Decrease In Global Methylation Research Articles

DNA methylation profiling identifies epigenetic signatures of early gastric cancer.

Research on the DNA methylation status of gastric cancer (GC) has primarily focused on identifying invasive GC to develop biomarkers for diagnostic. However, DNA methylation in noninvasive GC remains unclear. We conducted a comprehensive DNA methylation profiling study of differentiated-type intramucosal GCs (IMCs). Illumina 850K microarrays were utilized to assess the DNA methylation profiles of formalin-fixed paraffin-embedded tissues from eight patients who were Epstein-Barr virus-negative and DNA mismatch repair proficient, including IMCs and paired adjacent nontumor mucosa. Gene expression profiling microarray data from the GEO database were analyzed via bioinformatics to identify candidate methylation genes. The final validation was conducted using quantitative real-time PCR, the TCGA methylation database, and single-sample gene set enrichment analysis (GSEA). Genome-wide DNA methylation profiling revealed a global decrease in methylation in IMCs compared with nontumor tissues. Differential methylation analysis between IMCs and nontumor tissues identified 449 differentially methylated probes, with a majority of sites showing hypomethylation in IMCs compared with nontumor tissues (66.1% vs 33.9%). Integrating two RNA-seq microarray datasets, we found one hypomethylation-upregulated gene: eEF1A2, overlapped with our DNA methylation data. The mRNA expression of eEF1A2 was higher in twenty-four IMC tissues than in their paired adjacent nontumor tissues. GSEA indicated that the functions of eEF1A2 were associated with the development of IMCs. Furthermore, TCGA data indicated that eEF1A2 is hypomethylated in advanced GC. Our study illustrates the implications of DNA methylation alterations in IMCs and suggests that aberrant hypomethylation and high mRNA expression of eEF1A2 might play a role in IMCs development.

Genome-wide DNA methylation profiling identifies epigenetic signatures of gastric cardiac intestinal metaplasia

BackgroundMeasuring the DNA methylome may offer the opportunity to identify novel disease biomarkers and insights into disease mechanisms. Although aberrant DNA methylation has been investigated in many human cancers and precancerous lesions, the DNA methylation landscape of gastric cardiac intestinal metaplasia (IM) remains unknown. Therefore, we aimed to investigate the genome-wide DNA methylation landscape and to search for potential epigenetic biomarkers of gastric cardiac IM.MethodsHistopathologic profiling was performed on a total of 118 gastric cardiac biopsies from cancer-free individuals. Genome-wide DNA methylation analysis was performed on 11 gastric cardiac mucosal biopsies (IM = 7; normal = 4) using Illumina 850K microarrays. Transcriptional relevance of any candidate epigenetic biomarker was validated by qRT-PCR.ResultsThe detection rate of gastric cardiac IM was 23% (27/118) in cancer-free individuals. Genome-wide DNA methylation profiling showed a global decrease in methylation in IM compared with normal tissues (median methylation = 0.64 and 0.70 for gastric cardiac IM and normal tissues, respectively). Differential methylation analysis between gastric cardiac IM and normal tissues identified 38,237 differentially methylated probes (DMPs) with a majority of sites showing hypermethylation in IM compared with normal tissues (56.3% vs. 43.7%). Subsequent analysis revealed a significant enrichment of hypermethylated DMPs in promoter and CpG islands (p < 0.001 for both, Pearson χ2 test). For DMPs located in promoter CpG islands showing extreme hypermethylation, the candidate gene with the largest number of DMPs (n = 7) was mapped to HOXA5. Accordingly, mRNA expression of HOXA5 was significantly reduced in IM compared to normal tissue.ConclusionsOur results suggest the implication of alterations in DNA methylation in gastric cardiac IM and highlight that HOXA5 hypermethylation may be a promising epigenetic biomarker, emphasizing the role of aberrant HOXA5 expression in the pathogenesis of gastric cardiac IM.

All roads lead to Rome: the many ways to pluripotency.

Cell pluripotency, spatial restriction, and development are spatially and temporally controlled by epigenetic regulatory mechanisms that occur without any permanent loss or alteration of genetic material, but rather through modifications "on top of it." These changes modulate the accessibility to transcription factors, either allowing or repressing their activity, thus shaping cell phenotype. Several studies have demonstrated the possibility to interact with these processes, reactivating silenced genes and inducing a high plasticity state, via an active demethylating effect, driven by ten-eleven translocation (TET) enzymes and an overall decrease of global methylation. In agreement with this, TET activities have been shown to be indispensable for mesenchymal to epithelial transition of somatic cells into iPSCs and for small molecule-driven epigenetic erasure. Beside the epigenetic mechanisms, growing evidences highlight the importance of mechanical forces in supporting cell pluripotency, which is strongly influenced by 3D rearrangement and mechanical properties of the surrounding microenvironment, through the activation of specific mechanosensing-related pathways. In this review, we discuss and provide an overview of small molecule ability to modulate cell plasticity and define cell fate through the activation of direct demethylating effects. In addition, we describe the contribution of the Hippo signaling mechanotransduction pathway as one of the mechanisms involved in the maintenance of pluripotency during embryo development and its induction in somatic cells.

