infections (n = 19), the other with the stage of liver disease (n = 31). Pathway analysis of targeted genes using infection‐associated miRNAs revealed that the pathways related to cell death, DNA damage, recombination, and signal transduction were activated in HBV‐infected liver, and those related to immune response, antigen presentation, cell cycle, proteasome, and lipid metabolism were activated in HCV‐infected liver. The differences in the expression of infection‐associated miRNAs in the liver correlated significantly with those observed in Huh7.5 cells in which infectious HBV or HCV clones replicated. Out of the 31 miRNAs associated with disease state, 17 were down‐regulated in HCC, which up‐regulated cancer‐associated pathways such as cell cycle, adhesion, proteolysis, transcription, and translation; 6 miRNAs were up‐regulated in HCC, which down‐regulated anti‐tumor immune response. Conclusion: miRNAs are important mediators of HBV and HCV infection as well as liver disease progression, and therefore could be potential therapeutic target molecules. (HEPATOLOGY 2009.)

Different signaling pathways in the livers of patients with chronic hepatitis B or chronic hepatitis C.

The clinical manifestations of chronic hepatitis B (CH‐B) and chronic hepatitis C (CH‐C) are different. We previously reported differences in the gene expression profiles of liver tissue infected with CH‐B or CH‐C; however, the signaling pathways underlying each condition have yet to be clarified. Using a newly constructed cDNA microarray consisting of 9614 clones selected from 256,550 tags of hepatic serial analysis of gene expression (SAGE) libraries, we compared the gene expression profiles of liver tissue from 24 CH‐B patients with those of 23 CH‐C patients. Laser capture microdissection was used to isolate hepatocytes from liver lobules and infiltrating lymphoid cells from the portal area, from 16 patients, for gene expression analysis. Furthermore, the comprehensive gene network was analyzed using SAGE libraries of CH‐B and CH‐C. Supervised and nonsupervised learning methods revealed that gene expression was correlated more with the infecting virus than any other clinical parameters such as histological stage and disease activity. Pro‐apoptotic and DNA repair responses were predominant in CH‐B with p53 and 14‐3‐3 interacting genes having an important role. In contrast, inflammatory and anti‐apoptotic phenotypes were predominant in CH‐C. These differences would evoke different oncogenic factors in CH‐B and CH‐C. In conclusion, we describe the different signaling pathways induced in the livers of patients with CH‐B or CH‐C. The results might be useful in guiding therapeutic strategies to prevent the development of hepatocellular carcinoma in cases of CH‐B and CH‐C. (HEPATOLOGY 2006;44:1122–1138.)

Enhancement of tumor‐associated antigen‐specific T cell responses by radiofrequency ablation of hepatocellular carcinoma

Radiofrequency ablation (RFA) is one of the treatments for hepatocellular carcinoma (HCC) and is known to enhance host immune response. However, the epitopes to which enhanced immune responses occur, the impact on patient prognosis, and the functions and phenotype of T cells induced are still unclear. To address these issues, we analyzed immune responses before and after RFA in 69 HCC patients using 11 tumor‐associated antigen (TAA)‐derived peptides that we identified to be appropriate to analyze HCC‐specific immune responses. The immune responses were analyzed using enzyme‐linked immunospot (ELISPOT) assay and tetramer assays using peripheral blood mononuclear cells. An increase in the number of TAA‐specific T cells detected by interferon‐γ ELISPOT assays occurred in 62.3% of patients after RFA. The antigens and their epitope to which enhanced T cell responses occur were diverse, and some of them were newly induced. The number of TAA‐specific T cells after RFA was associated with the prevention of HCC recurrence, and it was clarified to be predictive of HCC recurrence after RFA by univariate and multivariate analyses. The number of TAA‐specific T cells after RFA was inversely correlated with the frequency of CD14+HLA‐DR−/low myeloid‐derived suppressor cells (MDSCs). The modification of T cell phenotype was observed after RFA. The number of TAA‐specific T cells at 24 weeks after RFA was decreased. Conclusion: Although RFA can enhance various TAA‐specific T cell responses and the T cells induced contribute to the HCC recurrence‐free survival of patients, besides immunosuppression by MDSCs, the memory phenotype and lifetime of TAA‐specific T cells are not sufficient to prevent HCC recurrence completely. Additional treatments by vaccine or immunomodulatory drugs might be useful to improve the immunological effect of RFA. (Hepatology 2013;1448–1457)

Protein expression profile characteristic to hepatocellular carcinoma revealed by 2D-DIGE with supervised learning

