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To identify methylation-silenced genes in prostate cancers, a microarray analysis for genes up-regulated by treatment with a demethylating agent, 5-aza-2′-deoxycytidine, was performed using three rat prostate cancer cell lines. Eight genes (Aebp1, Dysf, Gas6, LOC361288, Nnat, Ocm, RGD1308119, and Tgfbr2) were re-expressed at 16-fold or more, and their promoter CpG islands were shown to be densely methylated in the cancer cell lines. From the eight genes, Tgfbr2, a key mediator of transforming growth factor-β (TGF-β) signaling that has been strongly implicated in human and rat prostate carcinogenesis, was selected, and its silencing in primary samples was analyzed further. Tgfbr2 was methylated and markedly down-regulated in three of seven 3,2′-dimethyl-4-aminobiphenyl–induced invasive adenocarcinomas in the dorsolateral lobe of the rat prostate. In humans, marked down-regulation of TGFBR2 protein was observed in 12 of 20 high-grade prostatic intraepithelial neoplasia and 36 of 60 prostate cancers. DNA methylation of the human TGFBR2 promoter CpG islands repressed transcription, if present, but neither methylation nor mutation were detected in 27 human prostate cancers analyzed. Methylation silencing of rat Tgfbr2 was associated with histone H3 lysine 9 trimethylation, whereas decreased expression of human TGFBR2 was mainly due to decreased transcription activity, sometimes in concert with histone deacetylation and H3 lysine 27 trimethylation. The identification of methylation silencing of Tgfbr2 in rat prostate cancers, in accordance with TGFBR2 down-regulation in human prostate cancers, will enable us to analyze how aberrant methylation is induced in vivo and identify factors that promote and suppress the induction of aberrant methylation. [Cancer Res 2008;68(7):2112–21]

Supplementary Figure 4 from Inflammatory Processes Triggered by Helicobacter pylori Infection Cause Aberrant DNA Methylation in Gastric Epithelial Cells

Supplementary Figure 2 from Inflammatory Processes Triggered by Helicobacter pylori Infection Cause Aberrant DNA Methylation in Gastric Epithelial Cells

Supplementary Table 3 from Methylation Silencing of Transforming Growth Factor-β Receptor Type II in Rat Prostate Cancers

Supplementary Figure Legends 1-8 from Inflammatory Processes Triggered by Helicobacter pylori Infection Cause Aberrant DNA Methylation in Gastric Epithelial Cells

Supplementary Figure 3 from Inflammatory Processes Triggered by Helicobacter pylori Infection Cause Aberrant DNA Methylation in Gastric Epithelial Cells

Supplementary Table 5 from Methylation Silencing of Transforming Growth Factor-β Receptor Type II in Rat Prostate Cancers

Supplementary Tables1-4 from Inflammatory Processes Triggered by Helicobacter pylori Infection Cause Aberrant DNA Methylation in Gastric Epithelial Cells

Supplementary Figure 5 from Inflammatory Processes Triggered by Helicobacter pylori Infection Cause Aberrant DNA Methylation in Gastric Epithelial Cells

Supplementary Figure 1 from Inflammatory Processes Triggered by Helicobacter pylori Infection Cause Aberrant DNA Methylation in Gastric Epithelial Cells

Supplementary Table 6 from Methylation Silencing of Transforming Growth Factor-β Receptor Type II in Rat Prostate Cancers

Supplementary Table 2 from Methylation Silencing of Transforming Growth Factor-β Receptor Type II in Rat Prostate Cancers

Supplementary Table 1 from Methylation Silencing of Transforming Growth Factor-β Receptor Type II in Rat Prostate Cancers

Supplementary Table 4 from Methylation Silencing of Transforming Growth Factor-β Receptor Type II in Rat Prostate Cancers

Supplementary Figures 6-8 from Inflammatory Processes Triggered by Helicobacter pylori Infection Cause Aberrant DNA Methylation in Gastric Epithelial Cells

Chronic inflammation is deeply involved in various human disorders, such as cancer, neurodegenerative disorders, and metabolic disorders. Induction of epigenetic alterations, especially aberrant DNA methylation, is one of the major mechanisms, but how it is induced is still unclear. Here, we found that expression of TET genes, methylation erasers, was downregulated in inflamed mouse and human tissues, and that this was caused by upregulation of TET-targeting miRNAs such as MIR20A, MIR26B, and MIR29C, likely due to activation of NF-κB signaling downstream of IL-1β and TNF-α. However, TET knockdown induced only mild aberrant methylation. Nitric oxide (NO), produced by NOS2, enhanced enzymatic activity of DNA methyltransferases (DNMTs), methylation writers, and NO exposure induced minimal aberrant methylation. In contrast, a combination of TET knockdown and NO exposure synergistically induced aberrant methylation, involving genomic regions not methylated by either alone. The results showed that a vicious combination of TET repression, due to NF-κB activation, and DNMT activation, due to NO production, is responsible for aberrant methylation induction in human tissues.

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.

