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Dysregulation of transcriptional programs that are tightly regulated by DNA methylation during placental and fetal development at different gestational stages, may cause recurrent miscarriage. Here, we examined genome-wide DNA methylation in chorionic villi and decidual tissues from patients suffering RM and from healthy women who had undergone artificial abortion (n = 5 each). We found that 13,426 and 5816 CpG sites were differentially methylated in chorionic villi and decidua, respectively. DNA methylation profiles of chorionic villi, but not decidua, in RM patients was clearly distinct from AA controls. Among the differentially methylated genes, the enhancer region of SPATS2L was significantly more highly methylated in RM patients (n = 19) than AA controls (n = 19; mean methylation level, 52.0%-vs.-28.9%, P SPATS2L in extravillous trophoblast cells decreased their invasion and migration abilities. Our data indicate that particularly the chorionic villi in RM patients exhibit distinct DNA methylation profiles compared with normal pregnancies and that this changed DNA methylation status may impede the progression of embryo development via the altered expression of genes such as SPATS2L in the villi.

Serous ovarian cancer is the most frequent type of epithelial ovarian cancer. Despite the use of surgery and platinum‐based chemotherapy, many patients suffer from recurrence within 6 months, termed platinum resistance. Currently, the lack of relevant molecular biomarkers for the prediction of the early recurrence of serous ovarian cancers is linked to the poor prognosis. To identify an effective biomarker for early recurrence, we analyzed the genome‐wide DNA methylation status characteristic of early recurrence after treatment. The patients in The Cancer Genome Atlas (TCGA) dataset who showed a complete response after the first therapy were categorized into 2 groups: early recurrence serous ovarian cancer (ERS, recurrence ≤12 months, n = 51) and late recurrence serous ovarian cancer (LRS, recurrence >12 months, n = 158). Among the 12 differently methylated probes identified between the 2 groups, we found that ZNF671 was the most significantly methylated gene in the early recurrence group. A validation cohort of 78 serous ovarian cancers showed that patients with ZNF671 DNA methylation had a worse prognosis (P

Despite recent advances in clinical treatment, pancreatic cancer remains a highly lethal malignancy. In order to improve the survival rate of patients with pancreatic cancer, the development of non-invasive diagnostic methods using effective biomarkers is urgently needed. Here, we developed a highly sensitive method to detect DNA methylation in cell-free (cf)DNA samples based on the enrichment of methyl-CpG binding (MBD) protein coupled with a digital PCR method (MBD-ddPCR). Five DNA methylation markers for the diagnosis of pancreatic cancer were identified through DNA methylation microarray analysis in 37 pancreatic cancers. The sensitivity and specificity of the five markers were validated in another independent cohort of pancreatic cancers (100% and 100%, respectively; n = 46) as well as in The Cancer Genome Atlas data set (96% and 90%, respectively; n = 137). MBD-ddPCR analysis revealed that DNA methylation in at least one of the five markers was detected in 23 (49%) samples of cfDNA from 47 patients with pancreatic cancer. Further, a combination of DNA methylation markers and the KRAS mutation status improved the diagnostic capability of this method (sensitivity and specificity, 68% and 86%, respectively). Genome-wide MBD-sequencing analysis in cancer tissues and corresponding cfDNA revealed that more than 80% of methylated regions were overlapping; DNA methylation profiles of cancerous tissues and cfDNA significantly correlated with each other (R = 0.97). Our data indicate that newly developed MBD-ddPCR is a sensitive method to detect cfDNA methylation and that using five marker genes plus KRAS mutations may be useful for the detection of pancreatic cancers.

Spinal and bulbar muscular atrophy (SBMA) is a polyglutamine‐mediated neuromuscular disease caused by a CAG repeat expansion in the androgen receptor (AR) gene. While transcriptional dysregulation is known to play a critical role in the pathogenesis of SBMA, the underlying molecular pathomechanisms remain unclear. DNA methylation is a fundamental epigenetic modification that silences the transcription of various genes that have a CpG‐rich promoter. Here, we showed that DNA methyltransferase 1 (Dnmt1) is highly expressed in the spinal motor neurons of an SBMA mouse model and in patients with SBMA. Both genetic Dnmt1 depletion and treatment with RG108, a DNA methylation inhibitor, ameliorated the viability of SBMA model cells. Furthermore, a continuous intracerebroventricular injection of RG108 mitigated the phenotype of SBMA mice. DNA methylation array analysis identified hairy and enhancer of split 5 (Hes5) as having a CpG island with hyper‐methylation in the promoter region, and the Hes5 expression was strongly silenced in SBMA. Moreover, Hes5 over‐expression rescued the SBMA cells possibly by inducing Smad2 phosphorylation. Our findings suggest DNA hyper‐methylation underlies the neurodegeneration in SBMA.

