Naoko Hattori

Molecular Pathways: Involvement of Helicobacter pylori–Triggered Inflammation in the Formation of an Epigenetic Field Defect, and Its Usefulness as Cancer Risk and Exposure Markers

Infection-associated cancers account for a large proportion of human cancers, and gastric cancer, the vast majority of which is associated with Helicobacter pylori infection, is a typical example of such cancers. Epigenetic alterations are known to occur frequently in gastric cancers, and H. pylori infection has now been shown to induce aberrant DNA methylation in gastric mucosae. Accumulation of aberrant methylation in gastric mucosae produces a field for cancerization, and methylation levels correlate with gastric cancer risk. H. pylori infection induces methylation of specific genes, and such specificity is determined by the epigenetic status in normal cells, including the presence of H3K27me3 and RNA polymerase II (active or stalled). Specific types of inflammation, such as that induced by H. pylori infection, are important for methylation induction, and infiltration of monocytes appears to be involved. The presence of an epigenetic field defect is not limited to gastric cancers and is observed in various types of cancers. It provides translational opportunities for cancer risk diagnosis incorporating life history, assessment of past exposure to carcinogenic factors, and cancer prevention. Clin Cancer Res; 18(4); 923–9. ©2011 AACR.

Large-Scale Characterization of DNA Methylation Changes in Human Gastric Carcinomas with and without Metastasis

Purpose: Metastasis is the leading cause of death for gastric carcinoma. An epigenetic biomarker panel for predicting gastric carcinoma metastasis could have significant clinical impact on the care of patients with gastric carcinoma. The main purpose of this study is to characterize the methylation differences between gastric carcinomas with and without metastasis. Experimental Design: Genome-wide DNA methylation profiles between 4 metastatic and 4 nonmetastatic gastric carcinomas and their surgical margins (SM) were analyzed using methylated-CpG island amplification with microarray. The methylation states of 73 candidate genes were further analyzed in patients with gastric carcinoma in a discovery cohort (n = 108) using denatured high performance liquid chromatography, bisulfite-sequencing, and MethyLight. The predictive values of potential metastasis-methylation biomarkers were validated in cohorts of patients with gastric carcinoma in China (n = 330), Japan (n = 129), and Korea (n = 153). Results: The gastric carcinoma genome showed significantly higher proportions of hypomethylation in the promoter and exon-1 regions, as well as increased hypermethylation of intragenic fragments when compared with SMs. Significant differential methylation was validated in the CpG islands of 15 genes (P < 0.05) and confirmed using bisulfite sequencing. These genes included BMP3, BNIP3, CDKN2A, ECEL1, ELK1, GFRA1, HOXD10, KCNH1, PSMD10, PTPRT, SIGIRR, SRF, TBX5, TFPI2, and ZNF382. Methylation changes of GFRA1, SRF, and ZNF382 resulted in up- or downregulation of their transcription. Most importantly, the prevalence of GFRA1, SRF, and ZNF382 methylation alterations was consistently and coordinately associated with gastric carcinoma metastasis and the patients overall survival throughout discovery and validation cohorts in China, Japan, and Korea. Conclusion: Methylation changes of GFRA1, SRF, and ZNF382 may be a potential biomarker set for prediction of gastric carcinoma metastasis. Clin Cancer Res; 20(17); 4598–612. ©2014 AACR.

Epigenetic regulation of autophagy by the methyltransferase EZH2 through an MTOR-dependent pathway

Macroautophagy is an evolutionarily conserved cellular process involved in the clearance of proteins and organelles. Although the autophagy regulation machinery has been widely studied, the key epigenetic control of autophagy process still remains unknown. Here we report that the methyltransferase EZH2 (enhancer of zeste 2 polycomb repressive complex 2 subunit) epigenetically represses several negative regulators of the MTOR (mechanistic target of rapamycin [serine/threonine kinase]) pathway, such as TSC2, RHOA, DEPTOR, FKBP11, RGS16 and GPI. EZH2 was recruited to these genes promoters via MTA2 (metastasis associated 1 family, member 2), a component of the nucleosome remodeling and histone deacetylase (NuRD) complex. MTA2 was identified as a new chromatin binding protein whose association with chromatin facilitated the subsequent recruitment of EZH2 to silenced targeted genes, especially TSC2. Downregulation of TSC2 (tuberous sclerosis 2) by EZH2 elicited MTOR activation, which in turn modulated subsequent MTOR pathway-related events, including inhibition of autophagy. In human colorectal carcinoma (CRC) tissues, the expression of MTA2 and EZH2 correlated negatively with expression of TSC2, which reveals a novel link among epigenetic regulation, the MTOR pathway, autophagy induction, and tumorigenesis.