Chronic inflammation is involved in the development of colon cancer by inducing mutations and aberrant DNA methylation in colon epithelial cells. Furthermore, there is growing evidence that colonic microbiota modulates the inflammation response in the host and influences colon tumorigenesis. However, the influence of colonic microbiota on aberrant DNA methylation remains unknown. Here, we show the effect of colonic microbes on DNA methylation and tumorigenicity using a mouse model of human ulcerative colitis. Mice treated with azoxymethane (AOM) and dextran sulfate sodium (DSS) showed an increase in degree of colitis, as estimated by body weight, occult blood, and stool consistency/diarrhea at 2 weeks after treatment, but treatment with antibiotics markedly reduced the severity of the colitis. Although mucosal hyperplasia and increased inflammation‐related genes were observed in the colonic epithelial cells of the AOM/DSS‐treated mice, treatment with antibiotics abrogated these changes. In addition, treatment with antibiotics significantly decreased the number of mucosal nodules from 5.9 ± 5.3 to 0.2 ± 0.6 (P

The recent development of next-generation sequencing technology for extensive mutation analysis, and beadarray technology for genome-wide DNA methylation analysis has made it possible to obtain integrated pictures of genetic and epigenetic alterations, using the same cancer samples. In this study, we aimed to characterize such a picture in esophageal squamous cell carcinomas (ESCCs). Base substitutions of 55 cancer-related genes and copy number alterations (CNAs) of 28 cancer-related genes were analyzed by targeted sequencing. Forty-four of 57 ESCCs (77%) had 64 non-synonymous somatic mutations, and 24 ESCCs (42%) had 35 CNAs. A genome-wide DNA methylation analysis using an Infinium HumanMethylation450 BeadChip array showed that the CpG island methylator phenotype was unlikely to be present in ESCCs, a different situation from gastric and colon cancers. Regarding individual pathways affected in ESCCs, the WNT pathway was activated potentially by aberrant methylation of its negative regulators, such as SFRP1, SFRP2, SFRP4, SFRP5, SOX17, and WIF1 (33%). The p53 pathway was inactivated by TP53 mutations (70%), and potentially by aberrant methylation of its downstream genes. The cell cycle was deregulated by mutations of CDKN2A (9%), deletions of CDKN2A and RB1 (32%), and by aberrant methylation of CDKN2A and CHFR (9%). In conclusion, ESCCs had unique methylation profiles different from gastric and colon cancers. The genes involved in the WNT pathway were affected mainly by epigenetic alterations, and those involved in the p53 pathway and cell cycle regulation were affected mainly by genetic alterations. © 2016 Wiley Periodicals, Inc.

Epigenetic alterations underlie various human disorders, including cancer, and this has resulted in the development of drugs targeting epigenetic alterations. Although DNA demethylating agents are one of the major epigenetic drugs, only two compounds-5-azacytidine (5-aza-CR, azacitidine) and 5-aza-2'-deoxycytidine (5-aza-dC, decitabine)-have obtained clinical approval. Here, we aimed to establish a detection system for DNA demethylating agents suitable for a high-throughput screening (HTS) in mammalian cells. We inserted luciferase and EGFP reporter genes under the UCHL1 promoter, which is methylation-silenced in human colon cancers and can be readily demethylated to drive strong expression. Methylated UCHL1 promoter was introduced into HCT116 colon cancer cells, and transfectants with methylated exogenous UCHL1 promoter were obtained. By screening subclones from each of the epigenetically heterogeneous transfectant clones, we finally obtained three optimal subclones that expressed luciferase and EGFP after 5-aza-dC treatment with high signal-to-noise ratios. Nucleosomes with H3K9me2 were present around the exogenous UCHL1 promoter in all three subclones. Using one of the subclones (HML58-3), HTS was conducted using 19,840 small molecules. Two hit compounds were obtained, and these turned out to be 5-aza-dC and 5-aza-CR. The assay system constructed here demonstrates a robust response to DNA demethylating agents, along with high specificity, and will be useful for screening and biological assays in epigenetics.

Aims Primary central nervous system lymphoma (PCNSL) manifest aggressive clinical behaviour and have poor prognosis. Although constitutive activation of the nuclear factor-κB (NF-κB) pathway has been documented, knowledge about the genetic alterations leading to the impairment of the NF-κB pathway in PCNSLs is still limited. This study was aimed to unravel the underlying genetic profiles of PCNSL. Methods We conducted the systematic sequencing of 21 genes relevant to the NF-κB signalling network for 71 PCNSLs as well as the pyrosequencing of CD79B and MYD88 mutation hotspots in a further 35 PCNSLs and 46 glioblastomas (GBMs) for validation. Results The results showed that 68 out of 71 PCNSLs had mutations in the NF-κB gene network, most commonly affecting CD79B (83%), MYD88 (76%), TBL1XR1 (23%), PRDM1 (20%) and CREBBP1 (20%). These mutations, particularly CD79B and MYD88, frequently coincided within each tumour in various combinations, simultaneously affecting diverse pathways within the network. No GBMs had hotspot mutation of CD79B Y196 and MYD88 L265. Conclusions The prevalence of CD79B and MYD88 mutations in PCNSLs was considerably higher than reported in systemic diffuse large B-cell lymphomas. This observation could reflect the paucity of antigen stimuli from the immune system in the central nervous system (CNS) and the necessity to substitute them by the constitutive activation of CD79B and MYD88 that would initiate the signalling cascades. These hotspot mutations may serve as a genetic hallmark for PCNSL serving as a genetic marker for diagnose and potential targets for molecular therapy.