Hepatocellular carcinoma (HCC) is one of the most common and aggressive human malignancies. Although several major risks related to HCC, e.g., hepatitis B and/or hepatitis C virus infection, aflatoxin B1 exposure, alcohol drinking and genetic defects have been revealed, the molecular mechanisms leading to the initiation and progression of HCC have not been clarified. To reduce the mortality and improve the effectiveness of therapy, it is important to detect the proteins which are associated with tumor progression and may be useful as potential therapeutic or diagnosis targets. However, previous studies have not yet revealed the associations among HCC cells, histological grade and AFP. Here, we performed two-dimensional difference gel electrophoresis (2D-DIGE) combined with MS for 18 HCC patients. To focus not on individual proteins but on multiple proteins associated with pathogenesis, we introduce the supervised feature selection based on stochastic gradient boosting (SGB) for identifying protein spots that discriminate HCC/non HCC, histological grade of moderate/well and high alpha-fetoprotein (AFP)/low AFP level without arbitrariness. We detected 18, 25 and 27 protein spots associated with HCC, histological grade and AFP level, respectively. We confirmed that SGB is able to identify the known HCC-related proteins, e.g., heat shock proteins, carbonic anhydrase 2. Moreover, we identified the differentially expressed proteins associated with histological grade of HCC and AFP level and found that aldo-keto reductase 1B10 (AKR1B10) is related to well differentiated HCC, keratin 8 (KRT8) is related to both histological grade and AFP level and protein disulfide isomerase-associated 3 (PDIA3) is associated with both HCC and AFP level. Our pilot study provides new insights on understanding the pathogenesis of HCC, histological grade and AFP level.

Randomized, Phase II Study Comparing Interferon Combined with Hepatic Arterial Infusion of Fluorouracil plus Cisplatin and Fluorouracil Alone in Patients with Advanced Hepatocellular Carcinoma

Objective: This randomized phase II trial compared the response rates to treatment with interferon (IFN) combined with hepatic arterial infusion of fluorouracil (FU) plus cisplatin (CDDP) or FU alone in patients with advanced hepatocellular carcinoma (HCC). Methods: A total of 114 patients with measurable advanced HCC were enrolled and randomized into 2 groups. FU (300 mg/m2, days 1–5, days 8–12) with or without CDDP (20 mg/m2, days 1 and 8) was administered via the hepatic artery. IFNα-2b was administered 3 times per week for 4 weeks. Results: The response rates were 45.6% for the IFN/FU + CDDP group and 24.6% for the IFN/FU group. The response rate was significantly higher in the IFN/FU + CDDP group (p = 0.030). The median overall survival period was 17.6 months in the IFN/FU + CDDP group versus 10.5 months in the IFN/FU group (p = 0.522). The median progression-free survival period was 6.5 months in the IFN/FU + CDDP group versus 3.3 months in the IFN/FU group (p = 0.0048). Hematological toxicity was common, but no toxicity-related deaths were observed. Conclusion: These results show the clinical efficacy of adding CDDP to the hepatic arterial infusion of FU in combined chemotherapy regimens with IFN.

Beneficial effect of branched-chain amino acid supplementation on glycemic control in chronic hepatitis C patients with insulin resistance: implications for type 2 diabetes.

Branched-chain amino acids (BCAAs) improve disorders of albumin metabolism, quality of life, subjective symptoms, and prognosis in patients with chronic hepatitis. However, it remains unclear whether they improve insulin resistance. We examined the effects of BCAAs on glucose tolerance and insulin sensitivity in patients with chronic hepatitis C and insulin resistance. Individuals with a definitive diagnosis of chronic hepatitis C and insulin resistance were eligible for participation. Eligible participants were randomly assigned to the BCAA group or a control group. Participants were then crossed over to the other treatment for a further 12 weeks. Baseline clinical features, laboratory markers, fatty acid levels, and insulin sensitivity, assessed with oral glucose tolerance tests and a hyperinsulinemic euglycemic clamp, were also examined before and 12 and 24 weeks after the beginning of the study. Of the 27 patients who completed the study, 14 began in the BCAA group and 13 began as controls. There were no significant differences in glucose metabolism parameters or lipid profiles between the groups. HbA1c values were improved in 10 patients and worsened or remained unchanged in 17 patients. The only predictive variable for change in HbA1c was the baseline Matsuda index: the lower the index, the greater the improvement in HbA1c values. BCAA therapy did not have adverse effects on glucose tolerance or insulin sensitivity in patients with chronic hepatitis C and insulin resistance. Moreover, it had a therapeutic effect on HbA1c values in patients with marked peripheral (primarily muscle) insulin resistance.

