Hideyasu Takagi

Frequent epigenetic inactivation of SFRP genes in hepatocellular carcinoma

BackgroundActivation of the Wnt signaling pathway is frequently observed in hepatocellular carcinoma (HCC), though mutation of three of its components, CTNNB1, AXIN1, and AXIN2, is observed substantially less often.MethodsWe examined the relationship between Wnt signaling and epigenetic alteration of secreted frizzled-related protein (SFRP) genes in HCC.ResultsWe frequently detected the active form of β-catenin and accumulation of nuclear β-catenin in liver cancer cell lines. We detected methylation of SFRP family genes in liver cancer cell lines (SFRP1, 9/12, 75%; SFRP2, 7/12, 58%; SFRP4, 3/12, 25%; SFRP5, 7/12, 58%) and primary HCCs (SFRP1, 9/19, 47%; SFRP2, 12/19, 63%; SFRP5, 8/19, 42%), though methylation of SFRP4 was not found in primary HCCs. SFRP methylation also was detected in hepatitis B or C virus-associated chronic hepatitis (SFRP1, 6/37, 16%; SFRP2, 14/37, 38%; SFRP5, 5/37, 14%) and liver cirrhosis (SFRP1, 10/28, 36%; SFRP2, 9/28, 32%; SFRP5, 3/28, 11%), suggesting that methylation of these genes is an early event in liver carcinogenesis. Ectopic expression of SFRPs downregulated T-cell factor/lymphocyte enhancer factor (TCF/LEF) transcriptional activity in liver cancer cells, while overexpression of a β-catenin mutant and depletion of SFRP1 using siRNA synergistically upregulated TCF/LEF transcriptional activity.ConclusionsOur results confirm the frequent methylation and silencing of Wnt antagonist genes in HCC, and suggest that their loss of function contributes to activation of Wnt signaling during hepatocarcinogenesis.

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.

Interferon-α/β and Anti-Fibroblast Growth Factor Receptor 1 Monoclonal Antibody Suppress Hepatic Cancer Cells In Vitro and In Vivo

Background Hepatocellular carcinoma (HCC) is the most commonly occurring primary liver cancer and ranks as the fifth most frequently occurring cancer, overall, and the third leading cause of cancer deaths, worldwide. At present, effective therapeutic options available for HCC are limited; consequently, the prognosis for these patients is poor. Our aim in the present study was to identify a novel target for antibody therapy against HCC. Methodology/Principal Findings We used Western blot and flow cytometric and immunocytochemical analyses to investigate the regulation of FGFR1 expression by interferon-α/β in several human hepatic cancer cell lines. In addition, we tested the efficacy of combined treatment with anti-FGFR1 monoclonal antibody and interferon-α/β in a murine xenograft model of human HCC. We found that interferon-α/β induces expression of FGFR1 in human HCC cell lines, and that an anti-FGFR1 monoclonal antibody (mAb) targeting of the induced FGFR1 can effectively inhibit growth and survival of HCC cells in vitro and in vivo. Moreover, the combination of interferon-α, anti-FGFR1 mAb and peripheral blood mononuclear cells (PBMCs) exerted a significant antitumor effect in vitro. Conclusions Our results suggest that the combined use of an anti-FGFR1 antibody and interferon-α/β is a promising approach to the treatment of HCC.

The effect of forced expression of mutated K-RAS gene on gastrointestinal cancer cell lines and the IGF-1R targeting therapy.

Mutation in K‐RAS (K‐RAS‐MT) plays important roles in both cancer progression and resistance to anti‐epidermal growth factor receptor (EGFR) therapy in gastrointestinal tumors. Insulin‐like growth factor‐1 receptor (IGF‐1R) signaling is required for carcinogenicity and progression of many tumors as well. We have previously shown successful therapy for gastrointestinal cancer cell lines bearing a K‐RAS mutation using an anti‐IGF‐1R monoclonal antibody. In this study, we sought to evaluate effects of forced K‐RAS‐MT expression on gastrointestinal cancer cell lines representing a possible second resistance mechanism for anti‐EGFR therapy and IGF‐1R‐targeted therapy for these transfectants. We made stable transfectants of K‐RAS‐MT in two gastrointestinal cancer cell lines, colorectal RKO and pancreatic BxPC‐3. We assessed the effect of forced expression of K‐RAS‐MT on proliferation, apoptosis, migration, and invasion in gastrointestinal cancer cells. Then we assessed anti‐tumor effects of dominant negative IGF‐1R (IGF‐1R/dn) and an IGF‐1R inhibitor, picropodophyllin, on the K‐RAS‐MT transfectants. Overexpression of K‐RAS‐MT in gastrointestinal cancer cell lines led to more aggressive phenotypes, with increased proliferation, decreased apoptosis, and increased motility and invasion. IGF‐1R blockade suppressed cell growth, colony formation, migration, and invasion, and up‐regulated chemotherapy‐induced apoptosis of gastrointestinal cancer cells, even when K‐RAS‐MT was over‐expressed. IGF‐1R blockade inhibited the Akt pathway more than the extracellular signal‐regulated kinase (ERK) pathway in the K‐RAS‐MT transfectants. IGF‐1R/dn, moreover, inhibited the growth of murine xenografts expressing K‐RAS‐MT. Thus, K‐RAS‐MT might be important for progressive phonotype observed in gastrointestinal cancers. IGF‐1R decoy is a candidate molecular therapeutic approach for gastrointestinal cancers even if K‐RAS is mutated.

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.