The profiles of genetic and epigenetic alterations in cancer-related pathways are considered to be useful for selection of patients likely to respond to specific drugs, including molecular-targeted and epigenetic drugs. In this study, we aimed to characterize such profiles in gastric cancers (GCs).Genetic alterations of 55 cancer-related genes were analyzed by a benchtop next-generation sequencer. DNA methylation statuses were analyzed by a bead array with 485,512 probes.The WNT pathway was activated by mutations of CTNNB1 in 2 GCs and potentially by aberrant methylation of its negative regulators, such as DKK3, NKD1, and SFRP1, in 49 GCs. The AKT/mTOR pathway was activated by mutations of PIK3CA and PTPN11 in 4 GCs. The MAPK pathway was activated by mutations and gene amplifications of ERBB2, FLT3, and KRAS in 11 GCs. Cell-cycle regulation was affected by aberrant methylation of CDKN2A and CHFR in 13 GCs. Mismatch repair was affected by a mutation of MLH1 in 1 GC and by aberrant methylation of MLH1 in 2 GCs. The p53 pathway was inactivated by mutations of TP53 in 19 GCs and potentially by aberrant methylation of its downstream genes in 38 GCs. Cell adhesion was affected by mutations of CDH1 in 2 GCs.Genes involved in cancer-related pathways were more frequently affected by epigenetic alterations than by genetic alterations. The profiles of genetic and epigenetic alterations are expected to be useful for selection of the patients who are likely to benefit from specific drugs.

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

Combinations of histone modifications have significant biological roles, such as maintenance of pluripotency and cancer development, but cannot be analyzed at the single cell level. Here, we visualized a combination of histone modifications by applying the in situ proximity ligation assay, which detects two proteins in close vicinity (∼30 nm). The specificity of the method [designated as imaging of a combination of histone modifications (iChmo)] was confirmed by positive signals from H3K4me3/acetylated H3K9, H3K4me3/RNA polymerase II and H3K9me3/H4K20me3, and negative signals from H3K4me3/H3K9me3. Bivalent modification was clearly visualized by iChmo in wild-type embryonic stem cells (ESCs) known to have it, whereas rarely in Suz12 knockout ESCs and mouse embryonic fibroblasts known to have little of it. iChmo was applied to analysis of epigenetic and phenotypic changes of heterogeneous cell population, namely, ESCs at an early stage of differentiation, and this revealed that the bivalent modification disappeared in a highly concerted manner, whereas phenotypic differentiation proceeded with large variations among cells. Also, using this method, we were able to visualize a combination of repressive histone marks in tissue samples. The application of iChmo to samples with heterogeneous cell population and tissue samples is expected to clarify unknown biological and pathological significance of various combinations of epigenetic modifications.

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.

The accurate detection of the presence of lymph node metastases (LNM) of gastric cancers (GCs) is useful for the implementation of necessary and sufficient treatment, but current methods of detection are unsatisfactory. In the present study, we focused on DNA methylation markers since they have several advantages, including biological and chemical stability and informativeness even in the presence of contaminating cells. Using three metastatic lymph nodes and three primary GCs without LNM, methylation bead array analyses were performed, which enabled the interrogation of 485,577 CpG sites. A total of 31 CpG sites that were hypermethylated in the metastatic lymph nodes, compared with the GCs without LNM, were isolated. Using primary GCs with and without LNM (28 GCs with LNM and 10 without), their methylation levels were measured using quantitative PCR following treatment with sodium bisulfite or a methylation-sensitive restriction enzyme. Of the genomic regions around the 31 CpG sites, 10 regions demonstrated higher methylation levels in the GCs with LNM compared with the GCs without LNM (P

Aberrant hypermethylation of specific genes is present in esophageal squamous cell carcinomas (ESCCs). Such hypermethylation is also present in normal-appearing esophageal mucosae of ESCC patients and is considered to contribute to the formation of a field for cancerization. On the other hand, the presence of global hypomethylation in ESCCs or in their background esophageal mucosae is unknown. We collected 184 samples of esophageal mucosae (95 normal mucosae from healthy subjects, and 89 non-cancerous background mucosae from ESCC patients) and 93 samples of ESCCs. Methylation levels of repetitive elements (Alu, LINE1) and cancer/testis antigen genes (NY-ESO-1, MAGE-C1) were measured by bisulfite pyrosequencing and quantitative methylation-specific PCR, respectively. Methylation levels of Alu, LINE1, NY-ESO-1, and MAGE-C1 were significantly lower in ESCCs than in their background and normal mucosae. Also, in the background mucosae, a significant decrease of the Alu methylation level compared with the normal mucosae was present. In ESCCs, methylation levels of the two repetitive elements and the two cancer/testis antigen genes were correlated with each other. This is the first study to show the presence of global hypomethylation in ESCCs, and even in their non-cancerous background mucosae. Alu hypomethylation might reflect the severity of an epigenetic field for cancerization.