Global impact of somatic structural variation on the DNA methylome of human cancers

BackgroundGenomic rearrangements exert a heavy influence on the molecular landscape of cancer. New analytical approaches integrating somatic structural variants (SSVs) with altered gene features represent a framework by which we can assign global significance to a core set of genes, analogous to established methods that identify genes non-randomly targeted by somatic mutation or copy number alteration. While recent studies have defined broad patterns of association involving gene transcription and nearby SSV breakpoints, global alterations in DNA methylation in the context of SSVs remain largely unexplored.ResultsBy data integration of whole genome sequencing, RNA sequencing, and DNA methylation arrays from more than 1400 human cancers, we identify hundreds of genes and associated CpG islands (CGIs) for which the nearby presence of a somatic structural variant (SSV) breakpoint is recurrently associated with altered expression or DNA methylation, respectively, independently of copy number alterations. CGIs with SSV-associated increased methylation are predominantly promoter-associated, while CGIs with SSV-associated decreased methylation are enriched for gene body CGIs. Rearrangement of genomic regions normally having higher or lower methylation is often involved in SSV-associated CGI methylation alterations. Across cancers, the overall structural variation burden is associated with a global decrease in methylation, increased expression in methyltransferase genes and DNA damage response genes, and decreased immune cell infiltration.ConclusionGenomic rearrangement appears to have a major role in shaping the cancer DNA methylome, to be considered alongside commonly accepted mechanisms including histone modifications and disruption of DNA methyltransferases.

DNA methylation, transcriptome and genetic copy number signatures of diffuse cerebral WHO grade II/III gliomas resolve cancer heterogeneity and development

BackgroundDiffuse lower WHO grade II and III gliomas (LGG) are slowly progressing brain tumors, many of which eventually transform into a more aggressive type. LGG is characterized by widespread genetic and transcriptional heterogeneity, yet little is known about the heterogeneity of the DNA methylome, its function in tumor biology, coupling with the transcriptome and tumor microenvironment and its possible impact for tumor development.MethodsWe here present novel DNA methylation data of an LGG-cohort collected in the German Glioma Network containing about 85% isocitrate dehydrogenase (IDH) mutated tumors and performed a combined bioinformatics analysis using patient-matched genome and transcriptome data.ResultsStratification of LGG based on gene expression and DNA-methylation provided four consensus subtypes. We characterized them in terms of genetic alterations, functional context, cellular composition, tumor microenvironment and their possible impact for treatment resistance and prognosis. Glioma with astrocytoma-resembling phenotypes constitute the largest fraction of nearly 60%. They revealed largest diversity and were divided into four expression and three methylation groups which only partly match each other thus reflecting largely decoupled expression and methylation patterns. We identified a novel G-protein coupled receptor and a cancer-related ‘keratinization’ methylation signature in in addition to the glioma-CpG island methylator phenotype (G-CIMP) signature. These different signatures overlap and combine in various ways giving rise to diverse methylation and expression patterns that shape the glioma phenotypes. The decrease of global methylation in astrocytoma-like LGG associates with higher WHO grade, age at diagnosis and inferior prognosis. We found analogies between astrocytoma-like LGG with grade IV IDH-wild type tumors regarding possible worsening of treatment resistance along a proneural-to-mesenchymal axis. Using gene signature-based inference we elucidated the impact of cellular composition of the tumors including immune cell bystanders such as macrophages.ConclusionsGenomic, epigenomic and transcriptomic factors act in concert but partly also in a decoupled fashion what underpins the need for integrative, multidimensional stratification of LGG by combining these data on gene and cellular levels to delineate mechanisms of gene (de-)regulation and to enable better patient stratification and individualization of treatment.

Comprehensive Genomic Analysis of IDH Inhibitor-Treated AML Samples Delineates Molecular Mechanisms of Differentiation and Heterogeneous Patterns of Acquired Resistance