Visualization of multivalent histone modification in a single cell reveals highly concerted epigenetic changes on differentiation of embryonic stem cells

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.

Genetic and epigenetic alterations in normal tissues have differential impacts on cancer risk among tissues

Significance The relative importance of genetic and epigenetic alterations in normal tissues on cancer risk was clearly different between esophageal squamous cell and gastric cancers, implying a variety of differences in various types of cancers. The difference observed was well explained by known etiologies: tobacco mutagens for esophageal cancer and chronic inflammation for epigenetic alterations in gastric cancer. The study showed that, if epigenetic and genetic alterations in normal tissues are combined, reflecting their relative contributions, patients with cancer can be precisely discriminated, opening up an avenue to precision cancer risk diagnosis. The study also indicated that for effective cancer prevention, allocation of resources and efforts against genetic and epigenetic alterations should consider their relative contributions. Genetic and epigenetic alterations are both involved in carcinogenesis, and their low-level accumulation in normal tissues constitutes cancer risk. However, their relative importance has never been examined, as measurement of low-level mutations has been difficult. Here, we measured low-level accumulations of genetic and epigenetic alterations in normal tissues with low, intermediate, and high cancer risk and analyzed their relative effects on cancer risk in the esophagus and stomach. Accumulation of genetic alterations, estimated as a frequency of rare base substitution mutations, significantly increased according to cancer risk in esophageal mucosae, but not in gastric mucosae. The mutation patterns reflected the exposure to lifestyle risk factors. In contrast, the accumulation of epigenetic alterations, measured as DNA methylation levels of marker genes, significantly increased according to cancer risk in both tissues. Patients with cancer (high-risk individuals) were precisely discriminated from healthy individuals with exposure to risk factors (intermediate-risk individuals) by a combination of alterations in the esophagus (odds ratio, 18.2; 95% confidence interval, 3.69–89.9) and by only epigenetic alterations in the stomach (odds ratio, 7.67; 95% confidence interval, 2.52–23.3). The relative importance of epigenetic alterations upon genetic alterations was 1.04 in the esophagus and 2.31 in the stomach. The differential impacts among tissues will be critically important for effective cancer prevention and precision cancer risk diagnosis.

Establishment of a DNA methylation marker to evaluate cancer cell fraction in gastric cancer

AbstractBackgroundTumor samples are unavoidably ncontaminated with coexisting normal cells. Here, we aimed to establish a DNA methylation marker to estimate the fraction of gastric cancer (GC) cells in any DNA sample by isolating genomic regions specifically methylated in GC cells.MethodsGenome-wide and gene-specific methylation analyses were conducted with an Infinium HumanMethylation450 BeadChip array and by quantitative methylation-specific PCR, respectively. Purified cancer and noncancer cells were prepared by laser-capture microdissection. TP53 mutation data were obtained from our previous study using next-generation target sequencing.ResultsGenome-wide DNA methylation analysis of 12 GC cell lines, 30 GCs, six normal gastric mucosae, one sample of peripheral leukocytes, and four noncancerous gastric mucosae identified OSR2, PPFIA3, and VAV3 as barely methylated in normal cells and highly methylated in cancer cells. Quantitative methylation-specific PCR using 26 independent GCs validated that one or more of them was highly methylated in all of the GCs. Using four pairs of purified cells, we confirmed the three genes were highly methylated (85xa0% or more) in cancer cells and barely methylated (5xa0% or less) in noncancer cells. The cancer cell fraction assessed by the panel of the three genes showed good correlation with that assessed by the TP53 mutant allele frequency in 13 GCs (rxa0=xa00.77). After correction of the GC cell fraction, unsupervised clustering analysis of the genome-wide DNA methylation profiles yielded clearer clustering.ConclusionsA DNA methylation marker—namely, the panel of the three genes—is useful to estimate the cancer cell fraction in GCs.

Analysis of Gene-Specific DNA Methylation

Abstract DNA methylation at specific genomic regions can be analyzed by various techniques using bisulfite-mediated DNA conversion or methylation-sensitive restriction enzymes. Under appropriate conditions, the former technique specifically converts unmethylated cytosines into uracils. Methods taking advantage of this principle include bisulfite sequencing, methylation-specific PCR (MSP), real-time MSP, MethyLight, methylation-sensitive high-resolution melting analysis, and pyrosequencing, all of which are used widely. Combinations with leading-edge techniques, such as next-generation sequencing and digital PCR, have provided increased accuracy and a broader range of applications. The appropriate method should be selected considering factors, such as the amount of DNA required, flexibility in selection of CpG sites to analyze, degree of quantitation of the method, technical complexity, and the cost. This chapter reviews principles of methylation detection and characteristics of individual methods, and provides tips for bisulfite-mediated conversion, bisulfite sequencing, MSP, and quantitative MSP.