Pancreatic cancer is a highly lethal malignancy despite recent clinical advances. Since pancreatic cancer is often difficult to diagnose, there is an urgent need to develop noninvasive biomarkers for detection. Epigenetic alterations have been recognized as a common hallmark of many types of human cancers as well as pancreatic cancer. The cell-free DNA (cfDNA) in blood possesses tumor-related genetic alterations as well as epigenetic alterations. Therefore, invention of blood-based DNA methylation biomarkers and their high-sensitive detection system may be a promising diagnostic tool for pancreatic cancer. In order to establish the diagnostic biomarkers, first we performed genome-wide DNA methylation analysis of 38 endoscopic ultrasound-guided fine-needle aspiration specimens of pancreatic cancer using Illumina Infinium HumanMethylation450 BeadChip. We further analyzed the DNA methylation profiles of other types of tumors in the public database TCGA (The Cancer Genome Atlas), and identified five marker genes, which were specifically methylated in pancreatic cancers. Analysis of DNA methylation status of pancreas cancers comparing 10 normal tissues in TCGA data set revealed that 99% of tumors were methylated in at least one of the five markers (sensitivity, 98.9%; specificity, 44%). We next examined the DNA methylation status of these markers in the validation cohort (n=46) by bisulfite pyrosequencing or quantitative methylation-specific PCR and found that 42/46 (91.3%) cases were methylated in at least one marker. Finally, we analyzed methylation status in cfDNA from pancreatic cancer patients. We performed bisulfite sequencing using next-generation sequencer and detected methylation in only 5/13 (38.5%) samples in one of the markers. Since the amount of DNA from cancer tissue is very small, we need to develop more a sensitive technique to detect aberrant DNA methylation to diagnose cancer patients. Now we are developing a new sensitive method to detect DNA methylation in cfDNA. Citation Format: Keiko Shinjo, Keisuke Katsushima, Miho Suzuki, Genta Nagae, Hiroyuki Aburatani, Kenji Yamao, Yutaka Kondo. Development of a sensitive method to detect aberrant DNA methylation in cfDNA for pancreas cancer diagnosis [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 3331.

Given that treatment options for patients with glioblastoma are limited, much effort has been made to clarify the underlying mechanisms of gliomagenesis. Recent genome-wide genomic and epigenomic analyses have revealed that mutations in epigenetic modifiers occur frequently in gliomas and that dysregulation of epigenetic mechanisms is closely associated with glioma formation. Given that epigenetic changes are reversible, understanding the epigenetic abnormalities that arise in gliomagenesis might be key to developing more effective treatment strategies for glioma. In this review, we focus on the recent advancements in epigenetic research with respect to gliomas, consider how epigenetic mechanisms dynamically regulate tumor cells, including the cancer stem cell population, and discuss perspectives and challenges for glioma treatment in the near future.

Meningiomas are among the most common intracranial tumors and are mostly curable by surgical resection. However, some populations of meningiomas with benign histological profiles show malignant behavior. The reasons for this inconsistency are yet to be ascertained, and novel diagnostic criteria other than the histological one are urgently needed. The aim of the present study is to subclassify meningiomas from the viewpoint of gene methylation and to determine the subgroup with malignant characteristics. Thirty meningiomas were analyzed using microarrays for 6157 genes and were classified into three clusters on the basis of their methylation status; these were found to be independent of the histological grading. One of the clusters showed a high frequency of recurrence, with a marked accumulation of methylation in a subset of genes. We hypothesized that the aggressive meningiomas universally share characteristic methylation in certain genes; therefore, we chose the genes that strongly contributed to cluster formation. The quantified methylation values of five chosen genes (HOXA6, HOXA9, PENK, UPK3A and IGF2BP1) agreed well with microarray findings, and a scoring system consisting of the five genes significantly correlated with a high frequency of recurrence in an additional validation set of 32 patients. Of particular note is that three cases with malignant transformation already showed hypermethylation at histologically benign stage. In conclusion, a subgroup of meningiomas is characterized by aberrant hypermethylation of the subset of genes in the early stage of tumorigenesis, and our findings highlight the possibility of speculating potential malignancy of meningiomas by assessing methylation status.

Background and aims The majority of gastrointestinal stromal tumors (GISTs) have KIT mutations; however, epigenetic abnormalities that could conceivably potentiate the aggressiveness of GISTs are largely unidentified. Our aim was to establish epigenetic profiles associated with the malignant transformation of GISTs. Methods Methylation of four tumor suppressor genes, RASSF1A, p16, CDH1, and MGMT was analyzed in GISTs. Additionally, genome-wide DNA methylation profiles were compared between small, malignant-prone, and malignant GISTs using methylated GpG island amplification microarrays (MCAM) in a training set (n=40). Relationships between the methylation status of genes identified by MCAM and clinical features of the disease were tested in a validation set (n=75). Results Methylation of RASSF1A progressively increased from small to malignant GISTs. p16 was specifically methylated in malignant-prone and malignant GISTs. MCAM analysis showed that more genes were methylated in advanced than in small GISTs (average of 473 genes vs 360 genes, respectively, P =0.012). Interestingly, the methylation profile of malignant GISTs was prominently affected by their location. Two genes, REC8 and PAX3 , which were newly-identified via MCAM analysis, were differentially methylated in small and malignant GISTs in the training and validation sets. Patients with methylation of at least REC8 , PAX3 , or p16 had a significantly poorer prognosis ( P =0.034). Conclusion Our results suggest that GIST is not, in epigenetic terms, a uniform disease and that DNA methylation in a set of genes is associated with aggressive clinical behavior and unfavorable prognosis. The genes identified may potentially serve as biomarkers for predicting aggressive GISTs with poor survivability.

