Tumor Suppressor Gene Promoters Research Articles

Epigenetic inactivation of the CpG demethylase TET1 as a DNA methylation feedback loop in human cancers.

Promoter CpG methylation is a fundamental regulatory process of gene expression. TET proteins are active CpG demethylases converting 5-methylcytosine to 5-hydroxymethylcytosine, with loss of 5 hmC as an epigenetic hallmark of cancers, indicating critical roles of TET proteins in epigenetic tumorigenesis. Through analysis of tumor methylomes, we discovered TET1 as a methylated target, and further confirmed its frequent downregulation/methylation in cell lines and primary tumors of multiple carcinomas and lymphomas, including nasopharyngeal, esophageal, gastric, colorectal, renal, breast and cervical carcinomas, as well as non-Hodgkin, Hodgkin and nasal natural killer/T-cell lymphomas, although all three TET family genes are ubiquitously expressed in normal tissues. Ectopic expression of TET1 catalytic domain suppressed colony formation and induced apoptosis of tumor cells of multiple tissue types, supporting its role as a broad bona fide tumor suppressor. Furthermore, TET1 catalytic domain possessed demethylase activity in cancer cells, being able to inhibit the CpG methylation of tumor suppressor gene (TSG) promoters and reactivate their expression, such as SLIT2, ZNF382 and HOXA9. As only infrequent mutations of TET1 have been reported, compared to TET2, epigenetic silencing therefore appears to be the dominant mechanism for TET1 inactivation in cancers, which also forms a feedback loop of CpG methylation during tumorigenesis.

High expression of DNA methyltransferases in primary human medulloblastoma.

Epigenetic alterations have been implicated in cancer development. DNA methylation modulates gene expression, which is catalyzed by DNA methyltransferases (DNMTs). The objective of our study was to evaluate expression of DNMTs in medulloblastoma and analyze its correlation with clinical features. Nuclear expression of DNMT1, DNMT3A and DNMT3B was analyzed in human primary medulloblastoma of 44 patients using immunohistochemistry. Correlation of expression of DNMT levels with classical histological subtypes, novel molecular subgroups and survival of patients was analyzed. Elevated expression of DNMT1, DNMT3A and DNMT3B was observed in 63.64%, 68.18% and 72.73% of all cases, respectively. None of them showed a correlation with classical histology or survival. Concerning molecular subtypes, significantly higher expression of DNMT1 was observed in the SHH group compared to non-SHH samples (p = 0.02), but without significant difference in DNMT3A or DNMT3B levels between any subtypes. In conclusion, DNMT1, DNMT3A and DNMT3B are highly expressed in human medulloblastoma samples, suggesting that promoter hypermethylation may play a role in medulloblastoma development. Demethylation of tumor suppressor gene promoters may be considered as a possible future target in therapy of medulloblastoma.

Combination Treatment of AML Cells with Dnmt and HDAC Inhibitors: Synergistic Target Gene Downregulation Associated with Gene Body Demethylation