Allosteric inhibitors of mutant IDH1 or IDH2 induce differentiation of IDH-mutant AML myeloblasts, which in some patients (pts) can lead to a life-threatening differentiation syndrome (DS). The in vivo mechanism of how IDH inhibitors induce differentiation and occasionally DS is not fully understood. Furthermore, responders to the inhibitors often lose the differentiation effect, after median of 7 months. Although a recent study identified secondary mutations in the dimer-interface of IDH1/2 as a mechanism for acquired IDH-inhibitor resistance, given the rarity of this event, it is unlikely to represent the primary mechanism in the majority of pts.We conducted a comprehensive genomic analysis (DNA sequencing, single-cell DNA sequencing, RNA sequencing and cytosine methylation array) on longitudinally collected bone marrow specimens from IDH1/2-mutant AML pts treated with one of the IDH1/2 inhibitors (ivosidenib, enasidenib, IDH305 or AG881). In total, 146 samples from 39 pts collected at different time points (median 4 [IQR 3-5] samples per patient) were analyzed. 24 pts were IDH1-mutated receiving IDH1 inhibitor and 15 were IDH2-mutated receiving IDH2 inhibitor. 29 pts responded (CR, CRi, CRp or MLFS) and 10 had no response to the inhibitors. 8 of 29 responders developed DS. 22 of 29 responders relapsed after the median of 5 months.Cytosine methylation analysis revealed significant decrease in global methylation at response than baseline (mean beta value 0.54 at baseline vs. 0.45 at response, P < 0.001). This corroborated with a decrease in serum 2HG level, supporting the role of IDH inhibitors in reversing CpG island methylator phenotype (CIMP) likely through TET restoration. Motif enrichment analysis of differentially methylated promoter regions showed significant de-methylation of PU.1(SPI1) and C/EBP binding motifs at response. Consistent with this, RNA sequencing revealed that PU.1 and C/EBP target genes such as CSF2RA, CSF3R, IL34, TREM2, IL6R, CEBPE, GFI1, and KLF5 had significant upregulation at response. Expression of PU.1 itself was also significantly up-regulated upon response, along with other key hematopoietic differentiation genes and pathways such as GM-CSF, IL8-CXCR1 and IL3 pathways. Pts who developed DS had significantly higher upregulation of PU.1 and GM-CSF pathway compared to other DS-free responders, implicating the potential activation of these pathways in the mechanism of DS. At relapse, these pathways that mediated differentiation were all down-regulated when compared to that at response. Interestingly, methylation level remained suppressed at relapse (mean beta value 0.45 at response vs. 0.47 at relapse, P = 0.566), suggesting that the down-regulation of pro-differentiation signals at relapse is independent of methylation.Combined whole exome and targeted deep sequencing revealed the heterogeneous patterns of acquired mutations at relapse. Of 22 cases with baseline and relapse pairs, 18 acquired new driver mutations at relapse (Fig 1). The most frequently acquired mutations at relapse were in BCOR/BCORL1 (N = 6), hematopoietic differentiation genes (CEBPA [N=2] and RUNX1[N=4]), and RTK pathways (NRAS/KRAS [N=5] and PTPN11 [N=1]). We did not identify acquisition of dimer-interface IDH1/2 mutations at relapse. However, in 1 case, relapse was associated with acquisition of mutation in reciprocal IDH gene (IDH2-mutated case acquired IDH1 mutation). Singe-cell DNA sequencing of the double-mutant revealed that the reciprocal acquisition occurred in a different clone.Analysis of differentially expressed genes between baseline and relapse showed significant upregulation of genes in DNA damage response (DDR) pathways at relapse. Consistent with this, relapse samples showed significantly higher mutation burden than baseline, suggesting that accumulation of mutations are upregulating DDR pathway at relapse.In summary, IDH inhibitors induce differentiation and DS through upregulation of PU.1 and C/EBP target genes, which is associated with de-methylation of their binding motifs. The mechanisms of acquired resistance to IDH inhibitors are heterogeneous and involves various genes and pathways including BCOR/BCORL1, hematopoietic differentiation transcription factors (CEBPA, RUNX1), and RTK pathways (RAS, PTPN11). In a rare case, acquired mutation in reciprocal IDH gene was detected, occurring in a different clone. [Display omitted] DisclosuresDiNardo:Agios: Consultancy; Abbvie: Honoraria; Medimmune: Honoraria; Bayer: Honoraria; Celgene: Honoraria; Karyopharm: Honoraria. MacBeth:Celgene Corporation: Employment, Equity Ownership. Tosolini:Celgene Corporation: Employment. Frattini:Celgene Corporation: Employment, Equity Ownership. Kadia:Abbvie: Consultancy; Celgene: Research Funding; BMS: Research Funding; Pfizer: Consultancy, Research Funding; Jazz: Consultancy, Research Funding; Novartis: Consultancy; Amgen: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; BMS: Research Funding; Takeda: Consultancy; Novartis: Consultancy; Jazz: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Celgene: Research Funding; Takeda: Consultancy; Abbvie: Consultancy. Ravandi:Xencor: Research Funding; Astellas Pharmaceuticals: Consultancy, Honoraria; Sunesis: Honoraria; Amgen: Honoraria, Research Funding, Speakers Bureau; Sunesis: Honoraria; Bristol-Myers Squibb: Research Funding; Xencor: Research Funding; Abbvie: Research Funding; Bristol-Myers Squibb: Research Funding; Amgen: Honoraria, Research Funding, Speakers Bureau; Orsenix: Honoraria; Macrogenix: Honoraria, Research Funding; Seattle Genetics: Research Funding; Orsenix: Honoraria; Jazz: Honoraria; Jazz: Honoraria; Seattle Genetics: Research Funding; Abbvie: Research Funding; Macrogenix: Honoraria, Research Funding; Astellas Pharmaceuticals: Consultancy, Honoraria. Konopleva:Stemline Therapeutics: Research Funding.

Histone H3 lysine 4 methylation signature associated with human undernutrition

Chronically undernourished children become stunted during their first 2 years and thereafter bear burdens of ill health for the rest of their lives. Contributors to stunting include poor nutrition and exposure to pathogens, and parental history may also play a role. However, the epigenetic impact of a poor environment on young children is largely unknown. Here we show the unfolding pattern of histone H3 lysine 4 trimethylation (H3K4me3) in children and mothers living in an urban slum in Dhaka, Bangladesh. A pattern of chromatin modification in blood cells of stunted children emerges over time and involves a global decrease in methylation at canonical locations near gene start sites and increased methylation at ectopic sites throughout the genome. This redistribution occurs at metabolic and immune genes and was specific for H3K4me3, as it was not observed for histone H3 lysine 27 acetylation in the same samples. Methylation changes in stunting globally resemble changes that occur in vitro in response to altered methylation capacity, suggesting that reduced levels of one-carbon nutrients in the diet play a key role in stunting in this population. A network of differentially expressed genes in stunted children reveals effects on chromatin modification machinery, including turnover of H3K4me3, as well as posttranscriptional gene regulation affecting immune response pathways and lipid metabolism. Consistent with these changes, reduced expression of the endocytic receptor gene LDL receptor 1 (LRP1) is a driver of stunting in a mouse model, suggesting a target for intervention.

Revisiting the genomic hypomethylation hypothesis of aging.