Epigenetic fields for cancerization are involved in development of human cancers, especially those associated with inflammation and multiple occurrences. However, it is still unclear when such field defects are formed and what component of inflammation is involved in induction of aberrant DNA methylation. Here, in a mouse colitis model induced by dextran sulfate sodium (DSS), we identified three CpG islands specifically methylated in colonic epithelial cells exposed to colitis. Their methylation levels started to increase as early as 8 weeks after DSS treatment and continued to increase until colon cancers developed at 15 weeks. In contrast to the temporal profile of DNA methylation levels, infiltration of inflammatory cells spiked immediately after the DSS treatment and then gradually decreased. Exposure of cultured colonic epithelial cells to DSS did not induce DNA methylation and it was indicated that inflammation triggered by the DSS treatment was responsible for methylation induction. To clarify components of inflammation involved, severe combined immunodeficiency (SCID) mice that lack functional T- and B-cells were similarly treated. Even in SCID mice, DNA methylation, along with colon tumors, were induced at the same levels as in their background strain of mice (C.B17). Comparative analysis of inflammation-related genes showed that Ifng, Il1b and Nos2 had expression concordant with methylation induction whereas Il2, Il6, Il10, Tnf did not. These results showed that an epigenetic field defect is formed at early stages of colitis-associated carcinogenesis and that functional T and B cells are non-essential for the formation.

Aberrant DNA methylation accumulated in normal tissues, namely methylation burden, is closely associated with risk of carcinogenesis, especially in cancers related to chronic inflammation. Methylation burden is known to be influenced by multiple factors, such as genetic factors and strengths of carcinogens. However, the impact of the duration of exposure to a carcinogen is still unclear. Here, using a Mongolian gerbil model of Helicobacter pylori (H. pylori)-induced chronic gastritis, we aimed to clarify the impact of the duration of exposure on the methylation burden in normal gastric tissues (gastric mucosae). Following infection with H. pylori, DNA methylation levels at four CpG islands, HE6, SA9, SB5, and SD2, increased, depending upon the exposure duration. 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 DNA methylation, but not those with sparse DNA methylation, increased depending upon the infection period. DNA methylation levels at one of the four CpG islands, SA9, tended to be higher in the gastric mucosae of gerbils infected with H. pylori than in those of non-infected gerbils, even 50 weeks after eradication. These results showed, for the first time, that the methylation burden in normal tissues is influenced by the duration of exposure to a carcinogenic factor. Citation Format: Harumi Yamada, Hideyuki Takeshima, Tohru Niwa, Takeshi Toyoda, Satoshi Yamashita, Toshikazu Ushijima. The methylation burden is determined by the duration of exposure to carcinogenic factors [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 5340.

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. ''Vicious'' 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.

Altered patterns of DNA methylation associated with Helicobacter pylori (HP) infection of gastric epithelial cells are thought to contribute to gastric cancer risk. However, it is unclear whether this increased risk reflects an infection-associated inflammatory response or the infection itself. In this study, we sought to clarify mechanisms in a gerbil model of gastric cancer where we showed that HP infection is causally involved in induction of aberrant DNA methylation. By genome-wide screening, CpG islands that were aberrantly methylated in gerbil gastric cancer cell lines were isolated, and 10 islands were shown to be specifically methylated only in gastric mucosae infected with HP. By temporal analysis, methylation levels in gastric epithelial cells started to increase at 5 to 10 weeks after infection and reached high levels by 50 weeks. When HP was eradicated, methylation levels markedly decreased 10 and 20 weeks later, but they remained higher than those in gerbils that were not infected by HP. Expression levels of several inflammation-related genes (CXCL2, IL-1β, NOS2, and TNF-α) paralleled the temporal changes of methylation levels. Significantly suppressing inflammation with the immunosuppressive drug cyclosporin A did not affect colonization by HP but blocked the induction of altered DNA methylation. Our findings argue that DNA methylation alterations that occur in gastric mucosae after HP infection are composed of transient components and permanent components, and that it is the infection-associated inflammatory response, rather than HP itself, which is responsible for inducing the altered DNA methylation. Cancer Res; 70(4); 1430–40

To identify methylation-silenced genes in prostate cancers, a microarray analysis for genes up-regulated by treatment with a demethylating agent, 5-aza-2′-deoxycytidine, was performed using three rat prostate cancer cell lines. Eight genes (Aebp1, Dysf, Gas6, LOC361288, Nnat, Ocm, RGD1308119, and Tgfbr2) were re-expressed at 16-fold or more, and their promoter CpG islands were shown to be densely methylated in the cancer cell lines. From the eight genes, Tgfbr2, a key mediator of transforming growth factor-β (TGF-β) signaling that has been strongly implicated in human and rat prostate carcinogenesis, was selected, and its silencing in primary samples was analyzed further. Tgfbr2 was methylated and markedly down-regulated in three of seven 3,2′-dimethyl-4-aminobiphenyl–induced invasive adenocarcinomas in the dorsolateral lobe of the rat prostate. In humans, marked down-regulation of TGFBR2 protein was observed in 12 of 20 high-grade prostatic intraepithelial neoplasia and 36 of 60 prostate cancers. DNA methylation of the human TGFBR2 promoter CpG islands repressed transcription, if present, but neither methylation nor mutation were detected in 27 human prostate cancers analyzed. Methylation silencing of rat Tgfbr2 was associated with histone H3 lysine 9 trimethylation, whereas decreased expression of human TGFBR2 was mainly due to decreased transcription activity, sometimes in concert with histone deacetylation and H3 lysine 27 trimethylation. The identification of methylation silencing of Tgfbr2 in rat prostate cancers, in accordance with TGFBR2 down-regulation in human prostate cancers, will enable us to analyze how aberrant methylation is induced in vivo and identify factors that promote and suppress the induction of aberrant methylation. [Cancer Res 2008;68(7):2112–21]

