Daiya Takai

Comprehensive analysis of CpG islands in human chromosomes 21 and 22

CpG islands are useful markers for genes in organisms containing 5-methylcytosine in their genomes. In addition, CpG islands located in the promoter regions of genes can play important roles in gene silencing during processes such as X-chromosome inactivation, imprinting, and silencing of intragenomic parasites. The generally accepted definition of what constitutes a CpG island was proposed in 1987 by Gardiner-Garden and Frommer [Gardiner-Garden, M. & Frommer, M. (1987) J. Mol. Biol. 196, 261–282] as being a 200-bp stretch of DNA with a C+G content of 50% and an observed CpG/expected CpG in excess of 0.6. Any definition of a CpG island is somewhat arbitrary, and this one, which was derived before the sequencing of mammalian genomes, will include many sequences that are not necessarily associated with controlling regions of genes but rather are associated with intragenomic parasites. We have therefore used the complete genomic sequences of human chromosomes 21 and 22 to examine the properties of CpG islands in different sequence classes by using a search algorithm that we have developed. Regions of DNA of greater than 500 bp with a G+C equal to or greater than 55% and observed CpG/expected CpG of 0.65 were more likely to be associated with the 5′ regions of genes and this definition excluded most Alu-repetitive elements. We also used genome sequences to show strong CpG suppression in the human genome and slight suppression in Drosophila melanogaster and Saccharomyces cerevisiae. This finding is compatible with the recent detection of 5-methylcytosine in Drosophila, and might suggest that S. cerevisiae has, or once had, CpG methylation.

Genome structure-based screening identified epigenetically silenced microRNA associated with invasiveness in non-small-cell lung cancer†

MicroRNA (miRNA) expression is frequently altered in human cancers. To search for epigenetically silenced miRNAs in non‐small‐cell lung cancer (NSCLC), we mapped human miRNAs on autosomal chromosomes and selected 55 miRNAs in silico. We treated six NSCLC cell lines with the DNA methylation inhibitor 5‐aza‐2′‐deoxycytidine (5‐aza‐CdR) and determined the expressions of the 55 miRNAs. Fourteen miRNAs were decreased in the cancer cell lines and were induced after 5‐aza‐CdR treatment. After a detailed DNA methylation analysis, we found that mir‐34b and mir‐126 were silenced by DNA methylation. Mir‐34b was silenced by the DNA methylation of its own promoter, whereas mir‐126 was silenced by the DNA methylation of its host gene, EGFL7. A chromatin immunoprecipitation assay revealed H3K9me2 and H3K9me3 in mir‐34b and EGFL7, and H3K27me3 in EGFL7. The overexpression of mir‐34b and mir‐126 decreased the expression of c‐Met and Crk, respectively. The 5‐aza‐CdR treatment of lung cancer cell line resulted in increased mir‐34b expression and decreased c‐Met protein. We next analyzed the DNA methylation status of these miRNAs using 99 primary NSCLCs. Mir‐34b and mir‐126 were methylated in 41 and 7% of all the cases, respectively. The DNA methylation of mir‐34b was not associated with c‐Met expression determined by immunohistochemistry, but both mir‐34b methylation (p = 0.007) and c‐Met expression (p = 0.005) were significantly associated with lymphatic invasion in a multivariate analysis. The DNA methylation of mir‐34b can be used as a biomarker for an invasive phenotype of lung cancer.

Methylation-Sensitive Representational Difference Analysis and Its Application to Cancer Research

Abstract: Methylation‐sensitive representational difference analysis (MS‐RDA) was previously established to detect differences in the methylation status of two genomes. This method uses the digestion of genomic DNA with a methylation‐sensitive restriction enzyme, HpaII, and PCR to prepare ‘HpaII‐amplicons,’ followed by RDA. An HpaII‐amplicon prepared using betaine and reverse electrophoresis was enriched 3.6‐fold (compared with the HpaII‐amplicon prepared by the original method) with DNA fragments originating from CpG islands (CGIs). As for the specificity of MS‐RDA, it was shown that DNA fragments that are unmethylated in the tester and almost completely methylated in the driver are efficiently isolated. This indicated that genes that are in biallelic methylation or in monoallelic methylation with loss of the other allele are efficiently isolated. Further, by use of two additional methylation‐sensitive six‐base recognition restriction enzymes, SacII and NarI, more DNA fragments were isolated from CGIs in the 5′ regions of genes. After analysis of human lung, gastric, and breast cancers, 12 genes were seen to be silenced and additional genes seen to show decreased expression in association with methylation of genomic regions outside CGIs in the 5′ regions of genes. MS‐RDA is effective in identifying silenced genes in various cancers.