The genomic hypomethylation hypothesis of aging proposes that an overall decrease in global DNA methylation occurs with age, and it has been argued that the decrease in global DNA methylation could be an important factor in aging, resulting in the relaxation of gene expression regulation and abnormal gene expression. Since it was initially observed that DNA methylation decreased with age in 1974, 16 articles have been published describing the effect of age on global DNA methylation in various tissues from rodents and humans. We critically reviewed the publications on the effect of age on DNA methylation and the expression of the enzymes involved in DNA methylation to evaluate the validity of the hypomethylation hypothesis of aging. On the basis of the current scientific literature, we conclude that a decrease in the global methylation of the genome occurs in most if not all tissues/cells as an animal ages. However, age-related changes in DNA methylation in specific regions or at specific sites in the genome occur even though the global DNA methylation does not change.

Rainbow trout exposed to benzo[a]pyrene yields conserved microRNA binding sites in DNA methyltransferases across 500 million years of evolution

The objective of this study was to examine the regulation of DNA methylation following acute (24 h) and prolonged (14 d) exposure to low (1 ng/L) and high (10 ng/L) benzo[a]pyrene. However, with the recent release of the rainbow trout genome, we were able to conduct a more detailed analysis regarding the regulation of the enzymes involved in DNA methylation; DNA methyltransferases (DNMTs). Bioinformatic approaches were used to identify candidate microRNA (miRNA) that potentially bind to the DNMT1 and DNMT3a 3′UTR. Results indicated a significant decrease in global methylation in both liver and muscle, with an associated decrease in DNA methyltransferase activity and DNMT3a transcript abundance. There was a significant increase in one specific candidate miRNA (miR29a) that was predicted to bind to DNMT3a. Taking a comparative genomics approach, the binding sites of miR29a to the DNMT3a 3′UTR was compared across species, spanning fish to mammals, and revealed a highly conserved binding motif that has been maintained since the vertebrate ancestor, approximately 500 million years ago. This research establishes that miRNA act as an essential mediator between the environment and DNA methylation patterns via DNMTs, which is further confirmed by a genomic regulatory mechanism that has been deeply conserved throughout evolution.

1725P - Impact of global epigenetics machinery on clinical outcome of colorectal cancer patients treated with fluoropyrimidine-based therapy

Background: The pathogenesis of colorectal cancer (CRC) is complex and influenced by many factors related to genetic, epigenetics and chronic inflammatory processes. Methods: This is a prospective study, conducted on 102 Egyptian patients, diagnosed with CRC. Blood samples were collected at baseline and after 3 & 6 months of receiving fluoropyrimidine (FP) based therapy. DNA methylation was measured by LC/MS/MS spectroscopy, acetylated histones (H3) and (H4) were measured by ELISA and RNA expression of FP metabolizing enzymes (TS, TP and DPD), DNA methyl transferases (DNMT3A and B) in addition to inflammatory markers (COX2, IL6, and IL1B) by qRT-PCR. Results: The median age of the studied patients was 46 years, 47% of them were ≤ 45 years. Forty patients (38.8%) had rectal cancer, they exhibited significant H3 hyperacetylation and upregulation of COX2 and IL1B along with significantly lower median overall survival compared to colonic patients (14.6 versus 23 months respectively). FP therapy produced significant decrease in global methylation, acetylated H3 & H4 levels, downregulation in TP and DNMT3B but significant upregulation in TS and DPD over treatment time. Significant positive correlations were found between global methylation and IL1B (r2= 0.25, P = 0.01), acetylated H3 with DPD and COX2 (r2= 0.28, P = 0.02 and r2= 0.27, P = 0.03 respectively) and 5 methylated cytosine content (5MC) with DNMT3A and IL6 (r2= 0.25, P = 0.04 and r2= 0.34, P = 0.004 respectively). Overexpression of COX2 > 17 had a significant poor prognostic effect on overall and event free survivals (HR = 0.58, P = 0.003 and HR = 0.72, P = 0.008 respectively). Also patients who had global methylation> 30 showed significant reduced event free survival by 39% (P = 0.04). Conclusions: Global methylation and H3 acetylation regulated COX2, IL6 and IL1B which were not affected by the therapy, however H3 upregulated TS and DPD. Rectal cancer patients showed significant H3 hyperacetylation, upregulation of COX2 and IL1B along with significant lower overall survival. Legal entity responsible for the study: National Cancer Institute, Cairo University Funding: National Cancer Institute, Cairo University Disclosure: All authors have declared no conflicts of interest.

Reduction in promotor methylation utilizing EGCG (epigallocatechin-3-gallate) restores RXRα expression in human colon cancer cells

Silencing of regulatory genes through hypermethylation of CpG islands is an important mechanism in tumorigenesis. In colon cancer, RXRα, an important dimerization partner with other nuclear transcription factors, is silenced through this mechanism. We previously found that colon tumors in ApcMin/+ mice had diminished levels of RXRα protein and expression levels of this gene were restored by treatment with a green tea intervention, due to reduced promoter methylation of RXRα. We hypothesized that CIMP+ cell lines, which epigenetically silence key regulatory genes would also evidence silencing of RXRα and EGCG treatment would restore its expression. We indeed found EGCG to restore RXRα activity levels in the human cell lines, in a dose dependent manner and reduced RXRα promoter methylation. EGCG induced methylation changes in several other colon cancer related genes but did not cause a decrease in global methylation. Numerous epidemiological reports have shown the benefits of green tea consumption in reducing colon cancer risk but to date no studies have shown that the risk reduction may be related to the epigenetic restoration by tea polyphenols. Our results show that EGCG modulates the reversal of gene silencing involved in colon carcinogenesis providing a possible avenue for colon cancer prevention and treatment.