Germ cell tumors constitute a heterogeneous group that displays a broad spectrum of morphology. They often arise in testes; however, extragonadal occurrence, in particular brain, is not uncommon, and whether they share a common pathogenesis is unknown. We performed whole exome sequencing in 41 pairs of central nervous system germ cell tumors (CNS GCTs) of various histology and their matched normal tissues. We then performed targeted sequencing of 41 selected genes in a total of 124 CNS GCTs, 65 testicular germ cell tumors (tGCTs) and 8 metastatic GCTs to the CNS. The results showed that mutually exclusive mutations of genes involved in the MAPK pathway were most common (48.4 %), typically in KIT (27.4 %), followed by those in the PI3K pathway (12.9 %), particularly in MTOR (6.5 %), among the 124 CNS GCTs. Pure germinomas and non-germinomatous germ cell tumors (NGGCTs), as well as CNS and testicular GCTs, showed similar mutational profiles, suggesting that GCTs share a common molecular pathogenesis. Mutated MTOR identified in CNS GCTs upregulated phosphorylation of the AKT pathway proteins including AKT and 4EBP1 in nutrient-deprived conditions and enhanced soft-agar colony formation; both events were suppressed in a dose-dependent manner by addition of the MTOR inhibitor pp242. Our findings indicate that the dominant genetic drivers of GCTs regardless of the site of origin are activation of the MAPK and/or PI3K pathways by somatic point mutations. Mutated MTOR represents a potential target for novel targeted therapies for refractory GCTs.

Background In advanced gastric cancer (AGC), most clinical trials are designed on the basis of protein expression or gene amplification of specific genes. Recently, next-generation sequencing (NGS) allowed us to comprehensively profile the tumor gene status. This study aimed to elucidate the profiling between gene alterations and protein expression in AGC to aid in future clinical trials on AGC. Patients and methods Formalin-fixed, paraffin-embedded tumor samples from 121 stage III/IV gastric cancer patients were examined for protein expression of tyrosine kinase receptors (RTKs; ERBB2, EGFR, c-MET, and FGFR2) using immunohistochemistry (IHC). Furthermore, 409 cancer-related genes were sequenced to detect mutations and copy number variations using NGS. Results Most ERBB2 overexpression (IHC 3+) cases (80.0%) had ERBB2 amplification and did not have other RTK amplification or oncogene mutations. However, one-fourth of MET overexpression cases (25.0%) had ERBB2 alterations. EGFR and FGFR2 overexpression cases had ERBB2 alterations or other gene alterations such as KRAS or PIK3CA. On the other hand, most of the four RTK amplification cases (88.2%) were mutually exclusive with each amplification. However, RTK amplification did not simply correlate with protein overexpression, whereas cases with RTK high-level amplification had protein overexpression and rarely showed other co-existing gene alterations. Conclusion AGC involves a complicated arrangement of protein expression and gene alterations. Comprehensive analyses of NGS and IHC will be necessary to design the optimal therapy for treating the appropriate population of patients in future clinical trials.

Gastric cancer (GC) is highly influenced by aberrant methylation, and accumulation of aberrant methylation in gastric mucosae produces an epigenetic field for cancerization. Nevertheless, the individual driver genes involved in such field cancerization are still unclear. Here, we aimed to demonstrate that FAT4, a novel tumor suppressor identified by exome sequencing of GC, is methylation-silenced and that such methylation is involved in epigenetic field cancerization for GC. A transcription start site was determined by the 5′ rapid amplification of complementary DNA ends method. DNA methylation was analyzed by bisulfite sequencing with use of a next-generation sequencer or quantitative methylation-specific PCR. Gene expression was analyzed by quantitative reverse transcription PCR. A single transcription start site was identified for FAT4 in gastric epithelial cells, and a CpG island was located in the FAT4 promoter region. FAT4 was highly methylated in two of 13 GC cell lines and was not expressed in them. Removal of FAT4 methylation by a DNA demethylating agent (5-aza-2′-deoxycytidine) restored its expression in the two cell lines. In primary GC samples, FAT4 was methylated in 12 of 82 GCs (14.6 %). FAT4 methylation was associated with the presence of the CpG island methylator phenotype but not with prognosis, tumor invasion, lymph node metastasis, or histological types. In noncancerous gastric mucosae, high FAT4 methylation levels were associated with the presence of GC and Helicobacter pylori infection. FAT4 was methylation-silenced in GCs. Its methylation in gastric mucosae was associated with H. pylori infection and likely contributed to epigenetic field cancerization.