Background: Hypermethylation of CpG-rich tumor suppressor gene promoters constitutes a prototypical cancer-specific epigenetic abnormality. Recently, methylome-wide studies have provided evidence of extensive aberrant DNA methylation also in gene bodies and intergenic regions; their functional role is still under debate. DNA hypomethylating agents (HMAs) such as 5-aza-CdR (DAC) and 5-aza-CR are thought to act particularly by reactivating hypermethylated promoters of epigenetically silenced genes, and this activity is enhanced by combining HMAs with histone deacetylase inhibitors (HDACi). However, transcriptome-wide studies have shown that HMAs can also downregulate mRNAs. Recently, Yang et al. (Cancer Cell 2014) showed that gene body methylation can be strikingly reversed by DAC in human colon cancer cells, with remarkable downregulation of genes involved in metabolic processes regulated by c-MYC, implicating gene body methylation as a drug target. Therefore, we asked whether HMA-induced reversal not only of promoter but also of gene body methylation occurs in AML cells, and whether gene body demethylation is preferentially associated with gene repression rather than activation. We further asked whether the synergism between HMAs and HDACi in gene reactivation might also be observed for gene repression.Materials and Methods: U937 cells were treated with 3x24h pulses of DAC (50nM), alone or in combination with the HDACi panobinostat (Pano, 8nM) or Valproic acid (VPA, 1mM) for 24h with low-cytotoxic (>80% viability), <25% growth-inhibitory concentrations. Transcriptome and methylome profiles (biological triplicates) were generated using Affymetrix Human Gene 2.0 ST and Infinium HumanMethylation450 BeadChip arrays. Data were analyzed using R package RnBeads applying standard methods.Results: Transcriptome analyses revealed that DAC, Pano or VPA alone induced 207, 267 and 179 transcripts, with downregulation of 28, 45 and 22 transcripts, respectively (FDR<0.01; ≥2-fold change). When DAC was combined with either Pano (D+P) or VPA (D+V), a striking synergistic effect was observed for upregulated transcripts (1069 and 680). Remarkably, a synergistic effect on downregulated transcripts was even stronger (667 and 233 transcripts). As expected, DAC treatment resulted in extensive DNA demethylation: 295.6K CpGs (89% of 331.8K CpGs hypermethylated >30% in untreated U937) became >10% demethylated (FDR<0.01), with demethylation of additional 8.7K CpGs (2.6%) and 5.7K CpGs (1.7%) with D+P and D+V, respectively.Most of the differentially expressed transcripts mapped to demethylated CpGs located in both promoters and gene bodies. We focussed on up- and downregulated transcripts exclusively associated with either promoter- or gene body-demethylated CpGs. The ratio of transcripts mapping to promoter- vs. gene body-demethylated CpGs doubled from up- to downregulated transcripts (all 3 treatments). Thus, for upregulated transcripts these ratios were 1: 1.9 (DAC), 1: 3.5 (D+P) and 1: 3.4 (D+V); for downregulated transcripts 1: 4.0 (DAC), 1: 7.0 (D+P) and 1: 6.9 (D+V). Also, the combination treatments resulted in a clear shift to more transcripts mapping to gene body-demethylated CpGs than DAC treatment alone. Demethylated CpGs located in shore regions of gene bodies were particularly enriched in downregulated transcripts upon combination treatment, suggesting a possible mechanism of their regulation.Of the 667 transcripts downregulated by D+P, the 227 gene-body demethylation-associated transcripts included various putative or bona fide oncogenes (such as c-MYC) and epigenetic modifiers. Six potential candidates were chosen for validation by RT-qPCR. Gene ontology analysis revealed an enrichment for genes involved in RNA processing and transcription.Conclusions: The combination of a HMA with two different HDACi resulted not only in synergistic gene reactivation but also in massive, synergistic downregulation of genes including oncogenes, which was associated predominantly with gene body demethylation. Taken together, these findings have implications not only regarding the mechanisms of action of combined epigenetic treatment, but also for a better understanding of clinical responses in trials where this approach is tested, such as the randomized DECIDER AML trial (NCT00867672), by analyzing in vivo epigenetic drug effects on primary blasts. DisclosuresLübbert:Janssen: Honoraria, Other: travel support; Celgene: Honoraria, Other: travel support; TEVA: Other: travel support.

DNA Methyltransferases Inhibitors from Natural Sources.

DNA methyltransferases (DNMTs) catalyze the methylation at cytosine-C5 mainly in a CpG dinucleotide context. Although DNA methylation is essential for fundamental processes like embryonic development or differentiation, aberrant expression and/or activities of DNMTs are involved in several pathologies, from neurodegeneration to cancer. DNMTs inhibition can arrest tumor growth, cells invasiveness and induce differentiation, whereas their increased expression is shown in numerous cancer types. Moreover, hypermethylated promoters of tumor suppressor genes lead to their silencing. Hence, the use of specific inhibitors of DNMT might reactivate those genes and stop or even reverse the aberrant cell processes. To date, the only approved DNMTs inhibitors for therapy belong to the nucleoside-based family of drugs, but they display relevant side effects as well as high chemical instability. Thus, there is a keen interest actually exists to develop novel, potent and safe inhibitors possessing a nonnucleoside structure. Increasing literature evidence is highlighting that natural sources could help the researchers to achieve this goal. Indeed, several polyphenols, flavonoids, antraquinones, and others are described able to inhibit DNMTs activity and/or expression, thus decreasing the methylation/silencing of different genes involved in tumorigenesis. These events can lead to re-expression of such genes and to cell death in diverse cancer cell lines. Epigallocatechin-3-gallate (1) and laccaic acid A (11) resulted the most effective DNMT1 inhibitors with submicromolar IC50 values, acting as competitive inhibitors. Compound 1 and 11 both displayed gene demethylation and re-activation in several cancers. However, all of the natural compounds described in this review showed important results, from gene reactivation to cell growth inhibition. Moreover, some of them displayed interesting activity even in rodent cancer models and very recently entered clinical trials.

