Naoshi Nishida

Aberrant Methylation of Multiple Tumor Suppressor Genes in Aging Liver, Chronic Hepatitis, and Hepatocellular Carcinoma

Aberrant DNA methylation is an important epigenetic alteration in hepatocellular carcinoma (HCC). However, the molecular processes underlying the methylator phenotype and the contribution of hepatitis viruses are poorly understood. The current study is a comprehensive methylation analysis of human liver tissue specimens. A total of 176 liver tissues, including 77 pairs of HCCs and matching noncancerous liver and 22 normal livers, were analyzed for methylation. Methylation of 19 epigenetic markers was quantified, and the results were correlated with different disease states and the presence or absence of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections. Based on methylation profiles, the 19 loci were categorized into 3 groups. Normal liver tissues showed methylation primarily in group 1 loci (HIC‐1, CASP8, GSTP1, SOCS1, RASSF1A, p16, APC), which was significantly higher than group 2 (CDH1, RUNX3, RIZ1, SFRP2, MINT31) and group 3 markers (COX2, MINT1, CACNA1G, RASSF2, MINT2, Reprimo, DCC) (P < 0.0001). Noncancerous livers demonstrated increased methylation in both group 1 and group 2 loci. Methylation was significantly more abundant in HCV‐positive livers compared with normal liver tissues. Conversely, HCC showed frequent methylation at each locus investigated in all 3 groups. However, the group 3 loci showed more dense and frequent methylation in HCV‐positive cancers compared with both HBV‐positive cancers and virus‐negative cancers (P < 0.0001). Conclusion: Methylation in HCC is frequent but occurs in a gene‐specific and disease‐specific manner. Methylation profiling allowed us to determine that aberrant methylation is commonly present in normal aging livers, and sequentially progresses with advancing stages of chronic viral infection. Finally, our data provide evidence that HCV infection may accelerate the methylation process and suggests a continuum of increasing methylation with persistent viral infection and carcinogenesis in the liver. (HEPATOLOGY 2008.)

Sensitive detection of circulating hepatocellular carcinoma cells in peripheral venous blood.

Background. This study was performed to develop a sensitive method for the detection of circulating hepatocellular carcinoma (HCC) cells in peripheral blood, in advance of the diagnosis of distant metastasis of HCC by conventional means.

Genetic and Epigenetic Signatures in Human Hepatocellular Carcinoma: A Systematic Review

Hepatocellular carcinoma (HCC) is the third most common cause of cancer deaths worldwide, and the incidence of this fatal disease is still on rise. The majority of HCCs emerge in the background of a chronic liver disease, such as chronic hepatitis and liver cirrhosis. The current understanding is that majority of HCCs evolve as a consequence of chronic inflammation and due to the presence of infection with hepatitis viruses. These underlying pathogenic stimuli subsequently induce a spectrum of genetic and epigenetic alterations in several cancer-related genes, which are involved in cell-cycle regulation, cell growth and adhesion. Such widespread genomic alterations cause disruption of normal cellular signaling and finally lead to the acquisition of a malignant phenotype in HCC. In general, the type of gene alterations, such as point mutations, deletion of chromosomal regions and abnormal methylation of gene promoters differ according to the individual targeted gene. In HCC, incidence of genetic alterations is relatively rare and is limited to a subset of few cancer-specific genes, such as the tumor suppressor p53, RB genes and oncogenes such as the CTNNB1. In contrast, epigenetic changes that involve aberrant methylation of genes and other post-transcriptional histone modifications occur far more frequently, and some of these epigenetic alterations are now being exploited for the development of molecular diagnostic signatures for HCC. In addition, recent findings of unique microRNA expression profiles also provide an evidence for the existence of novel mechanisms for gene expression regulation in HCC. In this review article, we will review the current state of knowledge on the activation of various oncogenic pathways and the inactivation of tumor suppressor pathways in HCC that result in the disruption of cancer-related gene function. In addition, we will specifically emphasize the clinical implication of some of these genetic and epigenetic alterations in the management of hepatocarcinogenesis.