Silencing of HTR1B and reduced expression of EDN1 in human lung cancers, revealed by methylation-sensitive representational difference analysis

Aberrantly hypermethylated genes in human lung cancers were searched for by a genome scanning technique, methylation-sensitive-representational difference analysis (MS-RDA). A total of 59 DNA fragments were isolated as those methylated more heavily in either/both of two lung squamous cell carcinoma cell lines, EBC-1 and LK-2, than in a primary culture of normal human bronchial epithelium, NHBE. Thirty-four DNA fragments, whose hypermethylation was confirmed in primary squamous cell carcinomas, were sequenced. By database searches, 17 of them were shown to be located within 2 kb of putative CpG islands, and five of the 17 DNA fragments had transcribed regions of known genes in their vicinities. By RT–PCR of the five genes in the carcinoma cell lines and NHBE, decreased expression of HTR1B (5-hydroxytryptamine receptor 1B) and EDN1 (endothelin-1) was observed. Sequencing after bisulfite modification showed that the CpG island in the promoter region of HTR1B was hypermethylated, while that of EDN1 was not. Demethylation and re-expression of HTR1B were observed after treatment of LK-2 cells with 5-aza-2′-deoxycytidine. In primary lung cancers, decreased mRNA expression of HTR1B was observed in 11 of 20 cases, and that of EDN1 was in 16 of 20 cases. Immunohistochemical analysis of endothelin-1 confirmed that its immunoreactivity was reduced in squamous cell carcinoma cells compared with that in normal bronchial epithelial cells. Considering that endothelin-1 induces apoptosis in melanoma cells and that silencing of endothelin receptor B is observed in prostate cancers, its reduced expression was speculated to confer a growth advantage to lung cancer cells. MS-RDA was shown to isolate DNA fragments that are hypermethylated and silenced, such as HTR1B, and those whose expressions are altered and the methylation statuses outside the promoter region are altered, such as EDN1.

Identification of G0S2 as a gene frequently methylated in squamous lung cancer by combination of in silico and experimental approaches

Epigenetic changes can lead to abnormal expression of genes in cancer, and several genes have been reported to have aberrant promoter DNA methylation in non‐small‐cell lung cancer (NSCLC). We identified aberrantly methylated genes in NSCLC by combination of in silico and experimental approaches. We first applied bioinformatics, and from microarray datasets, we selected genes with low expression and having functions related to cancer. Next, combined bisulfite restriction analysis was carried out in 10 pooled resected lung cancer tissues to screen for genes that were aberrantly methylated, and the methylation ratio (the fraction of methylated DNA in extracted DNA from a cancer tissue sample) was quantified using quantitative analysis of methylated alleles. We identified 8 methylated genes (ARPC1B, DNAH9, FLRT2, G0S2, IRS2, PKP1, SPOCK1 and UCHL1) previously unreported in NSCLC. Analyses of methylation profiles of 101 resected lung cancer tissue samples revealed quantitatively low methylation in whole, methylation ratios were almost less than 30% even in the methylated samples, and no significant correlation to prognosis after 2 years of follow‐up using hierarchical clustering. DNA methylation of G0S2 gene was significantly more frequent in squamous lung cancer (n = 18, mean of methylation ratios: 15%) compared with nonsquamous lung cancer (n = 83, mean of methylation ratios: 2.6%) (Mann‐Whitney U test, p < 0.001). DNA methylation of G0S2 can be an important biomarker for squamous lung cancer.

CpG island methylation of microRNAs is associated with tumor size and recurrence of non-small-cell lung cancer

We investigated whether the CpG island methylation of certain microRNAs was associated with the clinicopathological features and the prognosis of non‐small‐cell lung cancer. The methylation of mir‐152, ‐9‐3, ‐124‐1, ‐124‐2, and ‐124‐3 was analyzed in 96 non‐small‐cell lung cancer specimens using a combined bisulfite restriction analysis. The median observation period was 49.5 months. The methylation of mir‐9‐3, ‐124‐2, and ‐124‐3 was individually associated with an advanced T factor independent of age, sex, and smoking habit. Moreover, the methylation of multiple microRNA loci was associated with a poorer progression‐free survival in a univariate analysis. Our result enlightens the accumulation of aberrant DNA methylation which occurs in concordance with the tumor progression. (Cancer Sci 2011; 102: 2126–2131)

Expression of PRMT5 in lung adenocarcinoma and its significance in epithelial-mesenchymal transition.

