Mika Wakabayashi

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.

Interleukin-1β induced by Helicobacter pylori infection enhances mouse gastric carcinogenesis.

Interleukin-1β (Il1b) is considered to be involved in Helicobacter pylori (HP)-induced human gastric carcinogenesis, while the role of its polymorphisms in gastric cancer susceptibility remains controversial. Here, we aimed to clarify the role of HP infection-induced IL1B in gastric inflammation and carcinogenesis using Il1b(-/-) (Il1b-null) mice. In gastric mucosa of the Il1b(+/+) (WT) mice, HP infection induced Il1b expression and severe inflammation. In contrast, in Il1b-null mice, recruitment of neutrophils and macrophages by HP infection was markedly suppressed. In a carcinogenicity test, the multiplicity of gastric tumors was significantly suppressed in theIl1b-null mice (58% of WT; P<0.005). Mechanistically, HP infection induced NF-κB activation both in the inflammatory and epithelial cells in gastric mucosae, and the activation was attenuated in the Il1b-null mice. Accordingly, increased proliferation and decreased apoptosis of gastric epithelial cells induced by HP infection in the WT mice were attenuated in the Il1b-null mice. These results demonstrated that the IL1B physiologically induced by HP infection enhanced gastric carcinogenesis by affecting both inflammatory and epithelial cells.

Induction of aberrant trimethylation of histone H3 lysine 27 by inflammation in mouse colonic epithelial cells

A field for cancerization (field defect), where genetic and epigenetic alterations are accumulated in normal-appearing tissues, is involved in human carcinogenesis, especially cancers associated with chronic inflammation. Although aberrant DNA methylation is involved in the field defect and induced by chronic inflammation, it is still unclear for trimethylation of histone H3 lysine 27 (H3K27me3), which is involved in gene repression independent of DNA methylation and functions as a pre-mark for aberrant DNA methylation. In this study, using a mouse colitis model induced by dextran sulfate sodium (DSS), we aimed to clarify whether aberrant H3K27me3 is induced by inflammation and involved in a field defect. ChIP-on-chip analysis of colonic epithelial cells revealed that H3K27me3 levels were increased or decreased for 266 genomic regions by aging, and more extensively (23 increased and 3574 decreased regions) by colitis. Such increase or decrease of H3K27me3 was induced as early as 2 weeks after the initiation of DSS treatment, and persisted at least for 16 weeks even after the inflammation disappeared. Some of the aberrant H3K27me3 in colonic epithelial cells was carried over into colon tumors. Furthermore, H3K27me3 acquired at Dapk1 by colitis was followed by increased DNA methylation, supporting its function as a pre-mark for aberrant DNA methylation. These results demonstrated that aberrant H3K27me3 can be induced by exposure to a specific environment, such as colitis, and suggested that aberrant histone modification, in addition to aberrant DNA methylation, is involved in the formation of a field defect.

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.

Frequent involvement of chromatin remodeler alterations in gastric field cancerization.

A field for cancerization, or a field defect, is formed by the accumulation of genetic and epigenetic alterations in normal-appearing tissues, and is involved in various cancers, especially multiple cancers. Epigenetic alterations are frequently present in chronic inflammation-exposed tissues, but information on individual genes involved in the formation of a field defect is still fragmental. Here, using non-cancerous gastric tissues of cancer patients, we isolated 16 aberrantly methylated genes, and identified chromatin remodelers ACTL6B and SMARCA1 as novel genes frequently methylated in non-cancerous tissues. SMARCA1 was expressed at high levels in normal gastric tissues, but was frequently silenced by aberrant methylation in gastric cancer cells. Moreover, somatic mutations of additional chromatin remodelers, such as ARID1A, SMARCA2, and SMARCA4, were found in 30% of gastric cancers. Mutant allele frequency suggested that the majority of cancer cells harbored a mutation when present. Depletion of a chromatin remodeler, SMARCA1 or SMARCA2, in cancer cell lines promoted their growth. These results showed that epigenetic and genetic alterations of chromatin remodelers are induced at an early stage of carcinogenesis and are frequently involved in the formation of a field defect.