The recent development of next-generation sequencing technology for extensive mutation analysis, and beadarray technology for genome-wide DNA methylation analysis has made it possible to obtain integrated pictures of genetic and epigenetic alterations, using the same cancer samples. In this study, we aimed to characterize such a picture in esophageal squamous cell carcinomas (ESCCs). Base substitutions of 55 cancer-related genes and copy number alterations (CNAs) of 28 cancer-related genes were analyzed by targeted sequencing. Forty-four of 57 ESCCs (77%) had 64 non-synonymous somatic mutations, and 24 ESCCs (42%) had 35 CNAs. A genome-wide DNA methylation analysis using an Infinium HumanMethylation450 BeadChip array showed that the CpG island methylator phenotype was unlikely to be present in ESCCs, a different situation from gastric and colon cancers. Regarding individual pathways affected in ESCCs, the WNT pathway was activated potentially by aberrant methylation of its negative regulators, such as SFRP1, SFRP2, SFRP4, SFRP5, SOX17, and WIF1 (33%). The p53 pathway was inactivated by TP53 mutations (70%), and potentially by aberrant methylation of its downstream genes. The cell cycle was deregulated by mutations of CDKN2A (9%), deletions of CDKN2A and RB1 (32%), and by aberrant methylation of CDKN2A and CHFR (9%). In conclusion, ESCCs had unique methylation profiles different from gastric and colon cancers. The genes involved in the WNT pathway were affected mainly by epigenetic alterations, and those involved in the p53 pathway and cell cycle regulation were affected mainly by genetic alterations. © 2016 Wiley Periodicals, Inc.

N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced rat stomach carcinomas are considered to be a good model for differentiated-type human stomach carcinomas. However, as for their molecular basis, only infrequent mutations of Catnb (beta-catenin) and Trp53 (p53) have been observed. Here, we carried out a whole-genome analysis of loss of heterozygosity (LOH) using 21 stomach carcinomas induced by MNNG in F(1) hybrids of ACI and BUF rats, and also analyzed promoter methylation of four tumor-suppressor genes. LOH analysis was performed using 130 polymorphic markers covering rat chromosomes 1-20 with an average interval of 20 Mbp. Despite adapting conditions so that LOH could be detected with up to a 50% contamination of stromal cells, no LOH was detected at any loci. CpG islands in putative promoter regions of four tumor-suppressor genes, Cdh1 (E-cadherin), Cdkn2a (p16), Mlh1, and Rassf1a, were analyzed by methylation-specific polymerase chain reaction (PCR). However, no methylation was detected. In contrast, the promoter region of Pgc (pepsinogen C), which lacks a CpG island, was methylated in all 21-cancer samples. These results indicated that LOH spanning a chromosomal region larger than 30-40 Mbp or silencing of Cdh1, Cdkn2a, Mlh1, and Rassf1a, was not involved in MNNG-induced rat stomach carcinomas. The search for other genes involved in these carcinomas needs to be continued.

Japanese quail embryos normally have longitudinal black and brown stripes formed by colored feather buds on their back whereas an autosomal dominant mutation, black at hatch (Bh), disrupts this pigmentation pattern by causing overall black and brown coating in heterozygotes and homozygotes, respectively. These phenotypes of the Bh mutant embryos suggest that the Bh locus plays an important role in the pigment pattern formation of plumage, but its genetic origin, including cloning of the responsible gene, has been insufficiently studied. In this study, we adapted genetically directed representational difference analysis with elimination of excessive clones (GDRDA-WEEC) to Bh quails and isolated two genetic markers linked to the Bh locus as DNA fragments. Cytogenetic study by fluorescence in situ hybridization (FISH) of the DNA fragments used as probes demonstrated that the marker loci were located in the same region on the long arm of chromosome 1. Close genetic linkage between the Bh and the marker loci, and the chromosomal location of the latter suggested that the Bh locus is located on the long-arm of chromosome 1 of the Japanese quail.

The plumage on the dorsal trunk of normal quail embryos exhibits longitudinal black and brown stripes of pigments produced by melanocytes. However, this pigmentation pattern disappeared in Bh (black at hatch) heterozygous and homozygous embryos because of overall black and brown pigmentation of plumages, respectively. To investigate the mechanisms of the pigment pattern formation of plumage and clarify the roles of the Bh locus in the pattern formation, we examined the expression pattern of genes relating to melanocyte development (Mitf, MelEM antigen, Kitl, Kit and EdnrB2) and melanin pigment production (Dct, Tyrp1, Tyr and Mmp115) in Bh mutant and wild-type embryos throughout development. As a result, we found that MelEM antigen was expressed in melanoblasts committed to produce black pigment before apparent melanogenic gene expression, and that Bh heterozygotes and homozygotes showed abnormal expression patterns of the MelEM antigen. These results indicate that MelEM antigen is a good marker for melanoblasts committed to produce black pigment, and suggests that the Bh locus directs melanocytes to produce eumelanin in proper positions.

The observation of the molecular orientation of alkanoic acid-derivatized porphyrins in ultrathin films deposited on mica was carried out by atomic force microscopy. It was observed that the tetraacid derivative 5, 10, 15, 20-tetra(N-10-carboxydecyl-pyridinium-4-yl) porphyrin was arranged as a monolayer with the porphyrin macrocycle oriented coplanar to the mica surface. On the other hand, the diacid derivative protoporphyrin IX Zn(II) formed a bilayer with the hydrophobic part inside and the hydrophilic part in the periphery. Therefore, in this case, the porphyrin macrocycle is roughly perpendicular to the mica surface.

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.