Liver metastasis is a fatal step in the progression of colorectal cancer (CRC); however, the epigenetic evolution of this process is largely unknown. To decipher the epigenetic alterations during the development of liver metastasis, the DNA methylation status of 12 genes, including 5 classical CpG island methylator phenotype (CIMP) markers, was analyzed in 62 liver metastases and in 78 primary CRCs (53 stage I–III; 25 stage IV). Genome-wide methylation analysis was also performed in stage I–III CRCs and in paired primary and liver metastatic cancers. Methylation frequencies of MGMT and TIMP3 increased progressively from stage I–III CRCs to liver metastasis (P = 0.043 and P = 0.028, respectively). The CIMP-positive cases showed significantly earlier recurrence of disease than did CIMP-negative cases with liver metastasis (P = 0.030), whereas no such difference was found in stage I–III CRCs. Genome-wide analysis revealed that more genes were methylated in stage I–III CRCs than in paired stage IV samples (P = 0.008). Hierarchical cluster analysis showed that stage I–III CRCs and stage IV CRCs were clustered into two distinct subgroups, whereas most paired primary and metastatic cancers showed similar methylation profiles. This analysis revealed distinct methylation profiles between stage I–III CRCs and stage IV CRCs, which may reflect differences in epigenetic evolution during progression of the disease. In addition, most methylation status in stage IV CRCs seems to be established before metastasis.

Aberrant DNA methylation is involved in colon carcinogenesis. Although the CpG island methylator phenotype (CIMP) is defined as a subset of colorectal cancers (CRCs) with remarkably high levels of DNA methylation, it is not known whether epigenetic processes are also involved in CIMP-negative tumors. We analyzed the DNA methylation profiles of 94 CRCs and their corresponding normal-appearing colonic mucosa with 11 different markers, including the five classical CIMP markers. The CIMP markers were frequently methylated in proximal CRCs (p < 0.01); however, RASSF1A methylation levels were significantly higher in distal CRCs, the majority of which are CIMP-negative (p < 0.05). Similarly, methylation levels of RASSF1A and SFRP1 in the normal-appearing mucosae of distal CRC cases were significantly higher than those in the proximal CRC cases (p < 0.05). They were also positively correlated with age (RASSF1A, p < 0.01; SFRP1, p < 0.01). Microarray-based genome-wide DNA methylation analysis of 18 CRCs revealed that 168 genes and 720 genes were preferentially methylated in CIMP-negative distal CRCs and CIMP-positive CRCs, respectively. Interestingly, more than half of the hypermethylated genes in CIMP-negative distal CRCs were also methylated in the normal-appearing mucosae, indicating that hypermethylation in CIMP-negative distal CRCs is more closely associated with age-related methylation. By contrast, more than 60% of the hypermethylated genes in CIMP-positive proximal CRCs were cancer specific (p < 0.01). These data altogether suggest that CpG island promoters appear to be methylated in different ways depending on location, a finding which may imply the presence of different mechanisms for the acquisition of epigenetic changes during colon tumorigenesis.

Malignant pleural mesothelioma (MPM) is a fatal thoracic malignancy, the epigenetics of which are poorly defined. We performed high-throughput methylation analysis covering 6,157 CpG islands in 20 MPMs and 20 lung adenocarcinomas. Newly identified genes were further analyzed in 50 MPMs and 56 adenocarcinomas via quantitative methylation-specific PCR. Targets of histone H3 lysine 27 trimethylation (H3K27me3) and genetic alterations were also assessed in MPM cells by chromatin immunoprecipitation arrays and comparative genomic hybridization arrays. An average of 387 genes (6.3%) and 544 genes (8.8%) were hypermethylated in MPM and adenocarcinoma, respectively. Hierarchical cluster analysis showed that the two malignancies have characteristic DNA methylation patterns, likely a result of different pathologic processes. In MPM, a separate subset of genes was silenced by H3K27me3 and could be reactivated by treatment with a histone deacetylase inhibitor alone. Integrated analysis of these epigenetic and genetic alterations revealed that only 11% of heterozygously deleted genes were affected by DNA methylation and/or H3K27me3 in MPMs. Among the DNA hypermethylated genes, three (TMEM30B, KAZALD1, and MAPK13) were specifically methylated only in MPM and could serve as potential diagnostic markers. Interestingly, a subset of MPM cases (4 cases, 20%) had very low levels of DNA methylation and substantially longer survival, suggesting that the epigenetic alterations are one mechanism affecting progression of this disease. Our findings show a characteristic epigenetic profile of MPM and uncover multiple distinct epigenetic abnormalities that lead to the silencing of tumor suppressor genes in MPM and could serve as diagnostic or prognostic targets. [Cancer Res 2009;69(23):9073–82]