Exposure to persistent organic pollutants and sperm DNA methylation changes in Arctic and European populations.

Persistent organic pollutants (POPs), such as PCBs (polychlorinated biphenyls) and DDT [1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane], are environmental contaminants with potential endocrine disrupting activity. DNA methylation levels in peripheral blood lymphocytes have been associated with serum concentrations of POPs in Greenland Inuit and Korean populations. Greenland Inuits are characterized by the highest worldwide POP levels. In this cross-sectional study we evaluated the relationship between serum POP concentrations and DNA methylation levels in sperm of non-occupationally exposed fertile men from Greenland, Warsaw (Poland), and Kharkiv (Ukraine). Serum levels of PCB-153 [1,2,4-trichloro-5-(2,4,5-trichlorophenyl)benzene], as a proxy of the total PCBs body burden, and of p,p'-DDE [1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene], the main metabolite of DDT were measured. Sperm DNA methylation level was assessed globally by flow cytometric (FCM) immunodetection of 5-methyl-cytosines and at specific repetitive DNA sequences (Alu, LINE-1, Satα) by PCR-pyrosequencing after bisulfite conversion. Multivariate linear regression analysis was applied to investigate correlations between serum POP concentrations and DNA methylation. No consistent associations between exposure to POPs and sperm DNA methylation at repetitive DNA sequences were detected. A statistically significant global decrease in methylation was associated with exposure to either POP by FCM analysis. This is the first study to investigate environmental exposure to POPs and DNA methylation levels considering sperm as the target cells. Although POP exposure appears to have a limited negative impact on sperm DNA methylation levels in adult males, the global hypomethylation detected by one of the methods applied suggests that further investigation is warranted.

Valproic acid and 5-azacytidine promote an increase of stemness phenotype in human osteosarcomas

Osteosarcoma (OS) is the most common pediatric tumour of bone in the world. It leads to local invasion and early metastasis to lung. The prognosis is poor with the 5-year survival rate of 65% for patients with primary tumour and 20% for patients with metastasis. OS contain a small cell population with stem cell like properties, referred to as cancer stem cells characterized by CD133 expression [1,2]. Recent studies have implicated epigenetic alterations, including DNA methylation and histone modifications, as prominent factors that contribute to the phenotype of cancer progenitor or stem cells. Here, we examined the epigenetic effects of the HDAC inhibitor valproic acid (VPA) and demethylating agent, 5’azacytidine (DAC), on the stemness phenotype in osteosarcoma cell lines. Saos-2 and MG63 cell lines were treated with 0,5 mM VPA and 3µM DAC for 48 hours alone, and in combination. CD133 expression as well as stemness markers expression including OCT4, Sox2 and Nanog was analyzed by flow cytometry and real-time PCR. Vimentin and osteocalcin levels were also tested. Sarcospheres formation rate was assessed as spheres number/seed single cells number. Specific histone modifications including H3-trymethyl-k9, H3-acetyl-k9, H3-trymethil-k27 and global methylation were analysed by flow cytometry and immunofluorescence. Moreover, soft agar and invasion assays were performed. Our findings indicated that DAC or VPA and their combination induced an increase of stemness characteristics of OS cells in terms of high expression of CD133, OCT4, Sox2 and Nanog and high sarcospheres-forming efficiency. Interestingly, combined treatment with DAC and VPA induced an increase of CD133 expression in a synergistic manner in all cell lines. Vimentin resulted up-regulated after treatment, whereas, the level of osteocalcin remained similar before and after treatment. Furthermore, soft agar assay revealed major colony-forming efficiency in treated cells compared to untreated cells, whereas invasion potential decreased after drug treatment. Interestingly, histone modification analyses correlated with those typical of embryonic stemness with an increase of H3-acetyl-k9 and decrease of H3-trymethyl-k9, and H3-trymethil-k27 after drug treatment as well as global methylation decreased in treated cells. In conclusion, DAC and VPA induced an increase of stemness associated to an decrease of global methylation, increase of acetylation and decrease of H3-trymethyl-k9, and H3-trymethil-k27. This study was supported by MIUR-EU funds (PON 01_02834 and PRIN 2011/13)

