Eriko Okochi-Takada

Aberrant methylations in cancer cells: where do they come from?

Cancer epigenetics is rapidly moving into a translational phase, and knowledge on how aberrant DNA methylation is induced is becoming important. Aging, chronic inflammation, and viral infections are known to promote methylation of non‐core regions of promoter CpG islands (CGI). The non‐core methylation and ‘seeds of methylation’, scattered methylation in a CGI, are considered to serve as triggers for dense methylation of a promoter CGI, which permanently represses expression of its downstream gene. Decreased gene transcription is an important factor that promotes induction of dense methylation. The presence of the CGI methylator phenotype (CIMP), in which methylation of multiple CGI was observed, is under dispute. Some gastric cancer cell lines have increased rates of de novo methylation, and neuroblastoma cases with CIMP show qualitatively different prognosis from those without. This strongly supports the presence of CIMP, but it seems to contain multiple entities. Limited knowledge is available for epimutagens, the chemicals that induce DNA demethylation or methylation. We have developed an assay system to detect demethylating agents, and an assay system for methylating agents is necessary. Efforts in the field on how aberrant methylation is induced will lead to new cancer prevention, diagnostics, and therapeutics. (Cancer Sci 2005; 96: 206 –211)

Silencing of the UCHL1 gene in human colorectal and ovarian cancers

Aberrant DNA methylation is associated with many types of human cancers. To identify genes silenced in human colorectal cancers, we performed a microarray analysis for genes whose expression was induced by treatment of HCT116 human colon cancer cells with a demethylating agent, 5‐aza‐2′‐deoxycitidine (5‐aza‐dC). Seven known genes were identified as being upregulated (≥8‐fold) and expressed at more than twice as high as the average level. Among these was the UCHL1 gene (also known as PGP9.5), which is involved in regulation of cellular ubiquitin levels. A dense CpG island in its promoter region was completely methylated in HCT116 cells, and no mRNA was detected. 5‐Aza‐dC treatment of HCT116 cells induced dose‐dependent demethylation of the CpG island, and restored UCHL1 mRNA and protein expression. UCHL1 silencing was observed in 11 of 12 human colorectal cancer cell lines, and its methylation was detected in 8 of 17 primary colorectal cancers. Further, UCHL1 silencing was observed in 6 of 13 ovarian cancer cell lines, and its methylation was detected in 1 of 17 primary ovarian cancers. These results showed that UCHL1 is inactivated in human colorectal and ovarian cancers by its promoter methylation, and suggest that disturbance of cellular ubiquitin levels is present.

Silencing of tissue factor pathway inhibitor‐2 gene in malignant melanomas

To identify tumor‐suppressor genes inactivated by aberrant methylation of promoter CpG islands (CGIs) in human malignant melanomas, genes upregulated by treatment of cells with a demethylating agent, 5‐aza‐2′‐deoxycytidine (5‐aza‐dC), were searched for using oligonucleotide microarrays in melanoma cell lines, HMV‐I, MeWo and WM‐115. Seventy‐nine known genes with CGIs were identified as being upregulated (≥16‐fold), and 18 of them had methylation of their putative promoter CGIs in 1 or more of 8 melanoma cell lines. Among the 18 genes, TFPI‐2, which is involved in repression of the invasive potential of malignant melanomas, was further analyzed. Its expression was repressed in a melanoma cell line with its complete methylation, and was restored by 5‐aza‐dC treatment. It was unmethylated in cultured neonatal normal epidermal melanocyte, and was induced by ultraviolet B. In surgical melanoma specimens, TFPI‐2 methylation was detected in 5 of 17 metastatic site specimens (29%), while it was not detected in 20 primary site specimens (0%) (p = 0.009). By immunohistochemistry, the 5 specimens with promoter methylation lacked immunoreactivity for TFPI‐2. The results showed that TFPI‐2 is silenced in human malignant melanomas by methylation of its promoter CGI and suggested that its silencing is involved in melanoma metastasis.

Association between frequent CpG island methylation and HER2 amplification in human breast cancers

The presence of frequent methylation of CpG islands (CGIs), designated as the CpG island methylator phenotype in some cancers, is associated with distinct clinicopathological characteristics, including gene amplification, in individual tumor types. Amplification of HER2 in human breast cancers is an important prognostic and therapeutic target, but an association between HER2 amplification and frequent CGI methylation is unknown. To clarify the association, we here quantified methylation levels of promoter CGIs of 11 genes, which are unlikely to confer growth advantage to cells, in 63 human breast cancers. The number of methylated genes in a cancer did not obey a bimodal distribution, and the 63 cancers were classified into those with frequent methylation (n = 16), moderate methylation (n = 26) and no methylation (n = 21). The incidence of HER2 amplification was significantly higher in the cancers with frequent methylation (11 of 16) than in those with no methylation (2 of 21, P = 0.001). Also, the number of methylated genes correlated with the degree of HER2 amplification (r = 0.411, P = 0.002). Correlation analysis with clinicopathological characteristics and methylation of CDKN2A, BRCA1 and CDH1 revealed that frequent methylation had significant correlation with higher nuclear grades (P = 0.001). These showed that frequent methylation had a strong association with HER2 amplification in breast cancers and suggested that frequent methylation can be a determinant of various characteristics in a fraction of human breast cancers.