DNA methylation plasticity contributes to the natural history of metastasis

DNA methylation plasticity contributes to the natural history of metastasis

133 DNA methylation profiles of tumor suppressor genes promoters involved in papillary thyroid carcinoma

133 DNA methylation profiles of tumor suppressor genes promoters involved in papillary thyroid carcinoma

Abstract 1071: Age-dependent DNA methylation in normal breast epithelium and breast cancer

Abstract Breast cancer risk factors include age, genetic alterations, weight, diet and others. More recently accumulating evidence suggests that epigenetic alterations are frequent and play important role in breast cancer development and progression. Epigenetic alterations include changes in DNA methylation, histone modifications and miRNA expression in the absence of changes in DNA sequence. The epigenetic changes in cancer include global loss of DNA methylation that might lead to genomic instability and gain of methylation at many promoters of tumor suppressor genes that leads to gene silencing. Interestingly, work from our own laboratory and others have established that in many different tissues, DNA methylation changes due to age have similar patterns as to changes that occur in cancer. Therefore, the goal of this study is to identify and characterize age-dependent DNA methylation changes in normal breast epithelium and their contribution to breast cancer development. To differentiate between age-dependent and independent DNA methylation changes, we utilized DNA extracted from 27 primary human mammary epithelial cells (age range 33-82 years old) derived from adjacent or contralateral normal mammary tissue of breast cancer patients. We measured DNA methylation using a highly sensitive methodology developed in our laboratory called Digital Restriction Enzyme Analysis of Methylation (DREAM). We also studied DNA methylation in six different breast cancer cell lines by DREAM. We also used publically available 450K TCGA data for 685 breast tumor and 98 normal samples. Using bioinformatic approach, we have defined age dependent (Spearman R &amp;gt; |0.38|, p &amp;lt;0.05) and independent (Spearman R &amp;lt; |0.37|, p&amp;gt;0.05) sites that exhibit DNA methylation changes in normal breast epithelium. Interestingly, we found that the age-related genes are more seeded (exhibit low level of methylation) in their promoter CpG islands and are enriched for polycomb group target genes compared to the non-age related genes (p value = 0.032). Furthermore, these age-related genes (for example HOXD9, TDRD10, MYOD1, DPYS, GDA, GIPC2, FAM162B, LAMA1, PKDREJ, etc) have increased DNA methylation in tumors compared to the normal samples in TCGA with a methylation difference of up to 50%. These changes are significantly different compared to not-age related genes or random genes (p&amp;lt;0.0001). The age-related genes that gained DNA methylation in tumors in TCGA dataset, were also hypermethylated in breast cancer cell lines compared to immortalized human mammary epithelial cells. Thirty one percent of the common genes between TCGA data set and breast cancer cell lines, showed decreased mRNA expression in RNA-seq data available through the TCGA data portal. Therefore, in this study we have characterized age-dependent epigenetic changes in normal breast epithelium as potential breast cancer risk associated alterations. Citation Format: Shoghag B. Panjarian, Carolyn Slater, Jozef Madzo, Jaroslav Jelinek, Xiaowei Chen, Jean-Pierre Issa. Age-dependent DNA methylation in normal breast epithelium and breast 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 1071. doi:10.1158/1538-7445.AM2015-1071

Epigenetic regulation of the intestinal epithelium.

The intestinal epithelium is an ideal model system for the study of normal and pathological differentiation processes. The mammalian intestinal epithelium is a single cell layer comprising proliferative crypts and differentiated villi. The crypts contain both proliferating and quiescent stem cell populations that self-renew and produce all the differentiated cell types, which are replaced every 3-5days. The genetics of intestinal development, homeostasis, and disease are well defined, but less is known about the contribution of epigenetics in modulating these processes. Epigenetics refers to heritable phenotypic traits, including gene expression, which are independent of mutations in the DNA sequence. We have known for several decades that human colorectal cancers contain hypomethylated DNA, but the causes and consequences of this phenomenon are not fully understood. In contrast, tumor suppressor gene promoters are often hypermethylated in colorectal cancer, resulting in decreased expression of the associated gene. In this review, we describe the role that epigenetics plays in intestinal homeostasis and disease, with an emphasis on results from mouse models. We highlight the importance of producing and analyzing next-generation sequencing data detailing the epigenome from intestinal stem cell to differentiated intestinal villus cell.

Detection of G-type density in promoter sequence of colon cancer oncogenes and tumor suppressor genes.