Characteristic patterns of altered DNA methylation predict emergence of human hepatocellular carcinoma

We aimed to identify the specific subset of tumor suppressor genes (TSGs) that are methylation‐silenced during the earliest steps of hepatocarcinogenesis, and to further evaluate whether these genes can serve as predictive biomarkers of hepatocellular carcinoma (HCC) emergence. A total of 482 liver tissues including 177 pairs of HCCs and matched nontumor livers and 128 liver biopsies from chronic hepatitis C (CHC) patients were analyzed for quantitative methylation analysis in 24 TSG promoters and three MINT loci. The tumors were classified as early, less‐progressed, and highly progressed HCCs using histology and radiological approaches. A subset of TSGs that harbored distinctly high levels of methylation in early HCCs were selected. Based on the methylation profiles of these genes, Kaplan‐Meier analyses were performed to determine time‐to‐HCC occurrence in CHC patients. Subsequently, multivariate analysis was performed using age, gender, fibrosis stage, and number of methylated TSGs as covariates. Among TSGs analyzed, a subset of eight TSGs (HIC1, GSTP1, SOCS1, RASSF1, CDKN2A, APC, RUNX3, and PRDM2) demonstrated a distinct cluster by hierarchical clustering and receiver operating characteristic analyses. This subset of TSGs showed significantly higher methylation levels in the early HCCs (P < 0.0001). In the CHC patients, methylation frequencies in these TSGs were associated with shorter time‐to‐HCC occurrence (P < 0.0001), and number of methylated genes was an independent risk factor for HCC (hazard ratio = 5.21, 95% confidence interval = 2.25‐11.76, P = 0.0002). Conclusion: Epigenetic inactivation of a subset of TSGs plays a critical role in the earliest steps of hepatocarcinogenesis. Furthermore, epigenetic inactivation of these genes in CHC provides a prognostic value for determining the risk for developing HCC later in life. (HEPATOLOGY 2012;56:994–1003)

Disruption of the p16/Cyclin D1/Retinoblastoma Protein Pathway in the Majority of Human Hepatocellular Carcinomas

p16, cyclin D1 and retinoblastoma protein (pRB) regulate G1 to S transition and are commonly targeted in various cancers. However, few studies have simultaneously examined all components of the p16/cyclin D1/pRB pathway (RB pathway) in hepatocellular carcinoma (HCC). To clarify the role of the disruption of the RB pathway in HCC, we analyzed p16, pRB and cyclin D1 in 47 HCCs. Inactivation of p16 was detected in 30 of 47 HCCs (64%) by Western blot analysis and significantly correlated with hypermethylation of the promoter of this gene. pRB expression was found to be absent in 13 of 47 HCCs (28%) by immunohistochemistry. We found that 38 of 47 HCCs (81%) contained at least one inactivation in either pRB or p16. Furthermore, there was a significant inverse correlation between p16 and pRB inactivation (p = 0.041). Overexpression of cyclin D1 was detected in 5 of 47 HCCs (11%) by immunohistochemistry. The cases with cyclin D1 overexpression exhibited an advanced clinicopathological appearance and also contained inactivation of pRB and/or p16. These findings suggest that inactivation of pRB and/or p16 is a major event in human hepatocarcinogenesis, while cyclin D1 overexpression may confer additional growth advantages to the tumor in addition to pRB and/or p16 inactivation in HCC.

High copy amplification of the aurora-A gene is associated with chromosomal instability phenotype in human colorectal cancers

Chromosomal instability (CIN) is a common but not universal feature of colorectal cancer (CRC); however, the molecular basis for CIN is controversial and poorly understood. Among the many plausible mechanisms proposed for CIN include disruption of G1/S and G2/M checkpoint regulation, and alterations in the spindle checkpoint genes. However, mutations in individual growth regulatory genes are not commonly observed in CRC. Therefore, a more comprehensive analysis of the genes involved in each cell cycle checkpoint regulatory pathway might be required to evaluate a possible role for involvement in CIN. We investigated the presence of high copy amplification of the cyclin E, Aurora-A, Skp2 genes, mutation of ubiquitin ligase CDC4 and promoter methylation of Mad2L1, as well as the expression of the gene products in a panel of 11 human CRC cell lines as well as 48 human CRC specimens. In the cell lines with CIN, we found amplification of the Aurora-A, cyclin E and Skp2 genes, and a mutation in the CDC4 gene, all of which resulted in altered expression of the cognate proteins. In the human CRC tissues, amplification of Aurora-A was frequent (29%), while alterations were rarely observed in cyclin E, Skp2 or CDC4. Aurora-A amplification was strongly associated with a high fractional allelic loss score (p=0.0001), but not with microsatellite instability, nor with the promoter methylation phenotype in these tumors. Our data confirm involvement in the CDC4-cyclin E pathway in the development of the CIN phenotype in human CRC, and find that amplification of the Aurora-A is a common target for disruption of this pathway.

