Noriyuki Akutsu

Transcriptional silencing of hedgehog-interacting protein by CpG hypermethylation and chromatic structure in human gastrointestinal cancer.

Hedgehog‐interacting protein (HHIP) was identified as a putative antagonist of the Hh pathway and as a target of Hh signalling. Our aim was to clarify the expression profiles and epigenetic alterations of the HHIP gene in gastrointestinal cancer. The expression and promoter epigenetic status of HHIP in cancer cell lines and freshly resected gastrointestinal cancer tissues were examined using RT‐PCR, tissue microarray analysis, methylation‐specific PCR, and chromatin immunoprecipitation assay. Cells were treated with the demethylating agent 5‐aza‐2′‐deoxycytidine and/or histone deacetylase inhibitor trichostatin A. WST‐8 assays and in vitro invasion assays after treatment with HHIP‐specific siRNA were performed. HHIP expression levels were reduced in most of the gastrointestinal cancer cell lines and in a certain subset of cancer tissues, and these were correlated with promoter hypermethylation. A heterochromatic structure characterized by neither acetylated H3 nor acetylated H4, and histone H3 lysine 9 hypermethylation and histone H3 lysine 4 hypomethylation was observed in cancer cells in which the HHIP gene was aberrantly silenced. On the other hand, overexpression of the HHIP gene was also found in some cancer tissues and there were significant correlations between protein expression levels of HHIP and those of Sonic hedgehog (Shh), Indian hedgehog, Patched, and glioma‐associated oncogene homologue‐1. An association was found between lymph node metastasis and HHIP silencing in colorectal cancer tissues with strong Shh expression and between advanced TNM stage and HHIP silencing in diffuse‐type gastric cancer tissues with strong Shh expression. Down‐regulation of HHIP expression by siRNA resulted in a significant increase in colon cancer cell growth and invasion in vitro. Silencing of the HHIP gene due to hypermethylation and chromatin remodelling appears to be frequently involved in gastrointestinal tumourigenesis. Copyright

Genome-wide analysis of DNA methylation identifies novel cancer-related genes in hepatocellular carcinoma

Aberrant DNA methylation has been implicated in the development of hepatocellular carcinoma (HCC). Our aim was to clarify its molecular mechanism and to identify useful biomarkers by screening for DNA methylation in HCC. Methylated CpG island amplification coupled with CpG island microarray (MCAM) analysis was carried out to screen for methylated genes in primary HCC specimens [hepatitis B virus (HBV)-positive, n = 4; hepatitis C virus (HCV)-positive, n = 5; HBV/HCV-negative, n = 7]. Bisulfite pyrosequencing was used to analyze the methylation of selected genes and long interspersed nuclear element (LINE)-1 in HCC tissue (n = 57) and noncancerous liver tissue (n = 50) from HCC patients and in HCC cell lines (n = 10). MCAM analysis identified 332, 342, and 259 genes that were methylated in HBV-positive, HCV-positive, and HBV/HCV-negative HCC tissues, respectively. Among these genes, methylation of KLHL35, PAX5, PENK, and SPDYA was significantly higher in HCC tissue than in noncancerous liver tissue, irrespective of the hepatitis virus status. LINE-1 hypomethylation was also prevalent in HCC and correlated positively with KLHL35 and SPDYA methylation. Receiver operating characteristic curve analysis revealed that methylation of the four genes and LINE-1 strongly discriminated between HCC tissue and noncancerous liver tissue. Our data suggest that aberrant hyper- and hypomethylation may contribute to a common pathogenesis mechanism in HCC. Hypermethylation of KLHL35, PAX, PENK, and SDPYA and hypomethylation of LINE-1 could be useful biomarkers for the detection of HCC.

Mediastinal Yolk Sac Tumor Producing Protein Induced by Vitamin K Absence or Antagonist-II

Extragonadal yolk sac tumors (YSTs) are rare. We herein report the case of a 66-year-old man with mediastinal, lung and liver tumors. The largest mass was located in the liver and contained a high concentration of protein induced by vitamin K absence or antagonist-II (PIVKA-II) and alpha-fetoprotein. Therefore, the lesion was difficult to distinguish from hepatocellular carcinoma. Finally, YST was diagnosed based on the results of a liver biopsy. Although chemotherapy was effective, the patient died of respiratory failure. The autopsy revealed primary mediastinal YST. In the current report, we describe this case of PIVKA-II-producing YST and review previous cases of PIVKA-II-producing tumors other than hepatoma.

Dysregulation of miRNA in chronic hepatitis B is associated with hepatocellular carcinoma risk after nucleos(t)ide analogue treatment

Hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma (HCC). Nucleos(t)ide analogue (NA) therapy effectively reduces the incidence of HCC, but it does not completely prevent the disease. Here, we show that dysregulation of microRNAs (miRNAs) is involved in post-NA HCC development. We divided chronic hepatitis B (CHB) patients who received NA therapy into two groups: 1) those who did not develop HCC during the follow-up period after NA therapy (no-HCC group) and 2) those who did (HCC group). miRNA expression profiles were significantly altered in CHB tissues as compared to normal liver, and the HCC group showed greater alteration than the no-HCC group. NA treatment restored the miRNA expression profiles to near-normal in the no-HCC group, but it was less effective in the HCC group. A number of miRNAs implicated in HCC, including miR-101, miR-140, miR-152, miR-199a-3p, and let-7g, were downregulated in CHB. Moreover, we identified CDK7 and TACC2 as novel target genes of miR-199a-3p. Our results suggest that altered miRNA expression in CHB contributes to HCC development, and that improvement of miRNA expression after NA treatment is associated with reduced HCC risk.

Abstract 4306: Free IGFBP3 and the risk of liver cancer in a nested case-control study

Background: Insulin-like growth factor-1 (IGF1) is a potent mitogen, whereas IGF-binding protein-3 (IGFBP3) binds and inhibits IGF1. High circulating IGF1 and low IGFBP3 are associated with increased risk of several cancers. Here we examined relations of those serum levels with the risk of hepatoma in a prospective case-control study nested in the JACC Study. Methods: A baseline survey was conducted from 1988 to 1990 and 39,242 subjects donated blood samples. Those who had been diagnosed as hepatoma were regarded as cases for nested case-control studies. Ninety-one cases and 263 controls are eligible for the present analysis. A conditional logistic model was used to estimate odds ratios (OR) for incidence of hepatoma associated with serum IGF1 and IGFBP3 levels. Results: Both IGF1 and molar ratio of IGF1/IGFBP3 were not correlated with the risk of hepatoma. After adjustment for hepatitis-viral infection, body mass index, smoking, and alcohol intake, higher molar difference of (IGFBP3 - IGF1) was associated with decreased risk of hepatoma (p for trend Conclusions: Molar difference of (IGFBP3 - IGF1) might associate inversely with the incidence of hepatoma. Citation Format: Yasushi Adachi, Masanori Nojima, Mitsuru Mori, Yasutaka Matsunaga, Noriyuki Akutsu, Shigeru Sasaki, Takao Endo, Youichi Kurozawa, Kenji Wakai, Akiko Tamakoshi. Free IGFBP3 and the risk of liver cancer in a nested case-control study. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4306.