Framework analysis for the carcinogenic mode of action of nitrobenzene.

Nitrobenzene (CASRN: 98-95-3) has been shown to induce cancers in many tissues including kidney, liver, and thyroid, following chronic inhalation in animals. However, with a few exceptions, genotoxicity assays using nitrobenzene have given negative results. Some DNA binding/adduct studies have brought forth questionable results and, considering the available weight of evidence, it does not appear that nitrobenzene causes cancer via a genotoxic mode of action. Nitrobenzene produces a number of free radicals during its reductive metabolism, in the gut as well as at the cellular level, and generates superoxide anion as a by-product during oxidative melabolism. The reactive species generated during nitrobenzene metabolism are considered candidates for carcinogenicity. Furthermore, several lines of evidence suggest that nitrobenzene exerts its carcinogenicity through a non-DNA reactive (epigenetic) fashion, such as a strong temporal relationship between non-, pre-, and neoplastic lesions leading to carcinogenesis. In this report, we first describe the absorption, distribution, metabolism, and excretion of nitrobenzene followed by a summary of the available genotoxicity studies and the only available cancer bioassay. We subsequently refer to the mode of action framework of the U.S. Environmental Protection Agencys 2005 Guidelines for Carcinogen Risk Assessment as a basis for presenting possible modes of action for nitrobenzene-induced cancers of the liver, thyroid, and kidney, as supported by the available experimental data. The rationale(s) regarding human relevance of each mode of action is also presented. Finally, we hypothesize that the carcinogenic mode of action for nitrobenzene is multifactorial in nature and reflective of free radicals, inflammation, and/or altered methylation. †These authors contributed equally to this work.

Development of a novel approach, the epigenome-based outlier approach, to identify tumor-suppressor genes silenced by aberrant DNA methylation.

Identification of tumor-suppressor genes (TSGs) silenced by aberrant methylation of promoter CpG islands (CGIs) is important, but hampered by a large number of genes methylated as passengers of carcinogenesis. To overcome this issue, we here took advantage of the fact that the vast majority of genes methylated in cancers lack, in normal cells, RNA polymerase II (Pol II) and have trimethylation of histone H3 lysine 27 (H3K27me3) in their promoter CGIs. First, we demonstrated that three of six known TSGs in breast cancer and two of three in colon cancer had Pol II and lacked H3K27me3 in normal cells, being outliers to the general rule. BRCA1, HOXA5, MLH1, and RASSF1A had high Pol II, but were expressed only at low levels in normal cells, and were unlikely to be identified as outliers by their expression statuses in normal cells. Then, using epigenome statuses (Pol II binding and H3K27me3) in normal cells, we made a genome-wide search for outliers in breast cancers, and identified 14 outlier promoter CGIs. Among these, DZIP1, FBN2, HOXA5, and HOXC9 were confirmed to be methylated in primary breast cancer samples. Knockdown of DZIP1 in breast cancer cell lines led to increases of their growth, suggesting it to be a novel TSG. The outliers based on their epigenome statuses contained unique TSGs, including DZIP1, compared with those identified by the expression microarray data. These results showed that the epigenome-based outlier approach is capable of identifying a different set of TSGs, compared to the expression-based outlier approach.

Hereditary diffuse gastric cancer in a Japanese family with a large deletion involving CDH1

Abstract Hereditary diffuse gastric cancer (HDGC), characterized by susceptibility to gastric signet ring cell carcinomas (SRCCs) and caused by CDH1 germline mutations, is rare in the Japanese. We present here a Japanese family with HDGC identified by comparative genomic hybridization (CGH) analysis. A 55-year-old woman was treated with completion gastrectomy for multiple SRCCs, and pathological examination revealed approximately 200 foci of SRCC with loss of E-cadherin expression. Her 30-year-old son had surveillance endoscopy and was found to have multiple SRCCs. He underwent total gastrectomy, and 32 foci of SRCC with loss of E-cadherin expression were histologically found. Although no point mutations were detected in CDH1 by sequencing, CGH revealed a 275-kb deletion involving exons 7–16 of CDH1 in both patients. While only a few HDGCs have been reported in East Asia, patients with multiple SRCC may need to be offered appropriate genetic counseling and testing in this area.