The guanine rich locations are present in human genome. Previous studies have shown that the presence of G rich sequences and motifs may be significant for gene activity and function. We decided to focus our interest to identify G rich motifs in promoters of oncogenes and tumor suppressor genes. We used a set of 100 most common oncogenes and tumor suppressor genes (TSG) for this analysis. We collected 600nt long promoters with -500 and +100 TSS (transcription start site) from the oncogenes and TSG set. Using a computer program, we calculated the G densities using numbers and locations of G forms with 100nt moving widow. We included G numbers from 2 to 7 guanines. Analysis shows that G density increases from -500 to +100 and more from TSS. G density is found to be maximum within -/+100 of TSS. The results of G densities were compared with the expression data of the selected oncogenes and tumor suppressor genes in patients with colon cancer (n=174).

Tetraspanin Family Member, CD82, Regulates Expression of EZH2 via Inactivation of p38 MAPK Signaling in Leukemia Cells.

PurposeWe recently found that the tetraspanin family member, CD82, which is aberrantly expressed in chemotherapy-resistant CD34+/CD38− acute myelogenous leukemia (AML) cells, negatively regulates matrix metalloproteinase 9, and plays an important role in enabling CD34+/CD38− AML cells to adhere to the bone marrow microenvironment. This study explored novel functions of CD82 that contribute to AML progression.Materials and MethodsWe employed microarray analysis comparing the gene expression profiles between CD34+/CD38− AML cells transduced with CD82 shRNA and CD34+/CD38− AML cells transduced with control shRNA. Real-time RT-PCR and western blot analysis were performed to examine the effect of CD82 knockdown on the expression of the polycomb group member, enhancer of zeste homolog 2 (EZH2), in leukemia cells. A chromatin immunoprecipitation assay was performed to examine the effect of CD82 expression on the amount of EZH2 bound to the promoter regions of tumor suppressor genes in leukemia cells. We also utilized methylation-specific PCR to examine whether CD82 expression influences the methylation status of the tumor suppressor gene promoter regions in leukemia cells.ResultsMicroarray analysis revealed that levels of EZH2 decreased after shRNA-mediated depletion of CD82 in CD34+/CD38− AML cells. Moreover, the antibody-mediated blockade of CD82 in leukemia cells lowered EZH2 expression via activation of p38 MAPK signaling, decreased the amount of EZH2 bound to the promoter regions of the tumor suppressor genes, and inhibited histone H3 lysine 27 trimethylation in these promoter regions, resulting in upregulation of the tumor suppressors at both the mRNA and protein levels.

New Insights on the Mechanism of Quinoline-based DNA Methyltransferase Inhibitors

Among the epigenetic marks, DNA methylation is one of the most studied. It is highly deregulated in numerous diseases, including cancer. Indeed, it has been shown that hypermethylation of tumor suppressor genes promoters is a common feature of cancer cells. Because DNA methylation is reversible, the DNA methyltransferases (DNMTs), responsible for this epigenetic mark, are considered promising therapeutic targets. Several molecules have been identified as DNMT inhibitors and, among the non-nucleoside inhibitors, 4-aminoquinoline-based inhibitors, such as SGI-1027 and its analogs, showed potent inhibitory activity. Here we characterized the in vitro mechanism of action of SGI-1027 and two analogs. Enzymatic competition studies with the DNA substrate and the methyl donor cofactor, S-adenosyl-l-methionine (AdoMet), displayed AdoMet non-competitive and DNA competitive behavior. In addition, deviations from the Michaelis-Menten model in DNA competition experiments suggested an interaction with DNA. Thus their ability to interact with DNA was established; although SGI-1027 was a weak DNA ligand, analog 5, the most potent inhibitor, strongly interacted with DNA. Finally, as 5 interacted with DNMT only when the DNA duplex was present, we hypothesize that this class of chemical compounds inhibit DNMTs by interacting with the DNA substrate.

ROS-mediated DNA methylation pattern alterations in carcinogenesis.