Recent studies have implicated epigenetics in the pathophysiology of diabetes. Furthermore, DNA methylation, which irreversibly deactivates gene transcription, of the insulin promoter, particularly the cAMP response element, is increased in diabetes patients. However, the underlying mechanism remains unclear. We aimed to investigate insulin promoter DNA methylation in an over-nutrition state. INS-1 cells, the rat pancreatic beta cell line, were cultured under normal-culture-glucose (11.2 mmol/l) or experimental-high-glucose (22.4 mmol/l) conditions for 14 days, with or without 0.4 mmol/l palmitate. DNA methylation of the rat insulin 1 gene (Ins1) promoter was investigated using bisulfite sequencing and pyrosequencing analysis. Experimental-high-glucose conditions significantly suppressed insulin mRNA and increased DNA methylation at all five CpG sites within the Ins1 promoter, including the cAMP response element, in a time-dependent and glucose concentration-dependent manner. DNA methylation under experimental-high-glucose conditions was unique to the Ins1 promoter; however, palmitate did not affect DNA methylation. Artificial methylation of Ins1 promoter significantly suppressed promoter-driven luciferase activity, and a DNA methylation inhibitor significantly improved insulin mRNA suppression by experimental-high-glucose conditions. Experimental-high-glucose conditions significantly increased DNA methyltransferase activity and decreased ten-eleven-translocation methylcytosine dioxygenase activity. Oxidative stress and endoplasmic reticulum stress did not affect DNA methylation of the Ins1 promoter. High glucose but not palmitate increased ectopic triacylglycerol accumulation parallel to DNA methylation. Metformin upregulated insulin gene expression and suppressed DNA methylation and ectopic triacylglycerol accumulation. Finally, DNA methylation of the Ins1 promoter increased in isolated islets from Zucker diabetic fatty rats. This study helps to clarify the effect of an over-nutrition state on DNA methylation of the Ins1 promoter in pancreatic beta cells. It provides new insights into the irreversible pathophysiology of diabetes.

Pancreatic cancer is highly lethal malignancy despite the recent clinical advancement. Since pancreatic cancer is often difficult to diagnose, there is an urgent need to develop minimally invasive biomarkers for detection. Epigenetic alterations have emerged as a common hallmark of many types of human cancers as well as pancreatic cancer. The presence of cell-free DNA (cfDNA) in blood stream has been known and cfDNA possesses tumor-related genetic alterations as well as epigenetic alterations. Therefore, invention of blood-based DNA methylation biomarkers and their high-sensitive detection system might be a promising diagnostic tool for pancreatic cancer. In order to establish the diagnostic biomarkers, first, we performed genome-wide DNA methylation analysis of 38 EUS-FNA specimens of pancreatic cancer with clinical stage III-IV using Illumina Infinium HumanMethylation450 BeadChip. We further analyzed the DNA methylation profiles of other types of tumors in the public database, TCGA (The Cancer Genome Atlas), and identified five marker genes, which were specifically methylated in pancreatic cancers. In addition, analysis of DNA methylation status of pancreas cancers comparing 10 normal tissues in TCGA data set revealed that 99% of tumors were methylated at least one of the five markers (sensitivity, 98.9%; specificity, 44%). We validated these methylation markers in different FNA sample set (n = 19) by bisulfite pyrosequencing or quantitative methylation specific PCR and found that 18/19 (95%) cases were methylated in at least one marker (AUC = 0.95). Finally, we performed methylation analysis in cfDNA from pancreatic cancer patients. From our preliminary data, we could detect aberrant DNA methylation in cfDNA from pancreatic cancer patients. We identified potential DNA methylation biomarkers for the diagnosis of pancreatic cancers and these markers might be applicable for detecting cancer in cfDNA. Citation Format: Keiko Shinjo, Keisuke Katsushima, Genta Nagae, Hiroyuki Aburatani, Kenji Yamao, Yutaka Kondo. Development of DNA methylation biomarkers for pancreatic cancer diagnosis. [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 4448.

Pancreatic cancer is highly lethal malignancy despite the recent clinical advancement. Since pancreatic cancer is often difficult to diagnose, there is an urgent need to develop novel biomarkers for early detection with blood tests. Epigenetic alterations have emerged as a common hallmark of many types of human cancers as well as pancreatic cancer. Studies have shown that epigenetic dysregulation is associated with multiple steps during carcinogenesis. Therefore, invention of blood-based DNA methylation biomarkers and their high-sensitive detection system may be a promising diagnostic tool for pancreatic cancer. In order to establish the diagnostic system, first, we performed genome-wide DNA methylation analysis of 38 EUS-FNA specimens of pancreatic cancer with clinical stage III-IV using Illumina Infinium HumanMethylation450 BeadChip. We further analyzed the DNA methylation profiles of other types of tumors in the public database, TCGA (The Cancer Genome Atlas), and identified five marker genes, which were specifically methylated in pancreatic cancers. In addition, analysis of DNA methylation status of pancreas cancers comparing 10 normal tissues in TCGA data set revealed that 99% of tumors were methylated at least one of the five markers (sensitivity, 98.9%; specificity, 44%). We next examined the DNA methylation status in the markers in the validation cohort (n=19) by bisulfite pyrosequencing and quantitative methylation specific PCR and found that 18/19 (95%) cases were methylated in at least one marker (AUC=0.95). Currently, our preliminary data showed that more than 95% of cancer patients have aberrant DNA methylation in circulating free DNA (cfDNA). Our results indicate that detection of DNA methylation in the cfDNA might be a promising and potent tool for diagnosis of pancreatic cancers. Citation Format: Keiko Shinjo, Yutaka Kondo. Use of DNA methylation in circulating free DNA as a potent biomarker for pancreatic cancer detection. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B14.