Methylation silencing of angiopoietin-like 4 in rat and human mammary carcinomas

Aberrant DNA methylation is deeply involved in the development and progression of human breast cancers, but its inducers and molecular mechanisms are still unclear. To reveal such inducers and clarify the molecular mechanisms, animal models are indispensable. Here, to identify genes silenced by promoter DNA methylation in rat mammary carcinomas, we took a combined approach of methylated DNA immunoprecipitation (MeDIP)–CpG island (CGI) microarray analysis and expression microarray analysis after treatment with epigenetic drugs. MeDIP‐CGI microarray revealed that among 5031 genes with promoter CGI, 465 were methylated in a carcinoma cell line induced by 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP), but not in normal mammary epithelial cells. By treatment of the cell line with 5‐aza‐2′‐deoxycytidine and trichostatin A, 29 of the 465 genes were shown to be re‐expressed. In primary mammary carcinomas, five (Angptl4, Coro1a, RGD1304982, Tmem37 and Ndn) of the 29 genes were methylated in one or more of 25 samples. Quantitative expression analysis revealed that Angptl4 had high expression in normal mammary glands, but low expression in primary carcinomas. Also in humans, ANGPTL4 was unmethylated and expressed in normal mammary epithelial cells, but was methylated in 11 of 91 (12%) primary breast cancers. This is the first study to identify genes aberrantly methylated in rat mammary carcinomas, and Angptl4 is a novel methylation‐silenced gene both in rat and human mammary carcinomas. The combination of the MeDIP‐CGI microarray analysis and expression microarray analysis after treatment with epigenetic drugs was effective in reducing the number of methylated genes that are not methylation silenced. (Cancer Sci 2011; 102: 1337–1343)

Abstract LB-378: The outlier approach discriminates drivers from passengers among genes methylated in cancers

A large number of genes are methylated in cancers, and most of them are methylated as passengers of carcinogenesis, and this fact hampers identification of drivers, tumor-suppressor genes (TSGs), silenced by aberrant methylation of promoter CpG islands (CGIs). To overcome this issue, we focused on the rule that the vast majority of genes methylated in cancers lack, in normal cells, RNA polymerase II (Pol II) and have trimethylation of histone H3 lysine 27 (H3K27me3) in their promoter CGIs. However, approximately 5% of the genes methylated in cancers are against this general rule, constituting a group of “outliers”. It is expected that TSGs belong to this group of outliers since they are expressed or ready to be expressed in normal cells. Here, we aimed to demonstrate that TSGs belong to the group of outliers, and that searching for outliers enables us to identify TSGs. First, we demonstrated that some of known TSGs in breast and colon cancers had Pol II and lacked H3K27me3 in normal cells, being outliers of the rule. We then made a genome-wide search for outlier genes in breast cancers. Based on Pol II binding and H3K27me3 statuses in normal cells and DNA methylation statuses in five breast cancer cell lines, 14 outlier genes were identified from 280 methylated genes. Among these genes, four genes were confirmed to be methylated in primary breast cancer samples, and two (HOXA5 and FBN2) of these were known TSGs. Among the remaining two genes, DZIP1 was shown to suppress growth of breast cancer cells, suggesting it to be a novel TSG. These results showed that some of known TSGs are the outlier genes and that TSGs can be efficiently identified by searching for outliers against the rule, the outlier approach. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-378. doi:1538-7445.AM2012-LB-378

Abstract 141: Identification of a gene silenced in rat mammary carcinomas by combination of DNA methylation microarray and chemical genomic screening

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Aberrant DNA methylation is deeply involved in the development and progression of human breast cancers, but its inducers and molecular mechanisms are still unclear. To reveal such inducers and analyze molecular mechanisms of methylation induction, animal models are indispensable. However, genes methylation-silenced in the animal models of breast cancer are unknown, and identification of inducers and analysis of mechanisms are hampered. Here, to identify genes methylation-silenced in rat primary mammary carcinomas, we took a combined approach of methylated DNA immunoprecipitation (MeDIP)-CpG island (CGI) microarray and chemical genomic screening. MeDIP-CGI microarray analysis of a carcinoma cell line induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) revealed that, among 4,680 promoter CGIs analyzed, 664 were methylated in the cell line. Sixty-two of the 664 genes were re-expressed by treatment of the cell line with 5-aza-2’-deoxycytidine and trichostatin A. An unmethylated status in normal mammary glands was confirmed for 31 of the 62 genes by MeDIP-CGI microarray analyses of a pool of 4 normal mammary glands. Among 13 primary mammary carcinomas induced by PhIP, five of the 31 genes were methylated in one carcinoma or more. Among 12 primary carcinomas induced by 7,12-dimethylbenz[a]anthracene, methylation of all the five genes was also observed. RT-PCR analysis revealed that one of the five genes had high expression in normal mammary glands, but low expression in carcinomas, showing that this gene was methylation-silenced in rat primary mammary carcinomas. In humans, the gene was expressed in mammary epithelial cells that had unmethylated DNA, and was down-regulated in three cancer cell lines that had methylated DNA. The gene was methylated in about 9% of 80 primary human breast cancers, and it was suggested that aberrant DNA methylation of this gene was a common event between rat and human breast cancers. This is the first study of genome-wide DNA methylation analysis using rat mammary carcinomas, and the methylation-silenced gene, along with the other methylated genes, is expected to be useful as a marker in identifying inducers of aberrant DNA methylation and analyzing its molecular mechanisms. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 141.