Elevated levels of both reactive oxygen species (ROS) and DNA methylation are characteristic of various types of cancer cells. However, the relation between these two is not well understood. Here we will discuss the cause-consequence relationship between ROS and DNA methylation. Cancer research reveals that disregulation of DNA methylation results in regional CpG island hypermethylation and generalized genomic hypomethylation. ROS-induced oxidative stress is associated with both aberrant hypermethylation of tumor suppressor gene (TSG) promoter regions and global hypomethylation. The DNA oxidation structure, 8-hydroxy-2'-deoxyguanosine (8-OHdG), can induce DNA hypomethylation by inhibiting DNA methylation at nearby cytosine bases, while another DNA oxidation structure, 5-hydroxymethylcytosine (5hmC), may achieve active DNA demethylation processes, thus, causing DNA hypomethylation. Recently, it has been found that ROS can function as catalysts of DNA methylation, further accounting for TSG promoter hypermethylation. Moreover, ROS may induce site-specific hypermethylation via either the up-regulation of expression of DNA methyltransferases (DNMTs) or the formation of a new DNMT containing complex. In addition, these ROS-induced DNA methylation pattern alterations have been implicated with not only malignant transformation, but also the progression of numerous tumors. In conclusion, ROS can influence both aspects of DNA methylation changes through different mechanisms, which play an important role of epigenetic regulation in cancer cells. Therefore, the comprehension of mechanisms leading to epigenetic modifications associated with ROS may help better understand the carcinogenesis and progression, as well as aid in the development of potential biomarkers for better cancer diagnostics and novel therapeutic strategies.

Abstract PR03: Macrophage phenotype drives tumor program via epigenetic machinery carried in secreted microvesicles

Abstract Macrophage phenotypes are reported to regulate tumor progression, angiogenesis, and metastasis in breast cancer by producing soluble factors modulating these programs. Macrophages also communicate via secreted microvesicles (MVs) which are taken-up by neighboring epithelium. MVs contain mRNAs coding the epigenetic regulating machinery, DNA methyltranferases (DNMTs) and histone deacetylases (HDACs), which augment or silence expression via promoter CpG island methylation. Tie2-expressing monocytes (TEMs) is a subset of monocytes reported to augment tumor angiogenesis and metastasis. Recently, we found that increased levels of colony stimulating factor-1 (CSF1) can expand the TEM population in circulation, enabling an influx into breast tumors. Interestingly, we also found that expansion of TEMs by hypoxia was regulated by HIF-1α; and not HIF-2α, but only once they enter the tumor proper. We hypothesized that MVs secreted from M1 and M2 macrophages or TEMs contain epigenetic regulatory machinery which regulate CpG island methylation and gene expression of tumor suppressor genes (TSGs) and genes driving epithelial-to-mesenchymal transition (EMT). We differentiated M1, M2, and TEMs in vitro from CD14+ monocytes isolated from peripheral blood. These cell populations were confirmed using flow cytometry for CD68 and CD80 for M1, CD163 for M2, and CD14/Tie2 for TEMs, as well as M1 (IL-6 and TNFα) and M2 (IL-10 and mannose receptor-1) gene expression profiles. After, we collected MVs using high speed centrifugation techniques characterized by flow cytometry and isolated their nucleic acid content. Using qRT-PCR, we found differential presence of mRNAs for DNMTs and HDACs between M1, M2, and TEM MVs. We cultured these MVs with MCF-10A normal mammary epithelial cells or BEAS-2B normal lung epithelial cells (target cells) for 24 hours and demonstrated MV uptake using Syto RNASelect (RNA) and DiIC16(3) (lipid membrane) and confocal microscopy. After, we isolated RNA and DNA from the target cells and analyzed DNMTs, HDACs, and EMT mRNA expression as well as methyl-specific PCR for CpG island methylation in the promoters of EMT genes. We found that MVs from M1 macrophages increased DNMTs mRNA expression compared to MVs produced from M2 and quiescent macrophages (M0) as well as untreated target cells. To the contrary, HDACs mRNA expression in these target cells cultured with M1-derived MVs was abrogated compared to target cells cultured with MVs from M2 and M0 macrophages and untreated target cells. As a result of the differential MV-carrying DNMTs and HDACs mRNA transferred to the target cells, we found significant differences in CpG island promoter methylation and resultant gene expression in a signature of EMT genes, including TWIST, WNT5A, VIM, FOXC2, KRT19, STAT3, SNAI1 BMP1, TGFb, DSP, AKT1, NUDT13, and ZEB1. The regulation of EMT and tumor suppressor gene promoter methylation and gene expression in MDA-MB-231 human breast cancer cells, as well as the disparate regulation of methylation and gene expression patterns on these target cells as well endothelial cells (HUVEC) by MVs collected from CD14+/Tie2+ TEMs is ongoing. Our current and ongoing work, we establish that M1 and M2 macrophages, and TEMs, secrete MVs containing distinct epigenetic profiles which are taken-up by target cells to regulate promoter methylation and gene expression of TSGs and genes driving EMT. This program of macrophage function may be important in the progression of solid tumors via inhibition of TSGs and activation of a signature of EMT genes in normal epithelial cells as well as to direct the endothelium to support tumor progression. This abstract is also presented as Poster A31. Citation Format: Duaa Dakhlallah, Ivory Patterson, Amy C. Gross, Randall Evans, Tim D. Eubank. Macrophage phenotype drives tumor program via epigenetic machinery carried in secreted microvesicles. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr PR03. doi:10.1158/1538-7445.CHTME14-PR03