Book review: The Epigenetics Revolution

Epigenetics is a rapidly evolving science that is often only described in scientific literature or textbooks. In “The Epigenetics Revolution”, Nessa Carey eloquently bridges the spheres of academia and scientific journalism (Carey, 2012). The phrasing “revolution” is a dramatic use of English that effectively portrays a momentous shift in biological thinking. For the novice, it is a good introduction to epigenetics whilst it provides professional readers with a concise summary of historically significant experiments and translational context. The text is divided into an introduction and 16 chapters which handle various concepts pertaining to epigenetic theory, key experimental studies and the potential for future therapeutic application. Carey starts with some historical context; she illustrates the successes and limitations of the human genome project and quotes some of the arguably overdramatic claims made by various organizations of the time. The first two chapters approach the issue of cellular differentiation by explaining the work of John Gurdon and the concept of a genetic “switch” rather than a loss of unneeded material. The analogy that DNA is a script to be interpreted is a particularly articulate explanation for novice readers. From this, Carey explains the epigenetic underpinnings of pluripotency and explores the potential application of stem cell therapies in regenerative medicine. Chapters 3 and 4 give an overview of genetic and epigenetic molecular biology, including the genetic code, DNA replication, transcription, translation, chromatin, DNA methylation, and histone modifications. These biological concepts are supplemented with an explanation of twin studies to illustrate the profound effect of environmental experience on genetically identical individuals. She makes reference to specific biological signals that induce epigenetic modification e.g. the hormone receptor. In the absence of somatic mutation, Carey also explores how epigenetic modifications can induce cellular abnormalities. Overall, this section provides an excellent narrative that uses famous scientific work to illustrate basic epigenetic concepts which is perfectly suited to novice readers or undergraduate students. Chapter 6 focuses on the heritability of epigenetic modifications. Environmental events such as the Dutch famine have clear effects on the health outcomes of offspring. Carey explains that this only occurs in susceptible regions of the genome and that epigenetic reprogramming during early embryogenesis circumvents the passage of many undesirable or dangerous modifications. Generation X is a very interesting chapter that explains the history of sex determination; an epigenetic process that depends upon tight regulation of X chromosome genes, termed “X chromosome dosage compensation.” The last quarter of the book focuses on epigenetic pathology. Carey illustrates this section with two very different disease states; psychiatric illness and cancer. This is purposefully designed to portray the universal influence of epigenetics. Carey makes an intelligent case for the role of childhood experience in shaping the epigenetic landscape; specifically, the association between abuse and the development of mental illness. Another interesting section explores the current contention surrounding neuro-epigenetics. This implies that epigenetic theory isn't sacrosanct and provokes a good debate for the reader. Disappointingly, neurodegeneration is only briefly mentioned. On the contrary, the concept of “epigenetic” aging is thorough and chapter 13 eloquently explains the global decrease in methylation and changes to the telomere system that are associated with the development of cancer. The final chapter elegantly draws together the biological principles, historical events, and translational examples that were given throughout the book. Carey concludes by exploring the potential for future drug discovery and the limitations and dangers that such agents may present. There is a description of recent research that is beginning to answer many elusive questions, such as the mechanisms underpinning the circadian rhythm. This section builds upon basic concepts with more advanced theory which might become challenging for some inexperienced readers. With that said, it is brilliantly written and provides a comprehensive review of many historically significant feats of epigenetic biology. “The Epigenetics Revolution” is a concise text that is accessible to a wide audience. However, such a compromise presents drawbacks; for the absolute beginner, gene nomenclature and complex scientific concepts may be confusing at times. If one is familiar with the field, many of the examples are unoriginal e.g. the agouti mouse experiments, the classic Dutch famine and its correlation with obesity, the royal jelly etc. Surprisingly, there is no mention of the early work by nobel laureates Barbara McClintock or Paul Berg. Despite these relatively minor criticisms, “The Epigenetics Revolution” is an excellent scientific biography that charts the past, present, and optimistic future of the field. It is especially suitable for informed non-specialists and students of biological sciences. Carey enthusiastically portrays a revolutionary system that could unlock the answers to many of nature's best kept secrets.

Abstract 840: Occupational exposure to trichloroethylene and LINE-1 methylation

Abstract Trichloroethylene (TCE) is a volatile chlorinated organic compound that is commonly used in industrial settings as a degreaser. Epidemiologic studies suggest that TCE exposure is associated with an increased risk of kidney cancer and may also be associated with non-Hodgkin lymphoma. However, the mechanisms for these associations are still not clear. To evaluate the relationship between occupational exposure to trichloroethylene (TCE) and LINE-1 methylation, a measure of global methylation, we conducted a cross-sectional molecular epidemiology study of 79 workers exposed to TCE and 81 comparable unexposed controls in Guangdong, China. Personal air exposure measurements and urine samples were collected prior to phlebotomy. The urine TCE metabolite, trichloroacetic acid (TCA), was measured by liquid chromatography electrospray ionization tandem mass spectrometry in urine samples and LINE-1 methylation was assessed by pyrosequencing DNA isolated from peripheral blood. LINE-1 methylation levels [median (range) in percent] were 78.40 (75.80-81.20) in controls, 77.80 (76.10-81.00) in lower exposed, and 77.50 (74.20-80.20) in higher exposed workers based on median urine TCA level (p trend = 0.012). These results suggest that TCE may be associated with a decrease in global methylation measured by the LINE-1 assay. Gene-specific methylation is currently being conducted to follow up these findings. Citation Format: Wei Hu, Luoping Zhang, Christopher Kim, Xiaojiang Tang, Sungkyoon Kim4, Bryan Bassig, Wei-Jie Seow, Min Shen, Chuangyi Qiu, Yechen Ge, Boris Reiss, Mark Purdue, Lee Moore, Laiyu Li, Fei Yue, Hanlin Huang, Martyn T. Smith, Roel Vermeulen, Nathaniel Rothman, Qing Lan. Occupational exposure to trichloroethylene and LINE-1 methylation. [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 840. doi:10.1158/1538-7445.AM2015-840