Although protein arginine methyltransferase 5 (PRMT5) has been implicated in various cancers, its expression pattern in lung adenocarcinoma cell lines and tissues has not been elucidated enough. In this study, microarray analysis of 40 non-small-cell lung carcinoma cell lines showed that PRMT5 was a candidate histone methyltransferase gene that correlated with epithelial-mesenchymal transition. Immunocytochemical analysis of these cell lines indicated that the expression of PRMT5 was localized to the cytoplasm of E-cadherin-low and vimentin-high cell lines, whereas it was predominant in the nucleus and faint in the cytoplasm of E-cadherin-high and vimentin-low cell lines. Immunohistochemical analysis of lung adenocarcinoma cases (n = 130) revealed that the expression of PRMT5 was high in the cytoplasm of 47 cases (36%) and the nuclei of 34 cases (26%). The marked cytoplasmic expression of PRMT5 was frequently observed in high-grade subtypes (1 of 17 low grade, 21 of 81 intermediate grade, and 25 of 32 high grade; P < .0001) such as solid adenocarcinoma with the low expression of thyroid transcription factor 1 (the master regulator of lung) and low expression of cytokeratin 7 and E-cadherin (2 markers for bronchial epithelial differentiation), whereas the high nuclear expression of PRMT5 was frequently noted in adenocarcinoma in situ, a low-grade subtype (6 of 17 low grade, 25 of 81 intermediate grade, and 3 of 32 high grade; P = .0444). The cytoplasmic expression of PRMT5 correlated with a poor prognosis (P = .0089). We herein highlighted the importance of PRMT5 expression, especially its cytoplasmic expression, in the process of epithelial-mesenchymal transition and loss of the bronchial epithelial phenotype of lung adenocarcinoma.

High-grade lung adenocarcinoma with fetal lung-like morphology: clinicopathologic, immunohistochemical, and molecular analyses of 17 cases.

Low-grade lung adenocarcinoma of fetal lung type, which is well characterized by its unique clinicopathologic and molecular features, is recognized as a distinct variant of lung cancer. In contrast, high-grade lung adenocarcinoma with fetal lung–like morphology (HG-LAFM) has not been studied widely. To characterize this subset better, we analyzed 17 high-grade adenocarcinomas with at least focal component resembling a developing epithelium in the pseudoglandular phase of the fetal lung. These rare (ca. 0.4%) carcinomas occurred predominantly in elderly men with a heavy smoking history, who showed elevated serum &agr;-fetoprotein in 4 of 5 cases tested. Histologic examination revealed a fetal lung–like component as a focal finding accounting for 5% to 60% of the total tumor volume. It was invariably admixed with tissues having a morphology not resembling that of a fetal lung. A coexisting non–fetal lung–like element was quite heterogenous in appearance, showing various growth patterns. However, clear-cell (88%), hepatoid (29%), and large cell neuroendocrine carcinoma (24%) histology seemed overrepresented. HG-LAFM was characterized immunohistochemically by frequent expression of &agr;-fetoprotein (41%), glypican-3 (88%), SALL-4 (59%), neuroendocrine markers (82%), CDX-2 (35%), and p53 (65%). HG-LAFM was molecularly heterogenous in that EGFR or KRAS mutation was observed in 22% of cases tested for both. Our data indicate that HG-LAFMs might form a coherent subgroup of lung adenocarcinomas. However, the uniformly focal nature of the fetal lung–like element, widely diverse coexisting non–fetal lung–like histology, and inhomogenous molecular profiles lead us to believe that HG-LAFM is best regarded as a morphologic pattern showing characteristic association with several clinicopathologic parameters rather than a specific tumor entity.

New therapeutic key for cystic fibrosis: a role for lipoxins

One of the hallmarks of cystic fibrosis is the propensity of patients to develop lung infections with Pseudomonas aeruginosa, which eventually compromises lung function. New data suggest loss of CFTR impairs lipoxin production, thus preventing resolution of lung inflammation and creating an environment susceptible to further infection.

Disruption of the expression and function of microRNAs in lung cancer as a result of epigenetic changes

Two decades have passed since the discovery of microRNA (miRNA), which determines cell fate in nematodes. About one decade ago, the conservation of miRNA in humans was also discovered. At present, the loss of certain miRNAs and the overexpression of miRNAs have been demonstrated in many types of diseases, especially cancer. A key miRNA in lung cancer was reported soon after the initial discovery of a tumor-suppressive miRNA in a hematological malignancy. Various causes of miRNA disruption are known, including deletions, mutations, and epigenetic suppression as well as coding genes. The recent accumulation of knowledge regarding epigenetic transcriptional suppression has revealed the suppression of several miRNAs in lung cancer in response to epigenetic changes, such as H3K9 methylation prior to DNA methylation and H3K27 methylation independent of DNA methylation. In this review, recent knowledge of miRNA disruption in lung cancer as a result of epigenetic changes is discussed. Additionally, emerging cancer-specific changes in RNA editing and their impact on miRNA function are described.