Methylation and Hydroxymethylation Status of Tumor Suppressor Genes and Common Fragile Sites Genes in Acute Myeloid Leukemia

Background: 5-methylation (5-mC) is the predominant epigenetic mark in mammalian genomic DNA. When promoter region of certain gene is hypermethylated, the gene becomes transcription silent. Promoter of tumor suppressor genes (TSG) usually exists in CpG islands, and silencing of TSGs in cancer cells is often associated with hypermethylation. p15, CDH1 are frequently methylated in myeloid malignancies such as acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). Common Fragile Site (CFS) is a fragile site on the chromosomes easy to produce gap and break, and it contains putative TSGs. FHIT, WWOX and PARK2 are the CFS genes known to be frequently methylated in solid tumors, but their status of hematologic malignancies has not been fully elucidated yet. 5-hydroxymethylaiton (5-hmC) is a newly discovered epigenetic modification that is presumably generated by oxidation of 5-mC by the TET family of cytosine oxygenases. Techniques identifying 5-mC cannot distinguish between 5-mC and 5-hmC, therefore 5-hmC status of the genes have not fully elucidated yet too. Recently it has been demonstrated that mutation of epigenetic modifiers (DNMT3A, TET2, IDH1/2) play important role on AML pathogenesis. We tried to clarify 5-mC and 5-hmC status of TSG p15, CDH1 and CFS genes FHIT, WWOX and PARK2 by using new techniques and the relationships with expression levels of epigenetic modifiers in AML.Methods: BM samples obtained from 74 of AML patients are subjected to the study after informed consent. This study was approved by IRB of Gunma University Hospital. DNA, RNA were extracted from BM mononuclear cells. Methylation specific PCR (MSP) was carried out to assay 5-mC of p15, CDH1, WWOX, PARK2. Quantification of 5-mC and 5-hmC (except PARK2) was carried out by methylation sensitive restriction enzyme assay (MSRE) with glucosylation and Q-PCR. Total DNA 5-mC and 5-hmC were analyzed by ELISA. The mRNA expression levels of p15, CDH1, FHIT, WWOX, PARK2, DNMT1, 3A, TET2 were quantified by Q-PCR.Results: MSP revealed that p15, CDH1, WWOX and PARK2 were methylated in 43.1%, 94.3%, 35.7% and 36.9% of AML, respectively. PARK2 methylation was not found in t(15;17) APL, but in 32% of normal karyotype AML (NK-AML), in 67% of t(8;21) CBF-AML. In contrast, the p15 methylation was found in 83.3% of APL, 45.5% of NK-AML, 50% of CBF-AML. WWOX methylation was found in 42.9% of APL, in 16% of NK-AML and 66.7% of CBF-AML. Adverse karyotype AML (adv-AML) tended to show lower % of WWOX, PARK2 and p15 methylation with 15.8%, 21.1% and 18.8% compare to good risk karyotype. The frequency of the methylation of PARK2 and WWOX were varied among karyotypes and the methylation was mutually exclusive. ELISA demonstrated that mean % of total 5-mC DNA was 1.08% and ratio of 5-hmC in 5-mC was 0.95% in AML. Interestingly, 5-hmC was 0% in adv-AML although 5-mC existed (mean: 1.05%). Locus specific MSRE-QPCR demonstrated that mean % of 5-mC of p15, CDH1, WWOX and FHIT were 6.62%, 1.25%, 8.33%, 2.88%, respectively., In adv-AML, 5-hmC of CDH1, WWOX and FHIT were not detected, although 5-mC of these genes were detected (0.41%, 9.0%, 2.14%) in accordance with whole DNA analysis. In good and intermediate AML, 5-hmC of these genes was 3.44%, 1.07%, 2.69% ,respectively. RQ-PCR demonstrated that CDH1, p15, WWOX, PARK2 and epigenetic modifier DNMT1, DNMT3A and TET2 expression were not different among various karyotype risks, but only FHIT expression significantly higher in good risk group (p=0.047). The expression levels of the genes were not significantly different between mentylated and unmethylated. The ratio of 5-hmC/5-mC of the TSGs tended to be associated with the expression levels of the corresponding genes, but the association did not reach statistical significance. DNMT3A expression in AML with 5-mC PARK2 was higher than in other AML (p=0.016). Contrary to the intuition, DNMT3A expression was positively correlated with FHIT, PARK2 expression (r=0.776, p<0.001, r=0.689, p<0.001). CDH1 expression was positively correlated with DNMT1 and negatively correlated with TET2 expression (r=0.447, p=0.009, r=-0.349, p=0.022). OS and EFS were not different among the methylation status of these genes.Conclusion: CFS genes are selectively methylated in AML. MSRE-QPCR can distinguish 5-mC and 5-hmC and quantify the ratio of them with locus specific manner. The relationship between gene expression and 5-hmC, 5-mC should be pursued. DisclosuresNo relevant conflicts of interest to declare.