Possible involvement of T helper type 2 responses to Toll-like receptor ligands in IgG4-related sclerosing disease

We report a case of immunoglobulin G4 (IgG4)-related sclerosing disease involving the pancreas, liver and salivary glands. Massive infiltration of IgG4-expressing plasma cells was seen in the liver and submandibular lymph nodes. Interestingly, accumulation of IgG4-expressing plasma cells was also seen in the colon and terminal ileum. Peripheral blood mononuclear cells (PBMCs) isolated from this patient exhibited enhanced production of IgG4 and interleukin-10 upon stimulation with Toll-like receptor (TLR) ligands as compared with those from a healthy control. In contrast, production of tumour necrosis factor α and interferon γ by PBMCs from this patient was markedly reduced. Since colonic mucosa is always exposed to TLR ligands derived from commensal organisms, the results of immunological studies suggest that enhanced T helper type 2 responses to intestinal microflora may underlie the immunopathogenesis in this patient with IgG4-related sclerosing disease.

Infrequent alterations of the p16INK4A gene in liver cancer

We examined the genomic status of the p16INK4A (inhibitor of cyclin‐dependent kinase 4 A) and cyclin‐dependent kinase 4 (CDK4) genes in 62 human hepatocellular carcinomas (HCCs), 5 cholangiocellular carcinomas and 6 cell lines derived from human liver cancers. Although no samples showed the homozygous deletion of the p16INK4A gene, we detected intragenic mutations of the p16INK4A gene in 3 HCCs and one HCC cell line, which led to an amino‐acid substitution or a frameshift. In 2 HCC samples with mis‐sense mutations of the p16INK4A gene, loss of heterozygosity on 9p22 was also detected, suggesting that the loss of function of p16 was induced during hepatocarcinogenesis. On the other hand, amplification or rearrangement of the CDK4 gene was not detected in any samples examined in this study. These results indicated that the mutations or deletions of the p16INK4A gene are not frequent, but may play a role in a sub‐set of human HCC.

Serum miR-21, miR-29a, and miR-125b Are Promising Biomarkers for the Early Detection of Colorectal Neoplasia.

Purpose: Circulating microRNAs (miRNA) are emerging as promising diagnostic biomarkers for colorectal cancer, but their usefulness for detecting early colorectal neoplasms remains unclear. This study aimed to identify serum miRNA biomarkers for the identification of patients with early colorectal neoplasms. Experimental Design: A cohort of 237 serum samples from 160 patients with early colorectal neoplasms (148 precancerous lesions and 12 cancers) and 77 healthy subjects was analyzed in a three-step approach that included a comprehensive literature review for published biomarkers, a screening phase, and a validation phase. RNA was extracted from sera, and levels of miRNAs were examined by real-time RT-PCR. Results: Nine miRNAs (miR-18a, miR-19a, miR-19b, miR-20a, miR-21, miR-24, miR-29a, miR-92, and miR-125b) were selected as candidate biomarkers for initial analysis. In the screening phase, serum levels of miR-21, miR-29a, and miR-125b were significantly higher in patients with early colorectal neoplasm than in healthy controls. Elevated levels of miR-21, miR-29a, and miR-125b were confirmed in the validation phase using an independent set of subjects. Area under the curve (AUC) values for serum miR-21, miR-29a, miR-125b, and their combined score in discriminating patients with early colorectal neoplasm from healthy controls were 0.706, 0.741, 0.806, and 0.827, respectively. Serum levels of miR-29a and miR-125b were significantly higher in patients who had only small colorectal neoplasms (≤5 mm) than in healthy subjects. Conclusions: Because serum levels of miR-21, miR-29a, and miR-125b discriminated patients with early colorectal neoplasm from healthy controls, our data highlight the potential clinical use of these molecular signatures for noninvasive screening of patients with colorectal neoplasia. Clin Cancer Res; 21(18); 4234–42. ©2015 AACR.

Recent Advancements in Comprehensive Genetic Analyses for Human Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) typically develops in the liver with chronic hepatitis and cirrhosis, and activation of oncogenes and inactivation of tumor suppressor genes occurs during carcinogenesis via genetic and epigenetic mechanisms. Recent advancements in the development of analyses for examining the cancer genome have revealed information regarding genetic alterations in HCC tissues. According to previous studies, the incidence of recurrent genetic alterations in individual genes was thought to be relatively rare and limited to a subset of a few cancer-specific genes such as tumor suppressor p53, RB genes and oncogenes such as CTNNB1. However, recent whole-genome analyses and exome sequencing of tumor DNA have revealed numerous novel alterations of cancer-related genes and pathways critical for HCC development. In addition, various risk factors for HCC, such as the presence or absence of hepatitis B and C virus, may affect the mutation profile of the corresponding cancer genome. On the other hand, genome-wide association studies have also identified important single-nucleotide polymorphisms involved in HCC development, which may allow detection of a group at high risk of HCC emergence. Such analyses will clarify how this malignancy can be treated, diagnosed and prevented more effectively.