Inactivation of methylcytosine dioxygenase, ten-eleven translocation (TET) is known to be associated with aberrant DNA methylation in cancers. Tumors with a CpG island methylator phenotype (CIMP), a distinct subgroup with extensive DNA methylation, show characteristic features in the case of colorectal cancer. The relationship between TET inactivation and CIMP in colorectal cancers is not well understood. The expression level of TET family genes was compared between CIMP-positive (CIMP-P) and CIMP-negative (CIMP-N) colorectal cancers. Furthermore, DNA methylation profiling, including assessment of the TET1 gene, was assessed in colorectal cancers, as well as colon polyps. The TET1 was silenced by DNA methylation in a subset of colorectal cancers as well as cell lines, expression of which was reactivated by demethylating agent. TET1 methylation was more frequent in CIMP-P (23/55, 42%) than CIMP-N (2/113, 2%, P < 0.0001) colorectal cancers. This trend was also observed in colon polyps (CIMP-P, 16/40, 40%; CIMP-N, 2/24, 8%; P = 0.002), suggesting that TET1 methylation is an early event in CIMP tumorigenesis. TET1 methylation was significantly associated with BRAF mutation but not with hMLH1 methylation in the CIMP-P colorectal cancers. Colorectal cancers with TET1 methylation have a significantly greater number of DNA methylated genes and less pathological metastasis compared to those without TET1 methylation (P = 0.007 and 0.045, respectively). Our data suggest that TET1 methylation may contribute to the establishment of a unique pathway in respect to CIMP-mediated tumorigenesis, which may be incidental to hMLH1 methylation. In addition, our findings provide evidence that TET1 methylation may be a good biomarker for the prediction of metastasis in colorectal cancer. Cancer Prev Res; 8(8); 702–11. ©2015 AACR.

Inactivation of methylcytosine dioxygenase, ten-eleven translocation (TET) is known to be associated with aberrant DNA methylation in cancers. Tumors with a CpG island methylator phenotype (CIMP), a distinct subgroup with extensive DNA methylation, show characteristic features in the case of colorectal cancer (CRC). The relationship between TET inactivation and CIMP in CRCs is not well understood. The expression level of TET family genes was compared between CIMP-positive (CIMP-P) and CIMP-negative (CIMP-N) CRCs. Furthermore, DNA methylation profiling, including assessment of the TET1 gene, was assessed in CRCs, as well as colon polyps. The TET1 was silenced by DNA methylation in a subset of CRCs as well as cell lines, expression of which was reactivated by demethylating agent. TET1 methylation was more frequent in CIMP-P (24/55, 44%) than CIMP-N (3/113, 3%, P Citation Format: Norihisa Ichimura, Keiko Shinjo, Fumiharu Ohka, Keisuke Katsushima, Akira Hatanaka, Masayuki Tojo, Yasuhiro Shimizu, Eiichiro Yamamoto, Hiromu Suzuki, Yutaka Kondo. Aberrant TET1 methylation closely associated with CpG island methylator phenotype in colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1054. doi:10.1158/1538-7445.AM2015-1054

Despite the clinical advancement, pancreatic cancer remains one of the most deadly cancers. Pancreatic cancers are often difficult to diagnose and there is no reliable screening biomarker for early detection of this cancer. Epigenetic alterations have emerged as a common hallmark of many types of human cancers as well as pancreatic cancers. Studies have shown that epigenetic dysregulation is associated with multiple steps during carcinogenesis. Therefore, detection of aberrant DNA methylation from blood samples may be a promising diagnostic tool for pancreatic cancers. First, we analyzed KRAS mutation status of 100 samples obtained by Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA), which is a safe and accurate technique to perform the pancreatic tissue biopsy, and found that more than 80% of the cells in the samples harbors KRAS mutation indicating high amount of cancer cells in the specimens. Next, we performed genome-wide DNA methylation analysis of 38 EUS-FNA specimens using Illumina Infinium HumanMethylation450 BeadChip. We further analyzed the deposited DNA methylation profiles of other tumor types in the public databases, such as TCGA, and identified 8 marker genes with high frequency and specificity of DNA methylation in pancreatic cancers. We validated the DNA methylation status of these genes by bisulfite pyrosequencing and quantitative methylation specific PCR. These markers were frequently methylated in EUS-FNA samples (95% of samples are methylation positive in at least one marker gene). Now we are analyzing the DNA methylation status in pancreatic cancers and corresponding blood samples in order to identify the most effective set of markers for diagnose pancreatic cancers. Our results suggest that analysis of DNA methylation in the circulating free DNA might be a safe and potent tool for evaluating and staging pancreatic cancers. Citation Format: Keiko Shinjo, Fumiharu Ohka, Keisuke Katsushima, Akira Hatanaka, Norihisa Ichimura, Zhao Juan, Kenji Yamao, Yutaka Kondo. DNA methylation in circulating free DNA as a new biomarker for pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1069. doi:10.1158/1538-7445.AM2015-1069