BACKGROUND: Intracranial germ cell tumors (iGCTs) are rare in the Western countries, however they are the second most common brain tumors in patients under 14 in Japan. Unlike other common pediatric brain tumors, the biology of iGCTs is largely unknown. METHODS: We performed a whole exome sequencing in a large series of iGCTs to elucidate their molecular pathogenesis. A total of 198 germ cell tumors (GCTs) including 133 iGCTs (69 pure germinomas, 56 NGGCTs and 8 metastatic tumors) as well as 65 testicular germ cell tumors (tGCTs) (39 seminomas and 26 non-seminoma GCTs) were collected from 13 centers participating in the Intracranial Germ Cell Tumor Consortium in Japan. Somatic mutations in all coding exons were investigated by whole exome sequencing (WES) in 41 tumors and the matched normal DNAs. Based on the WES data, 41 candidate genes were selected according to the frequency and/or significance of the mutations found. All coding exons of these 41 genes spanning over 160kb were PCR-amplified in a further 157 GCTs and sequenced using the IonTorrent system. The results were integrated with the patients' clinical information that was available for 124 iGCT patients. RESULTS: On average, 15.4 non-synonymous somatic mutations were observed in each tumor, ranging from 1 to 140 by WES in 41 iGCTs. The combined WES and IonTorrent screenings showed that KIT was the most frequently mutated gene in both iGCTs (27%) and tGCTs (18%). MTOR was the second most frequently mutated also in both iGCTs (7%) and tGCTs (6%). RAS mutations (KRAS, HRAS, NRAS) were altogether found in 13% of iGCTs and 12% of tGCTs. These mutations were mutually exclusive to each other and also to KIT mutations. Collectively, the genes involved in the MAPK pathway (e.g., KIT, RAS, NF1) and the PI3K/MTOR pathway (e.g., MTOR, PTEN) were mutated in 44% and 13% of all GCTs. Among the iGCTs, these alterations were significantly more common among pure germinomas than NGGCTs. The mutated MTOR protein was shown to have increased kinase activity, which was suppressed by specific MTOR inhibitors. CONCLUSIONS: Our comprehensive mutational genomic analysis of GCTs revealed that alterations of the MAPK and/or PI3K/MTOR pathways play a critical role in the pathogenesis of both iGCTs and tGCTs, although the extent of their involvement depends on the histopathological subtypes. Our findings will hopefully lead to the development of a targeted therapy for treatment-resistant iGCTs. SECONDARY CATEGORY: Tumor Biology.

Disappearance of the coloration pattern of the plumage and whole body hemorrhage is caused by an autosomal dominant black at hatch (Bh) mutation in the Japanese quail. In this study, we adopted genetically directed representational difference analysis (GDRDA) to obtain a genetic marker linked to Bh from our closed colony stock. A DNA pool of 10 wild-type (+/+) quails was subtracted from another DNA pool of 10 quails heterozygous for the Bh mutation (Bh/+). After analysis of 106 clones isolated by three series of GDRDA using three different restriction enzymes, one clone was shown to detect a restriction fragment length polymorphism (RFLP) between +/+ and Bh/+ quails. A genomic region flanking the polymorphic restriction site was cloned by inverse-PCR, and a PCR-RFLP maker linked to the Bh mutation was developed. The marker showed no recombination with the Bh mutation in 101 quails analyzed. This is the first report in which a genetic marker linked to a specific gene was successfully isolated by G...

Bh (black at hatch) is a mutation of Japanese quails which causes darkening or lightening of the plumage in heterozygotes or homozygotes, respectively. We chemically analyzed melanin pigments in feather germs of Bh mutant embryos and in feathers of adult animals. Dark brown dorsal feathers of wild-type adult animals had white barrings, but heterozygous ones lacked clear barrings. The feathers of wild-type and heterozygote animals contained both eumelanins and pheomelanins, the latter being more pheomelanic. On the dorsal skin of 10-day old wild-type embryos, longitudinal stripes from black and yellow rows of feather germs developed; two or three longitudinal rows of black feather germs and then two or three rows of yellow feather germs next to the short central feather germs. Heterozygous embryos appeared black in plumage pigmentation, due to the presence of 'gray' feather germs in rows of dorsal feather germs that corresponded to yellow rows in wild-type embryos. Homozygous dorsal feather germs did not develop the black and yellow longitudinal stripes, but were brown. Chemical analysis showed that embryos of each genotype contained both eumelanins and pheomelanins in the feather germs; however, the eumelanin content in homozygous feather germs was very low. These results suggest that the Bh mutation causes pheomelanic changes in feathers of quails.

Recent development of personal sequencers for extensive mutation analysis and bead array technology for comprehensive DNA methylation analysis have made it possible to obtain integrated pictures of genetic and epigenetic alterations on the same set of cancer samples. Here, we aimed to establish such pictures of gastric cancers (GCs). Comprehensive methylation analysis of 30 GCs revealed that the number of aberrantly methylated genes was highly variable among individual GCs. Extensive mutation analysis of 55 known cancer-related genes revealed that 19 of the 30 GCs had 24 somatic mutations of eight different genes (CDH1, CTNNB1, ERBB2, KRAS, MLH1, PIK3CA, SMARCB1, and TP53). Integration of information on the genetic and epigenetic alterations revealed that the GCs with the CpG island methylator phenotype (CIMP) tended to have mutations of oncogenes, CTNNB1, ERBB2, KRAS, and PIK3CA. This is one of the first studies in which both genetic and epigenetic alterations were extensively analyzed in the same set of samples. It was also demonstrated for the first time in GCs that the CIMP was associated with oncogene mutations.