Epigenetic repression of regulator of G‐protein signaling 2 by ubiquitin‐like with PHD and ring‐finger domain 1 promotes bladder cancer progression

Ubiquitin-like with PHD and ring-finger domain1 (UHRF1) binds to methylated promoters of tumor-suppressor genes and suppresses gene expression by forming complexes with DNA methyltransferases. Recent studies have shown that repression of regulator of G-protein signaling (RGS) 2 increases cancer cell growth. However, little is known about whether UHRF1 promotes bladder cancer progression by epigenetic silencing of RGS2. Here, we show that UHRF1 expression is increased in bladder cancer cell lines and in most bladder cancer tissues as compared with normal controls. UHRF1 overexpression increases bladder cancer cell proliferation, whereas inhibition of UHRF1 suppresses cell proliferation. In bladder cancer cells, UHRF1 inhibits RGS2 expression by increasing the methylation of CpG nucleotides of the RGS2 promoter. DNA methylation analysis showed tumor-specific TGS2 promoter methylation in 73% (38/52) of bladder tumors. High UHRF1 expression of correlated with aberrant TGS2 promoter methylation in bladder tumors, which results in the loss of TGS2 expression, as confirmed by demethylation analysis in cell lines. Functionally, re-expression of RGS2 partly abrogates UHRF1-induced bladder cell proliferation. Furthermore, Kaplan-Meier analysis showed that low TGS2 expression is significantly correlated with reduced overall survival in patients with bladder cancer. These results demonstrate that epigenetic repression of RGS2 by UHRF1 contributes to bladder cancer progression.

Suppression of TET1-Dependent DNA Demethylation Is Essential for KRAS-Mediated Transformation

Hypermethylation-mediated tumor suppressor gene (TSG) silencing is a central epigenetic alteration in RAS-dependent tumorigenesis. Ten-eleven translocation (TET) enzymes can depress DNA methylation by hydroxylation of 5-methylcytosine (5mC) bases to 5-hydroxymethylcytosine (5hmC). Here, we report that suppression of TET1 is required for KRAS-induced DNA hypermethylation and cellular transformation. In distinct nonmalignant cell lines, oncogenic KRAS promotes transformation by inhibiting TET1 expression via the ERK-signaling pathway. This reduces chromatin occupancy of TET1 at TSG promoters, lowers levels of 5hmC, and increases levels of 5mC and 5mC-dependent transcriptional silencing. Restoration of TET1 expression by ERK pathway inhibition or ectopic TET1 reintroduction in KRAS-transformed cells reactivates TSGs and inhibits colony formation. KRAS knockdown increases TET1 expression and diminishes colony-forming ability, whereas KRAS/TET1 double knockdown bypasses the KRAS dependence of KRAS-addicted cancer cells. Thus, suppression of TET1-dependent DNA demethylation is critical for KRAS-mediated transformation.

Mechanistic and biological significance of DNA methyltransferase 1 upregulated by growth factors in human hepatocellular carcinoma.

Dysregulation of growth factor signaling plays a pivotal role in controlling the malignancy phenotype and progression of hepatocellular carcinoma (HCC). However, the precise oncogenic mechanisms underlying transcription regulation of certain tumor suppressor genes (TSGs) by growth factors are poorly understood. In the present study, we report a novel insulin-like growth factor 1 (IGF1) pathway that mediates de novo DNA methylation and TSG (such as DLC1 and CHD5) silencing by upregulation of the DNA methyltransferase 1 (DNMT1) via an AKT/β-transducin repeat-containing protein (βTrCP)-mediated ubiquitin-proteasome pathway in HCC. Analysis of DNA methylation in CpG islands of target genes revealed high co-localization of DNMT1 and DNMT3B on the promoters of TSGs associated with enhanced CpG hypermethylation. Our results point to a novel epigenetic mechanism for growth factor-mediated repression of TSG transcription that involves DNA methylation.