Epigenetic alterations have emerged as a common hallmark of many types of human cancers including lung cancers. Therefore, detection of aberrant DNA methylation from blood samples may be a promising diagnostic tool for cancers. We have been developing a highly sensitive plasma-based approach to detect aberrant DNA methylation in lung cancer patients. First, we identified seven markers from genome-wide methylation analysis, which can diagnose lung cancer in tissue samples with high accuracy (sensitivity, 91%; specificity, 100%). Next, we evaluated DNA methylation status in tissue samples and corresponding plasma samples in each patient using pyrosequencing analysis and qMSP. However, DNA methylation status between tissue and plasma samples were not concordant by both methods (pyrosequencing, 0/19 cases; qMSP, 3/9 cases). Given the concentration of DNA in plasma originated from cancerous tissue might theoretically be very low, we need to use more sensitive and accurate methods. We used digital PCR and next generation sequencer to analyze the methylation status of plasma DNA. These methods are both highly sensitive. Now we invent a new technology using specific probes, which can recognize sequence information including methylated cytosine even in one DNA molecule. This new technology might be applicable as a practical tool for lung cancer diagnosis. Citation Format: Keiko Shinjo, Eisaku Kondo, Yutaka Kondo. New technology to detect DNA methylation in plasma sample from lung cancer patients. [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 416. doi:10.1158/1538-7445.AM2014-416

Tumor cell plasticity contributes to functional and morphologic heterogeneity. To uncover the underlying mechanisms of this plasticity, we examined glioma stem-like cells (GSC) where we found that the biologic interconversion between GSCs and differentiated non-GSCs is functionally plastic and accompanied by gain or loss of polycomb repressive complex 2 (PRC2), a complex that modifies chromatin structure. PRC2 mediates lysine 27 trimethylation on histone H3 and in GSC it affected pluripotency or development-associated genes (e.g., Nanog, Wnt1, and BMP5) together with alterations in the subcellular localization of EZH2, a catalytic component of PRC2. Intriguingly, exogenous expression of EZH2-dNLS, which lacks nuclear localization sequence, impaired the repression of Nanog expression under differentiation conditions. RNA interference (RNAi)–mediated attenuation or pharmacologic inhibition of EZH2 had little to no effect on apoptosis or bromodeoxyuridine incorporation in GSCs, but it disrupted morphologic interconversion and impaired GSC integration into the brain tissue, thereby improving survival of GSC-bearing mice. Pathologic analysis of human glioma specimens revealed that the number of tumor cells with nuclear EZH2 is larger around tumor vessels and the invasive front, suggesting that nuclear EZH2 may help reprogram tumor cells in close proximity to this microenvironment. Our results indicate that epigenetic regulation by PRC2 is a key mediator of tumor cell plasticity, which is required for the adaptation of glioblastoma cells to their microenvironment. Thus, PRC2-targeted therapy may reduce tumor cell plasticity and tumor heterogeneity, offering a new paradigm for glioma treatment. Cancer Res; 73(14); 4559–70. ©2013 AACR.

Pancreatic ductal adenocarcinoma (PDAC) is a highly invasive cancer with a poor prognosis. Although microRNA (miRNA) transcripts have a crucial role in carcinogenesis and development, little information is known regarding the aberrant DNA methylation of miRNAs in PDAC. Using methylated DNA immunoprecipitation-chip analysis, we found that miR-615-5p was hypermethylated in its putative promoter region, which silenced its expression in PDAC cell lines. In addition, the overexpression of miR-615-5p in pancreatic cancer cells suppressed cell proliferation, migration and invasion. Insulin-like growth factor 2 (IGF2) is an imprinted gene, and its abnormal expression contributes to tumor growth. Here, we identified IGF2 as a target of miR-615-5p using a luciferase reporter assay. IGF2 upregulation in PDAC tissues was not correlated with a loss of imprinting but was inversely correlated with miR-615-5p downregulation. In addition, miR-615-5p suppressed pancreatic cancer cell proliferation, migration and invasion by directly targeting IGF2, and this effect could be reversed by co-transfection with IGF2. Furthermore, the stable overexpression of miR-615-5p inhibited tumor growth in vivo and was correlated with IGF2 expression. Using RNA sequencing, we further identified miR-615-5p as potentially targeting other genes, such as the proto-oncogene JUNB, and interfering with the insulin signaling pathway. Taken together, our results demonstrate that miR-615-5p was abnormally downregulated in PDAC cells due to promoter hypermethylation, which limited its inhibition of IGF2 and other target genes, thereby contributing to tumor growth, invasion and migration. These data demonstrate a novel and important role of miR-615-5p as a tumor suppressor in PDAC.