Chronic inflammation triggered by Helicobacter pylori causes altered DNA methylation in stomach mucosae, which is deeply involved in gastric carcinogenesis. This study aimed to elucidate the correlation between altered mucosal DNA methylation levels and activity of H. pylori-related gastritis, because inflammatory activity shows particular correlations with the development of diffuse-type cancer.Methylation levels in stomach mucosae of 78 healthy volunteers were determined by real-time methylation-specific PCR or bisulfite pyrosequencing. Examined loci were the promoter CpG islands of six genes (FLNc, HAND1, THBD, p41ARC, HRASLS, and LOX) and the CpG sites of non-coding repetitive elements (Alu and Satα) that are reportedly altered by H. pylori infection. Activity of H. pylori-related gastritis was evaluated using two serum markers: H. pylori antibody titer and pepsinogen II.Methylation levels of the six CpG islands were consistently increased, and those of the two repetitive elements were consistently decreased in a stepwise manner with the activity of gastric inflammation as represented by serum marker levels. Each serum marker level was well correlated with the overall DNA methylation status of stomach mucosa, and these two serologic markers were additive in the detection of the mucosa with severely altered DNA methylation.Alteration in mucosal DNA methylation level was closely correlated with activity of H. pylori-related gastritis as evaluated by serum markers. The observed correlation between altered DNA methylation levels and activity of H. pylori-related gastritis appears to be one of the relevant molecular mechanisms underlying the development of diffuse-type cancer.

Tumor-suppressor genes on chromosome X can be inactivated by a single hit, any of the point mutations, chromosomal loss and aberrant DNA methylation. As aberrant DNA methylation can be induced frequently, we here aimed to identify a tumor-suppressor gene on chromosome X inactivated by promoter DNA methylation. Of 69 genes on chromosome X upregulated by treatment of a gastric cancer cell line with a DNA-demethylating agent, 5-aza-2'-deoxycytidine, 11 genes had low or no expression in the cell line and abundant expression in normal gastric mucosae. Among them, FHL1 was frequently methylation-silenced in gastric and colon cancer cell lines, and methylated in primary gastric (21/80) and colon (5/50) cancers. Knockdown of the endogenous FHL1 in two cell lines by two kinds of shRNAs significantly increased cell growth in vitro and sizes of xenografts in nude mice. Expression of exogenous FHL1 in a non-expressing cell line significantly reduced its migration, invasion and growth. Notably, a somatic mutation (G642T; Lys214Asn) was identified in one of 144 colon cancer specimens, and the mutant FHL1 was shown to lack its inhibitory effects on migration, invasion and growth. FHL1 methylation was associated with Helicobacter pylori infection and accumulated in normal-appearing gastric mucosae of gastric cancer patients. These data showed that FHL1 is a methylation-silenced tumor-suppressor gene on chromosome X in gastrointestinal cancers, and that its silencing contributes to the formation of an epigenetic field for cancerization.

In the vector control system using the slip frequency control method, the rotor resistance of an induction motor is used to calculate a slip frequency. Thus the change in temperature of the rotor resistance causes the deterioration of the torque control characteristic. This paper presents a new method of compensating for the rotor resistance change which is robust for the stator resistance change. A current control loop is composed of the λ-δ axes in which the λ axis is coincident to the stator current. In this method, the stator voltage error on the δ axis which is directly obtainable from this current control loop was used. The change in the stator voltage was able to be detected accurately, therefore the torque control accuracy was improved, particularly in the low-speed region. The experimental results of the current response and the compensation for the rotor resistance deviation also are shown. Moreover, although the mutual inductance has been treated as an invariable value, the value does change by a frequency and an exciting command. In this control method, an initial tuning of the equivalent mutual inductance was achieved by detecting the deviation component of the stator voltage on the δ axis at the no-load running condition. Furthermore, in the region with the constant power where the field weakening control was achieved, the excellent experimental results of the compensation for the deviation of the equivalent mutual inductance are shown.

Chronic inflammation is deeply involved in induction of aberrant DNA methylation, but it is unclear whether any type of persistent inflammation can induce methylation and how induction of cell proliferation is involved. In this study, Mongolian gerbils were treated with five kinds of inflammation inducers [Helicobacter pylori with cytotoxin-associated gene A (CagA), H.pylori without CagA, Helicobacter felis, 50% ethanol (EtOH) and saturated sodium chloride (NaCl) solution]. Two control groups were treated with a mutagenic carcinogen that induces little inflammation (20 p.p.m. of N-methyl-N-nitrosourea) and without any treatment. After 20 weeks, chronic inflammation with lymphocyte and macrophage infiltration was prominent in the three Helicobacter groups, whereas neutrophil infiltration was mainly observed in the EtOH and NaCl groups. Methylation levels of eight CpG islands significantly increased only in the three Helicobacter groups. By Ki-67 staining, cell proliferation was most strongly induced in the NaCl group, demonstrating that induction of cell proliferation is not sufficient for methylation induction. Among the inflammation-related genes, Il1b, Nos2 and Tnf showed increased expression specifically in the three Helicobacter groups. In human gastric mucosae infected by H.pylori, NOS2 and TNF were also increased. These data showed that inflammation due to infection of the three Helicobacter strains has a strong potential to induce methylation, regardless of their CagA statuses, and increased cell proliferation was not sufficient for methylation induction. It was suggested that specific types of inflammation characterized by expression of specific inflammation-related genes, along with increased cell proliferation, are necessary for methylation induction.