Gene expression profiling of hepatitis B- and hepatitis C-related hepatocellular carcinoma using graphical Gaussian modeling.

BACKGROUND & AIMS Gene expression profiling of hepatocellular carcinoma (HCC) and background liver has been studied extensively; however, the relationship between the gene expression profiles of different lesions has not been assessed. METHODS We examined the expression profiles of 34 HCC specimens (17 hepatitis B virus [HBV]-related and 17 hepatitis C virus [HCV]-related) and 71 non-tumor liver specimens (36 chronic hepatitis B [CH-B] and 35 chronic hepatitis C [CH-C]) using an in-house cDNA microarray consisting of liver-predominant genes. Graphical Gaussian modeling (GGM) was applied to elucidate the interactions of gene clusters among the HCC and non-tumor lesions. RESULTS In CH-B-related HCC, the expression of vascular endothelial growth factor-family signaling and regulation of T cell differentiation, apoptosis, and survival, as well as development-related genes was up-regulated. In CH-C-related HCC, the expression of ectodermal development and cell proliferation, wnt receptor signaling, cell adhesion, and defense response genes was also up-regulated. Many of the metabolism-related genes were down-regulated in both CH-B- and CH-C-related HCC. GGM analysis of the HCC and non-tumor lesions revealed that DNA damage response genes were associated with AP1 signaling in non-tumor lesions, which mediates the expression of many genes in CH-B-related HCC. In contrast, signal transducer and activator of transcription 1 and phosphatase and tensin homolog were associated with early growth response protein 1 signaling in non-tumor lesions, which potentially promotes angiogenesis, fibrogenesis, and tumorigenesis in CH-C-related HCC. CONCLUSIONS Gene expression profiling of HCC and non-tumor lesions revealed the predisposing changes of gene expression in HCC. This approach has potential for the early diagnosis and possible prevention of HCC.

Abstract 3915: α-Fetoprotein-producing hepatoma cells have increased Jagged1 gene copy numbers and their growth can be suppressed by gamma secretase inhibitor

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: Approximately 70% of patients with hepatocellular carcinoma (HCC) have abnormally high α-fetoprotein (AFP) levels, which increase with tumor progression and angiogenesis. We previously used cDNA microarray comparative genomic hybridization (aCGH) to demonstrate an association between hepatoma AFP-producing status and genomic copy number gain and loss. Although genomic copy number change is reported to be associated with some oncogenic factors, its relationship with molecular pathway aberration has not been elucidated. Here, we examined whether signal pathways are associated with both expression and genomic gain for the purpose of discovering clinically useful molecular targets in hepatoma patients. Method: Changes in DNA copy number of hepatoma cells were analyzed using bacterial artificial chromosome (BAC)-aCGH and compared with gene expression profiles. Candidate genes were identified, and real-time PCR was used to confirm gene expression changes in AFP-producing cells. siRNA was used to inhibit gene expression and the effect of this on hepatoma cell growth was determined. Confirmation of genomic alteration was performed by real-time PCR on clinical HCC samples. Growth of hepatoma cells was examined following the application of an inhibitor of the identified signal transduction-related gene. Results: The BAC clone was significantly amplified in some regions of chromosome 20p from hepatoma cells. This amplified region included the ligand for Notch signal transduction, Jagged1, which was shown to be up-regulated by microarray analysis and real-time PCR in AFP-producing hepatoma cells. Surgically treated AFP-up-regulated HCC samples were used to demonstrate the significant correlation between AFP and Jagged1 gene expression. Interestingly, tissue Jagged1 genomic gain and AFP expression were also correlated. The suppression of Jagged1 using siRNA in an AFP-producing cell line significantly repressed cell growth compared with an AFP-negative cell line. The inhibitor of the Notch pathway, gamma secretase inhibitor (GSI), is currently undergoing clinical trials for the treatment of Alzheimers disease, and its effectiveness has been shown as a cancer therapy for solid tumors. Here, continuous application of GSI to hepatoma cells resulted in a significant suppression of cell growth. Conclusion: The genomic amplification of Jagged1 contributes to the continuous mRNA expression that activates the Jagged1-Notch signaling pathway in hepatomas. GSI may be a promising candidate for use in the therapy of AFP-producing advanced HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3915. doi:10.1158/1538-7445.AM2011-3915