CpG island hypermethylation as a biomarker for the early detection of lung cancer.

Lung cancer is the most frequent cause of cancer-related deaths and causes over one million deaths worldwide each year. Despite significant strides in the diagnosis and treatment of lung cancer, the prognosis is extremely poor, with the overall 5-year survival rates still remaining around 15 %. This is largely due to occult metastatic dissemination, which appears in approximately two-thirds of patients at the time of detection. Thus, the development of efficient diagnostic methods to enable the early detection of cancer for these patients is clearly imperative.One promising approach is the identification of lung cancer-specific biomarkers at an early stage. The de novo methylation of CpG islands within the promoters of tumor suppressor genes is one of the most frequently acquired epigenetic changes during the pathogenesis of lung cancer and usually associated with transcriptional downregulation of a gene. The analysis of DNA methylation patterns in sputum, bronchial fluid, plasma, or serum could become a powerful tool for the accurate and early diagnosis of lung cancer with unparalleled specificity and sensitivity.

A Prospective Study of Tumor Suppressor Gene Methylation as a Prognostic Biomarker in Surgically Resected Stage I to IIIA Non–Small-Cell Lung Cancers

While retrospective analyses support an association between early tumor recurrence and tumor suppressor gene promoter methylation in early-stage non-small-cell lung cancers (NSCLCs), few studies have investigated this question prospectively. Primary tumor tissue from patients with resected pathologic stage I to IIIA NSCLCs was collected at the time of surgery and analyzed for promoter methylation via methylation-specific reverse transcriptase polymerase chain reaction (MethyLight). The primary objective was to determine an association between promoter methylation of 10 individual tumor suppressor genes (CDKN2A, CDH13, RASSF1, APC, MGMT, GSTP1, DAPK1, WIF1, SOCS3, and ADAMTS8) and recurrence-free survival (RFS), with the secondary objectives of determining association with overall survival (OS), and relation to clinical or pathologic features. A total of 107 patients had sufficient tumor tissue for successful promoter methylation analysis. Majority of patients were former/current smokers (88%) with lung adenocarcinoma (78%) and pathologic stage I disease (62%). Median follow-up was 4 years. When controlled for pathologic stage, promoter methylation of the individual genes CDKN2A, CDH13, RASSF1, APC, MGMT, GSTP1, DAPK1, WIF1, and ADAMTS8 was not associated with RFS. Promoter methylation of the same genes was not associated with OS except for DAPK1 which was associated with improved OS (p = 0.03). The total number of genes with methylated promoters did not correlate with RFS (p = 0.89) or OS (p = 0.55). Contrary to data established by previous retrospective series, tumor suppressor gene promoter methylation (CDKN2A, CDH13, RASSF1, APC, MGMT, GSTP1, DAPK1, WIF1, and ADAMTS8) was not prognostic for early tumor recurrence in this prospective study of resected NSCLCs.

Regulation of CBP and Tip60 coordinates histone acetylation at local and global levels during Ras-induced transformation

Cell transformation is clearly linked to epigenetic changes. However, the role of the histone-modifying enzymes in this process is still poorly understood. In this study, we investigated the contribution of the histone acetyltransferase (HAT) enzymes to Ras-mediated transformation. Our results demonstrated that lysine acetyltransferase 5, also known as Tip60, facilitates histone acetylation of bulk chromatin in Ras-transformed cells. As a consequence, global H4 acetylation (H4K8ac and H4K12ac) increases in Ras-transformed cells, rendering a more decompacted chromatin than in parental cells. Furthermore, low levels of CREB-binding protein (CBP) lead to hypoacetylation of retinoblastoma 1 (Rb1) and cyclin-dependent kinase inhibitor 1B (Cdkn1b or p27Kip1) tumour suppressor gene promoters to facilitate Ras-mediated transformation. In agreement with these data, overexpression of Cbp counteracts Ras transforming capability in a HAT-dependent manner. Altogether our results indicate that CBP and Tip60 coordinate histone acetylation at both local and global levels to facilitate Ras-induced transformation.