Analysis of DNA Methylation in Tissues Exposed to Inflammation

Induction of aberrant DNA methylation is one of the most important mechanisms mediating the effect of inflammation on cancer development. Aberrant methylation of promoter CpG islands of tumor suppressor genes can silence their downstream genes, and that in cancer tissues is associated with prognosis or therapeutic effects. In addition, aberrant methylation can occur in tissues exposed to specific types of inflammation, producing a so-called epigenetic field for cancerization, and its accumulation is correlated with cancer risk. Thus, aberrant methylation at specific loci is an important biomarker and mediator of the carcinogenic effect of inflammation. DNA methylation at specific genomic regions can be analyzed by various methods based upon bisulfite-mediated DNA conversion, which specifically converts unmethylated cytosines into uracils under appropriate conditions. Methylation-specific PCR (MSP), quantitative MSP, and bisulfite sequencing are widely used, and this chapter provides protocols for bisulfite-mediated conversion, quantitative MSP, and bisulfite sequencing.

Chapter 8 – Analysis of Gene-Specific DNA Methylation

DNA methylation at specific genomic regions can be analyzed by various techniques using bisulfite-mediated DNA conversion or methylation-sensitive restriction enzymes. Under appropriate conditions, the former technique specifically converts unmethylated cytosines into uracils. Methods taking advantage of this principle include bisulfite sequencing, methylation-specific PCR (MSP), real-time MSP, MethyLight, methylation-sensitive high-resolution melting analysis, and pyrosequencing, all of which are used widely. Combinations with leading-edge techniques, such as next-generation sequencing and digital PCR, have provided increased accuracy and a broader range of applications. The appropriate method should be selected considering factors, such as the amount of DNA required, flexibility in selection of CpG sites to analyze, degree of quantitation of the method, technical complexity, and the cost. This chapter reviews principles of methylation detection and characteristics of individual methods, and provides tips for bisulfite-mediated conversion, bisulfite sequencing, MSP, and quantitative MSP.

Abstract LB-245: Preclinical study of epigenetic drug-based differentiation therapy for neuroblastoma

Neuroblastoma (NBL) is the most common extracranial solid tumor in children. We previously reported that the CpG island methylator phenotype (CIMP) of NBL was strongly associated with poor prognosis, and also suggested that CIMP may be a possible target for DNA demethylation therapy. Differentiation therapy with 13-cis-retinoic acid has already been established as the standard for high-risk NBLs in the USA. In this study, we aimed to establish an “epigenetic drug-based differentiation therapy” using a combination of a DNA demethylating agent (5-aza-2’-deoxycytidine: 5-Aza-CdR) and a differentiation agent (tamibarotene: TBT), a new synthetic retinoid. Treatment with 5-Aza-CdR suppressed the growth of 12 NBL cell lines by increasing the number of cells in the S-phase. Genome-wide DNA methylation analysis revealed that 5-Aza-CdR treatment induced global DNA hypomethylation, and that genes related to cell death and neurological processes were enriched as hypomethylated genes, suggesting that DNA demethylation therapy might assist the differentiation agent in inducing neuron differentiation. TBT induced differentiation of five NBL cell lines along with induction of neural extension and upregulation of differentiation markers, such as HOXD4, NGFR, and NTRK1. Pretreatment with 5-Aza-CdR increased the expression levels of differentiation markers, indicating that 5-Aza-CdR enhanced TBT-induced differentiation in vitro. Finally, the tumor suppression effect of 5-Aza-CdR and TBT in vivo was investigated using a mouse xenograft model of KELLY and NB-1 cell lines. Although a synergistic effect of 5-Aza-CdR and TBT was not apparent, they could induce significant tumor regression without severe side-effects. From these data, we concluded that 5-Aza-CdR and TBT had antitumor activity in vitro and in vivo, and that epigenetic drug-based differentiation therapy is a promising therapeutic strategy for NBL. Citation Format: Naoko Hattori, Akiko Mori, Kana Kimura, Emi Kubo, Kiyoshi Asada, Hiroshi Kawamoto, Toshikazu Ushijima. Preclinical study of epigenetic drug-based differentiation therapy for neuroblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-245.