Background & Aims Cells of tumors associated with chronic inflammation frequently have altered patterns of DNA methylation, including hepatocellular carcinomas. Chronic hepatitis has also been associated with aberrant DNA methylation, but little is known about their relationship. Methods Pyrosequencing was used to determine the methylation status of cultured Huh7.5.1 hepatoma cells after hepatitis C virus (HCV) infection. We also studied mice with severe combined immunodeficiency carrying the urokinase-type plasminogen activator transgene controlled by an albumin promoter (urokinase-type plasminogen activator/severe combined immunodeficient mice), in which up to 85% of hepatocytes were replaced by human hepatocytes (chimeric mice). Mice were given intravenous injections of hepatitis B virus (HBV) or HCV, liver tissues were collected, and DNA methylation profiles were determined at different time points after infection. We also compared methylation patterns between paired samples of hepatocellular carcinomas and adjacent nontumor liver tissues from patients. Results No reproducible changes in DNA methylation were observed after infection of Huh7.5.1 cells with HCV. Livers from HBV- and HCV-infected mice had genome-wide, time-dependent changes in DNA methylation, compared with uninfected urokinase-type plasminogen activator/severe combined immunodeficient mice. There were changes in 160 ± 63 genes in HBV-infected and 237 ± 110 genes in HCV-infected mice. Methylation of 149 common genes increased in HBV- and HCV-infected mice; methylation of some of these genes also increased in hepatocellular carcinoma samples from patients compared with nontumor tissues. Expression of Ifng , which is expressed by natural killer cells, increased significantly in chimeric livers, in concordance with induction of DNA methylation, after infection with HBV or HCV. Induction of Ifng was reduced after administration of an inhibitor of natural killer cell function (anti−asialo GM1). Conclusions In chimeric mice with humanized livers, infection with HBV and HCV appears to activate a natural kill cell−dependent innate immune response. This contributes to the induction and accumulation of aberrant DNA methylation in human hepatocytes.

DNA methylation affects the aggressiveness of human malignancies. Cancers with CpG island methylator phenotype (CIMP), a distinct group with extensive DNA methylation, show characteristic features in several types of tumors. In this study, we initially defined the existence of CIMP in 41 lung adenocarcinomas (AdCas) through genome-wide DNA methylation microarray analysis. DNA methylation status of six CIMP markers newly identified by microarray analysis was further estimated in a total of 128 AdCas by bisulfite pyrosequencing analysis, which revealed that 10 (7.8%), 40 (31.3%) and 78 (60.9%) cases were classified as CIMP-high (CIMP-H), CIMP-low and CIMP-negative (CIMP-N), respectively. Notably, CIMP-H AdCas were strongly associated with wild-type epidermal growth factor receptor (EGFR), males and heavy smokers (P = 0.0089, P = 0.0047 and P = 0.0036, respectively). In addition, CIMP-H was significantly associated with worse prognosis; especially among male smokers, CIMP-H was an independent prognostic factor (hazard ratio 1.7617, 95% confidence interval 1.0030-2.9550, P = 0.0489). Compellingly, the existence of CIMP in AdCas was supported by the available public datasets, such as data from the Cancer Genome Atlas. Intriguingly, analysis of AdCa cell lines revealed that CIMP-positive AdCa cell lines were more sensitive to a DNA methylation inhibitor than CIMP-N ones regardless of EGFR mutation status. Our data demonstrate that CIMP in AdCas appears to be a unique subgroup that has distinct clinical traits from other AdCas. CIMP classification using our six-marker panel has implications for personalized medical strategies for lung cancer patients; in particular, DNA methylation inhibitor might be of therapeutic benefit to patients with CIMP-positive tumors.

Besides known genetic aberrations, epigenetic alterations have emerged as common hallmarks of many cancer types, including lung cancer. Epigenetics is a process involved in gene regulation, mediated via DNA methylation, histone modification, chromatin remodeling, and functional noncoding RNAs, which influences the accessibility of the underlying DNA to transcriptional regulatory factors that activate or repress expression. Studies have shown that epigenetic dysregulation is associated with multiple steps during carcinogenesis. Since epigenetic therapy is now in clinical use in hematopoietic diseases and undergoing trials for lung cancer, a better understanding of epigenetic abnormalities is desired. Recent technologies for high-throughput genome-wide analyses for epigenetic modifications are promising and potent tools for understanding the global dysregulation of cancer epigenetics. In this chapter, studies of epigenetic abnormality and its clinical implication in lung cancers are discussed.

Besides known genetic aberrations, epigenetic alterations have emerged as common hallmarks of many cancer types, including lung cancer. Epigenetics is a process involved in gene regulation, mediated via DNA methylation, histone modification, chromatin remodeling, and functional noncoding RNAs, which influences the accessibility of the underlying DNA to transcriptional regulatory factors that activate or repress expression. Studies have shown that epigenetic dysregulation is associated with multiple steps during carcinogenesis. Since epigenetic therapy is now in clinical use in hematopoietic diseases and undergoing trials for lung cancer, a better understanding of epigenetic abnormalities is desired. Recent technologies for high-throughput genome-wide analyses for epigenetic modifications are promising and potent tools for understanding the global dysregulation of cancer epigenetics. In this chapter, studies of epigenetic abnormality and its clinical implication in lung cancers are discussed.

Epigenetics mechanisms are essential for normal development and maintenance of tissue-specific gene expression patterns in normal cells. Certain forms of histone methylation induce local heterochromatin formation, which is readily reversible, whereas DNA methylation leads to stable long-term repression. The precise epigenomic landscape present in normal cells undergoes extensive distortion in cancer. These characteristic epigenetic mechanisms are thought to be associated with tumor formation and progression. The reversible nature of epigenetic changes has led to the emergence of the field of epigenetic therapy, which may lead to potent and promising cancer treatment.