Conventional Dose Hypomethylating Agents Induce CG Antigen Genes In Vivo

Abstract 2441 Background:Cancer testis or cancer germline (CG) antigens are a group of genes whose usual expression is limited to embryonic or germ cell tissues and which can also be aberrantly expressed in a variety of malignancies (Fratta E et al. Mol Oncol. 2011;5:164). The CG antigen genes MAGE-A1, XAGE-1, NY-ESO-1, and PRAME (among others) are epigenetically regulated. Most adult non-germline tissues demonstrate dense CpG island promoter methylation, with no gene expression. Endogenous expression of CG genes in epithelial malignancy can be immunogenic and a number of vaccine strategies are currently underway, however studies in hematologic malignancy have shown limited baseline expression, and thus there has been limited interest in them as a target in AML. Recently, several groups have shown that treatment with the hypomethylating agents, azacitidine(Aza) and decitabine(Dac) are sufficient to re-induce gene expression in myeloid cell lines, prompting interest in these genes as a therapeutic target in myeloid malignancy (Almstedt M et al. Leuk Res 2010;34:899). Re-expression of these genes has also been observed in MDS/AML patients treated with Dac, albeit at higher doses (15–270mg/m2) than those currently in standard use (Sigalotti L et al. Blood 2003;101:4644). Hypothesis:We hypothesized that conventional therapy with Aza(75mg/m2) and Dac(20mg/m2) induces CG promoter hypomethylation and gene expression, and that this may be sufficient to induce anti-CG antigen directed antibody responses. Methods:We obtained samples every 2–4 days from 2 AML patients during the first cycle of Aza or Dac treatment, and analyzed baseline and post-treatment samples for methylation (by pyrosequencing) and CG gene expression (by RT-PCR) of the above genes. We further tested serum from 10 patients for the presence of baseline and post treatment (following 1–10 cycles of Aza or Dac) anti-CG antibodies using an ELISA-based method. Results:Both AML patients treated with hypomethylating agents demonstrated a time dependent decrease in global methylation (by LINE-1 pyrosequencing), and CG antigen promoter methylation which nadired at day 6 post treatment. Baseline methylation of MAGE-A1 (90%), XAGE-1 (85%), NY-ESO-1 (92%) and PRAME (50–60%) was similar and high in both patients pre-treatment. Both Dac and Aza induced hypomethylation of all the CG genes studies, and this hypomethylation correlated with changes in LINE-1 methylation. Low level expression of MAGE-A1, XAGE-1, and PRAME were induced in a time dependent fashion following treatment with both Aza and Dac, however only limited re-expression of NY-ESO-1 was observed. ELISA testing for antibodies against a panel of 25 CG antigens (including MAGE-A1, XAGE-1 and NY-ESO-1) was performed on 10 patients with either MDS or AML. Following hypomethylating agents there was evidence for increased antibody levels, most notably against MAGE proteins, compared to baseline. Conclusion:Conventional hypomethylating agent therapy can induce CG gene expression, which may be sufficient to induce anti-CG antigen antibody responses. Augmentation of anti-tumor immune responses may be possible in myeloid malignancies using a combination of hypomethylating agents and CG antigen-directed vaccines. Disclosures:Odunsi:Ludwig Institute for Cancer Reserach: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract A39: EGCG, a green tea polyphenol, can reverse methylation related silencing of genes in human colon carcinomas

Abstract Silencing of regulatory genes through hypermethylation of CpG islands is an important mechanism in tumorigenesis. In colon cancer, RXRα, an important dimerization partner with other nuclear transcription factors, is silenced through this mechanism. Previous studies from our laboratory have shown that ApcMin/+ mice treated with the carcinogen azoxymethane (AOM) will develop colonic tumors but when treated with green tea a reduction in tumor numbers was found. We found that colon tumors had lower expression of RXR protein but expression levels were restored by treatment with green tea. We also found that the degree of promoter methylation of the RXR gene was reduced. To translate these findings to human colon cancer we treated CpG Island Methylator Phenotype sensitive and insensitive (CIMP+ &amp; CIMP-) human colon cancer cell lines with epigallocatechin gallate (EGCG - major polyphenolic compound in green tea). We hypothesized that CIMP+ cell lines which epigenetically silence key regulatory genes would demonstrate silencing of RXRα and that EGCG would restore its expression. To test this we used the following human colon cancer cell lines: HCT116, SW48, HCT15 - CIMP+; HT29, SW480, SW620 - CIMP- and IEC-6 - normal control. We found EGCG to restore RXR activity levels in the CIMP+ lines, in a dose dependent manner (0, 50, 100 &amp; 150 μM EGCG was tested for 48 and 72 hour durations). EGCG also reduced RXRα promoter methylation in one CpG island from a CIMP+ cell line compared to a CIMP-line. We found that EGCG produced methylation changes in several other colon cancer related genes but did not cause a decrease in global methylation. Numerous epidemiological reports have shown the benefits of green tea consumption in reducing colon cancer risk but to date no studies have shown that some of the risk reduction may be related to the epigenetic regulation of key genes by constituent tea polyphenols. Our results here show that EGCG, a common tea polyphenolic compound, can modulate the reversal of gene-silencing involved colon carcinogenesis and maybe a possible avenue for colon cancer treatment. Supported by USPHS grant CA96694 from the National Cancer Institute. Citation Information: Cancer Prev Res 2011;4(10 Suppl):A39.