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)

Degree of methylation burden is determined by the exposure period to carcinogenic factors

Aberrant DNA methylation accumulated in normal tissues, namely methylation burden, is associated with risk of carcinogenesis. The levels of methylation burden are known to be influenced by multiple factors, such as genetic factors and strengths of carcinogenic factors. However, the impact of the degree of exposure to a carcinogenic factor is still unclear. Here, using a Mongolian gerbil model of Helicobacter pylori (H. pylori)‐induced gastritis, we aimed to clarify the impact of the degree of exposure on methylation burden in normal gastric tissues. DNA methylation levels of four CpG islands, HE6, SA9, SB5, and SD2, increased by H. pylori infection, depending upon the infection period. After eradication of H. pylori, DNA methylation levels decreased, but tended to be higher in gastric mucosae with a longer infection period. DNA molecules with dense methylation, but not those with sparse methylation, increased depending upon the infection period. DNA methylation levels of one of the four CpG islands, SA9, tended to be higher in gastric mucosae of gerbils infected with H. pylori, even 50 weeks after eradication than in those of non‐infected gerbils. These results showed for the first time that the levels of methylation burden in normal tissues are influenced by the degree of exposure to a carcinogenic factor.

Abstract 5331: Repression ofTETgenes and enhancement of DNA methyltransferase activity are critical for induction of aberrant DNA methylation

Chronic inflammation is deeply involved in the development of human cancers by inducing epigenetic alterations, such as aberrant DNA methylation. Among various inflammatory cytokines, the expression of Il1b, Tnf, and Nos2, is associated with induction of aberrant DNA methylation. However, the molecular mechanisms by which these cytokines induce aberrant DNA methylation remain unclear. Here, we show that the activation of the NF-kB signaling pathway, downstream of IL-1β and TNF-α, caused up-regulation of specific miRNAs, such as miR-20a, miR-26b, and miR-29c, and that these miRNAs caused repression of the Tet methylcytosine dioxygenases (Tet) genes, Tet1, Tet2, and Tet3. TET repression by overexpression of one of these miRNAs in cultured cells appeared to be insufficient for induction of aberrant DNA methylation as detected by an Infinium MethylationEPIC BeadChip array. However, triple knockout of TET genes induced strong DNA methylation at thousands of genomic loci. At the same time, exposure to nitric oxide, produced by Nos2, enhanced enzymatic activity of DNA methyltransferases (DNMTs). Treatment of cultured cells with nitric oxide induced weak DNA methylation at hundreds of genomic loci. These results show that both the repression of TET genes and enhancement of DNMT activity, induced by chronic inflammation, are critical for induction of aberrant DNA methylation. 99Vicious99 combination of such dysregulations might have a synergistic effect on induction of aberrant DNA methylation. Citation Format: Hideyuki Takeshima, Tohru Niwa, Harumi Yamada, Satoshi Yamashita, Mika Wakabayashi, Toshikazu Ushijima. Repression of TET genes and enhancement of DNA methyltransferase activity are critical for induction of aberrant DNA methylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5331.

Abstract 2314: The combination of DNA methylation and H3K27me3 is a cancer-specific dual epigenetic modification