Background: Lung cancer is the leading cause of human cancer death worldwide. DNA methylation affects the aggressiveness of human malignancies. Cancers with CpG island methylator phenotype (CIMP), a distinct group with extensive DNA methylation, show characteristic features in several types of tumors. However, the existence and characteristics of CIMP in lung adenocarcinomas (AdCas) from the global point of view has remained elusive. The aim of this study is to clarify the significance of DNA methylation, especially the clinical impact of CIMP, in AdCas. Methods: Using genome-wide microarray analysis, DNA methylation profiling of 41 AdCas were initially examined. DNA methylation status of six CIMP markers newly identified by microarray analysis was further estimated in a validation set of 87 AdCas by bisulfate pyrosequencing. A total of 128 AdCas were examined for the significance of CIMP. In addition, responsiveness to DNA methylation inhibitor was examined in AdCa cell lines with different CIMP status. Results: Consensus clustering analysis based on DNA methylation showed that AdCas were classified into three subgroups, clusters 1, 2, and 3 (average of 767, 486, and 319 methylated genes in each subgroup, respectively; P 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 5003. doi:1538-7445.AM2012-5003

Background and aims: While the connection between chronic inflammation and tumorigenesis was suspected in hepatocellular carcinomas (HCCs), key factors linking these two processes were not completely understood. Ample evidence shows that accumulation of genetic and epigenetic abnormalities may be fundamental mechanisms underlying tumor formation. The aim of this study is to examine whether viral infection affects epigenetic profile in chronic hepatitis/liver cirrhosis (CH/LC) and HCC tissues. Methods: To evaluate the direct effects of viral infection, we examined DNA methyaltion changes in hepatitis C virus (HCV) infected cells in vitro. Genome-wide DNA methylation status was examined in liver tissues repopulated with human hepatocytes (humanized liver) in uPA/SCID mice, which were infected with hepatitis B virus (HBV-mice, 13 mice) or HCV-mice (14 mice), using methylated CpG island amplification-microarrays (MCAM, 6,157 CpG islands). Genome-wide DNA methylation status was also examined in 37 HCCs: 19 and 18 were associated with HCV and HBV infection, respectively (HCC-HBV and HCC-HCV). Further, quantitative pyrosequencing analysis was performed to examine the methylation status of selected genes. Result: MCAM analysis in humanized mouse liver revealed that larger number of genes were methylated in HCV-mice than HBV-mice (287 genes versus 157 genes, P=0.034, respectively). DNA methylation target genes in HBV-mice were also methylated in HCV-mice, while both HBV-mice and HCV-mice had own specific targets. Association between aberrant DNA methylation and infection period was more strongly observed in HBV-mice (r=0.769) than HCV-mice (r=0.175). Interestingly, both HBV and HCV affected DNA methylation not only in infected humanized liver (hepatocytes) but also surrounding mouse tissues (non-infected cells). In vitro analysis of HCV infection did not affect DNA methylaion in cultured cells for more than a month. Thses data suggest that HCV infection may affect DNA methylaiton via inflammatory responses. Consistently, various cytokines from mouse tissues were upregulated after HBV and HCV infections. In clinical samples, most of the DNA methylation target genes in HCC-HBV were also methylated in HCC-HCV, as was found in mouse model. In addition, many DNA methylation target genes in human HCCs were also identified in mouse model with MCAM analysis. Conclusion: We developed mouse model to assess DNA methylaton profiling after hepatitis viral infection. Although HBV protein has been known to induce aberrant DNA methylation, our data sugget that viral infection is a strong inducer of aberrant DNA methylation via inflammatory responses. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4811. doi:10.1158/1538-7445.AM2011-4811

Background: Lung cancer is the leading cause of cancer-related death worldwide. While targeted therapy, such as tyrosine kinase inhibitors (TKIs), has been used against the non small cell lung cancers (NSCLCs) patients with epidermal growth factor receptor (EGFR) mutation, not all the patients received benefit from TKIs. Epigenetic alterations have been recognized as one of the important mechanism of NSCLCs. The aim of this study is to clarify the significance of DNA methylation, especially the clinical impacts of CpG island methylator phenotype (CIMP), in NSCLCs. Methods: Using methylated CpG island amplification-microarray (MCAM), a genome-wide DNA methylation analysis, 41 lung adenocarcinomas (AdCas) were analyzed. DNA methylation of newly identified genes from MCAM analysis was further examined in another cohort of 86 AdCas with bisulfate pyrosequencing method. Results: Unsupervised hierarchical cluster analysis revealed that DNA methylation was extensively accumulated in one subgroup (n=4, 9.8%), which may represent CIMP tumors (651 genes vs. 354 genes, P=0.0004). DNA methylation status of newly identified 7 CIMP markers revealed that 8 (9.3%), 12 (14%), and 66 (76.7%) were classified into CIMP-high, CIMP-low, and CIMP-negative tumors, respectively. CIMP-high tumors showed no EGFR mutations and frequent exposure to smoking. In addition, these tumors had worse prognosis than the other tumors (P=0.035), suggesting that CIMP-high tumors have characteristic clinical features, which may represent an alternative mechanism to the EGFR mutation. Treatments of NSCLC cell lines showed that CIMP positive AdCa cell lines are sensitive to DNA demethylating agent regardless of EGFR status. Conclusions: Our data suggest that CIMP in AdCas appears to be a unique subgroup, which has distinguished clinical traits from the other AdCas. Applying DNA demethylating agent to CIMP-high tumors might be a new strategy for the treatment of NSCLCs without EGFR mutation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 61. doi:10.1158/1538-7445.AM2011-61