128 GLOBAL DNA HYPOMETHYLATION MARKS A PROSTATE CANCER FIELD EFFECT

You have accessJournal of UrologyProstate Cancer: Basic Research1 Apr 2011128 GLOBAL DNA HYPOMETHYLATION MARKS A PROSTATE CANCER FIELD EFFECT Jeffrey Tomaszewski, William Green, Abid Dar, Simone Harya, Rajiv Dhir, Federico Monzon-Bordonaba, and Denise O'Keefe Jeffrey TomaszewskiJeffrey Tomaszewski Pittsburgh, PA More articles by this author , William GreenWilliam Green Pittsburgh, PA More articles by this author , Abid DarAbid Dar Pittsburgh, PA More articles by this author , Simone HaryaSimone Harya Pittsburgh, PA More articles by this author , Rajiv DhirRajiv Dhir Pittsburgh, PA More articles by this author , Federico Monzon-BordonabaFederico Monzon-Bordonaba Houston, TX More articles by this author , and Denise O'KeefeDenise O'Keefe Pittsburgh, PA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.195AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Among a variety of environmental and genetic factors involved in prostate carcinogenesis, epigenetic aberrations such as gene hypermethylation and global DNA hypomethylation of repetitive sequences like LINE-1 retrotransposons are regularly associated with prostate cancer initiation, progression and metastasis. The primary aim of this study was to characterize global methylation in prostate cancer and evaluate the epigenetic microenvironment and spatial field effect in micro-dissected prostate tissue. METHODS We analyzed micro-dissected prostate cancer and normal-adjacent to tumor (NAT) from 66 patients with prostate adenocarcinoma, and normal prostate tissue from 8 cancer-free organ donors for LINE-1 methylation levels. The methylation levels were correlated with clinicopathologic parameters. RESULTS The mean global methylation index in prostate tumors was significantly less than methylation levels in age-matched cancer-free prostates (53.4% ± 15.4% vs. 81.4% ± 4.5%, respectively; p<0.01). Histopathologically normal prostate tissue from PCa patients had lower global methylation when compared to cancer-free prostates. Global methylation was significantly decreased in prostate tumor tissue compared to NAT (53.4% ± 15.4% vs. 69.5% ± 12.3%, respectively; p<0.05) (Figure 1). Global methylation was lowest in cancer tissue and gradually increased concomitant with distance from the tumor, indicating a marked field effect characterized by a decrease in global methylation (Figure 2). There were no significant differences in LINE-1 methylation when patients were stratified by age, Gleason grade, pathologic stage, biochemical recurrence, or lymph node status. CONCLUSIONS Significant hypomethylation of LINE-1 retrotransposons and a marked methylation field effect were observed in prostate cancer and cancer-adjacent benign tissue, respectively. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e53-e54 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jeffrey Tomaszewski Pittsburgh, PA More articles by this author William Green Pittsburgh, PA More articles by this author Abid Dar Pittsburgh, PA More articles by this author Simone Harya Pittsburgh, PA More articles by this author Rajiv Dhir Pittsburgh, PA More articles by this author Federico Monzon-Bordonaba Houston, TX More articles by this author Denise O'Keefe Pittsburgh, PA More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

DAMD to epigenetic silence

For many decades, cancer research focused on genetic changes that led to tumorigenesis. However, it is now established that epigenetics also plays an important role in cancer initiation and development (1, 2). The cancer epigenome is marked by a global decrease in methylation, leading to genomic instability and increased methylation at promoters, which in turn leads to gene silencing (3). The importance of this abnormal DNA methylation in silencing cancer-related genes has recently gained a great deal of attention. Detecting altered methylation in tumors on a genome-wide scale is not always easy, but its importance has led to the development of many assays including restriction enzyme digestion, ligation mediated PCR, bisulfite sequencing technologies such as methylation sensitive PCR, incubation of DNA with methylated DNA binding proteins or antibodies to methylcytosine among others. These approaches have their strengths and weaknesses and in this issue of PNAS, Diede et al. describe an approach employing physical properties of DNA called denaturation analysis of methylation differences (DAMD) (4). The method relies on the fact that methylated CpG dinucleotides have different melting characteristics compared to unmethylated CpG dinucleotides. Although the specific conditions described in the paper limit the analyses to CpG islands (i.e., areas with a high density of CpG dinucleotides), the method can be modified in such a way as to also examine DNA methylation differences in genomic regions that are less CpG rich. The authors use the altered melting behavior of methylated … 1 To whom correspondence should be addressed. E-mail: jones_p{at}ccnt.hsc.usc.edu.

Inactive X chromosome-specific reduction in placental DNA methylation

Genome-wide levels of DNA methylation vary between tissues, and compared with other tissues, the placenta has been reported to demonstrate a global decrease in methylation as well as decreased methylation of X-linked promoters. Methylation is one of many features that differentiate the active and inactive X, and it is well established that CpG island promoters on the inactive X are hypermethylated. We now report a detailed analysis of methylation at different regions across the X in male and female placenta and blood. A significant (P < 0.001) placental hypomethylation of LINE1 elements was observed in both males and females. Relative to blood placental promoter hypomethylation was only observed for X-linked, not autosomal promoters, and was significant for females (P < 0.0001) not males (P = 0.9266). In blood, X-linked CpG island promoters were shown to have moderate female methylation (66% across 70 assays) and low (23%) methylation in males. A similar methylation pattern in blood was observed for ∼20% of non-island promoters as well as 50% of the intergenic or intragenic CpG islands, the latter is likely due to the presence of unannotated promoters. Both intragenic and intergenic regions showed similarly high methylation levels in male and female blood (68 and 66%) while placental methylation of these regions was lower, particularly in females. Thus placental hypomethylation relative to blood is observed globally at repetitive elements as well as across the X. The decrease in X-linked placental methylation is consistently greater in females than males and implicates an inactive X specific loss of methylation in the placenta.