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Alterations of epigenetic modifications are promising targets of cancer therapy, and several epigenetic drugs are now used in the clinical field. At the same time, individual epigenetic modifications have physiological roles. Therefore, a cancer-specific combination of epigenetic modifications (dual modifications) is considered to be better as a therapeutic target. However, it is unclear whether or not such cancer-specific dual modification is present. In this study, we aimed to clarify whether cancer-specific dual modification is present by focusing on the combination of two major repressive modifications, DNA methylation and trimethylation of histone H3 lysine 27 (H3K27me3). DNA methylation and H3K27me3 analyses in human colon, breast, and prostate cancer cell lines by Infinium HumanMethylation450 and ChIP-on-chip, respectively, revealed that 24.7±4.1% of DNA methylated genes were co-localized with H3K27me3 in cancer cells. In contrast, only 11.8±7.1% of DNA methylated genes were co-localized with H3K27me3 in their normal counterpart cells. 29.9±17.4% of genes with the dual modifications had neither DNA methylation nor H3K27me3 in normal counterpart cells. Expression levels of genes with the dual modifications were as low as those of genes with only DNA methylation in cancer cells. Analysis of re-activation of genes with the dual modifications by a DNA demethylating reagent, 5-aza-2′-deoxycytidine (5-aza-dC), and/or an EZH2 inhibitor, GSK126, revealed that genes with the dual modifications, such as IGFBP7, SFRP1, and SLC6A15, were re-activated more efficiently by the combination treatment than by a single treatment with 5-aza-dC. In contrast, such a combination effect on gene re-activation was not observed for genes with only DNA methylation. Furthermore, the combination treatment by 5-aza-dC and GSK126 had an additive inhibitory effect on growth of cancer cells. These results showed that genes with the dual modifications, DNA methylation and H3K27me3, are increased in cancer cells, and the combination of a DNA demethylating reagent and an EZH2 inhibitor is useful for re-activation of these genes. Citation Format: Hideyuki Takeshima, Mika Wakabayashi, Naoko Hattori, Satoshi Yamashita, Toshikazu Ushijima. The combination of DNA methylation and H3K27me3 is a cancer-specific dual epigenetic modification. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2314. doi:10.1158/1538-7445.AM2014-2314

Abstract 5349: Aberrant trimethylation of histone H3 lysine 27 is induced by chronic inflammation in mouse colonic epithelial cells, and is involved in the formation of a field for cancerization.

A field for cancerization (field defect), where genetic and epigenetic alterations are accumulated in normal-appearing tissues, is involved in human carcinogenesis, especially cancers associated with chronic inflammation. While aberrant DNA methylation is involved in the field defect and induced by chronic inflammation [Ushijima and Hattori, Clin Cancer Res, 2012], it is still unclear for trimethylation of histone H3 lysine 27 (H3K27me3), which is involved in gene repression independent of DNA methylation and functions as a pre-mark for aberrant DNA methylation. In this study, using a mouse colitis model induced by dextran sulfate sodium (DSS), we aimed to clarify whether or not aberrant H3K27me3 is induced by inflammation and involved in a field defect. Analysis of H3K27me3 levels in colonic epithelial cells by chromatin immunoprecipitation combined with CpG island microarray analysis (ChIP-on-chip analysis) revealed that H3K27me3 levels were increased or decreased for 266 genomic regions by aging, and more extensively (27 increased and 3,782 decreased regions) by colitis. Among the 3,809 regions with alterations by DSS-induced colitis, 212 regions overlapped with those whose alterations were induced by aging. Analysis of the temporal profiles of H3K27me3 levels in the course of DSS treatment revealed that increase or decrease of H3K27me3 was induced as early as two weeks after the initiation of DSS treatment, and persisted at least for 16 weeks, even after the inflammation disappeared. Some of the aberrant H3K27me3 in colonic epithelial cells was carried over into colon tumors. Furthermore, H3K27me3 acquired at Dapk1 by colitis was followed by increased DNA methylation, supporting its function as a pre-mark for aberrant DNA methylation [Takeshima et al., Carcinogenesis, in press]. Regarding mechanisms of aberrant H3K27me3 induction, expression changes of polycomb-related genes including Ezh2, Kdm6a, and Kdm6b, were not involved. These results demonstrated that aberrant H3K27me3 can be induced by exposure to a specific environment, such as colitis, and suggested that aberrant histone modification, in addition to aberrant DNA methylation, is involved in the formation of a field defect. Citation Format: Hideyuki Takeshima, Daigo Ikegami, Mika Wakabayashi, Tohru Niwa, Young-Joon Kim, Toshikazu Ushijima. Aberrant trimethylation of histone H3 lysine 27 is induced by chronic inflammation in mouse colonic epithelial cells, and is involved in the formation of a field for cancerization. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5349. doi:10.1158/1538-7445.AM2013-5349