Hypermethylation of two CpG sites upstream of CASP8AP2 promoter influences gene expression and treatment outcome in childhood acute lymphoblastic leukemia

DNA hypermethylation of Caspase 8 associated protein 2 (CASP8AP2) and its role in childhood acute lymphoblastic leukemia (ALL) is unclear. We analyzed methylation status of CpG sites upstream of CASP8AP2 gene in 86 children with ALL by bisulfite sequencing and quantitative PCR. Methylation percentage of two CpG sites at positions of −1189 and −1176 was inversely correlated with mRNA expression (Spearman correlation: −0.333, P = 0.002). High methylation was associated with the existence of minimal residual disease (MRD) at day 78 (P = 0.035), The patients in high methylation group had a poor treatment outcome. The combination of methylation level and MRD at day 33 might improve current risk stratification.     

DNA methylation patterns in lung carcinomas

The genome of epithelial tumors is characterized by numerous chromosomal aberrations, DNA base sequence changes, and epigenetic abnormalities. The epigenome of cancer cells has been most commonly studied at the level of DNA CpG methylation. In squamous cell carcinomas of the lung, CpG methylation patterns undergo substantial changes relative to normal lung epithelium. Using a genome-scale mapping technique for CpG methylation (MIRA-chip), we characterized CpG island methylation and methylation patterns of entire chromosome arms at a level of resolution of ～100 bp. In individual stage I lung carcinomas, several hundred and probably up to a thousand CpG islands become methylated. Interestingly, a large fraction (almost 80%) of the tumor-specifically methylated sequences are targets of the Polycomb complex in embryonic stem cells. Homeobox genes are particularly overrepresented and all four HOX gene loci on chromosomes 2, 7, 12, and 17 are hotspots for tumor-associated methylation because of the presence of multiple methylated CpG islands within these loci. DNA hypomethylation at CpGs in squamous cell tumors preferentially affects repetitive sequence classes including SINEs, LINEs, subtelomeric repeats, and segmental duplications. Since these epigenetic changes are found in early stage tumors, their contribution to tumor etiology as well as their potential usefulness as diagnostic or prognostic biomarkers of the disease should be considered.     

Differential methylation hybridization profiling identifies involvement of STAT1-mediated pathways in breast cancer

Many cancer-related genes are regulated by an epigenetic mechanism through modification of the methylation status of CpG sites at the promoter. This study was carried out at a genome-wide scale to mine genes in which the methylation of CpG sites is altered in breast cancer tissues. Differential methylation hybridization analysis was conducted using a chromosomal DNA mixture of ten normal and cancer tissue sets. A CpG microarray harboring 237,220 CpG sites of the whole genome was interrogated and the resulting methylation level differences, as well as the RNA expression differences, between the normal and cancer sets for selected genes were verified in breast cell lines by methylation-specific PCR and real-time PCR analyses. As a result, we identified and verified novel genes that were hypermethylated in breast cancer, such as NRN1, CA5B and RPIA. Pathway analysis of the genes with altered methylation patterns identified the involvement of a differentiation-related network of genes whose activity may be heavily regulated by STAT1 in breast tumorigenesis. Our results suggest that epigenetic dysregulation of cellular processes relevant to STAT1-dependent cellular differentiation may be intimately involved in breast carcinogenesis. These findings lend credence to the possibility of using tumor-specific alterations in methylation patterns as biomarkers in estimating prognosis and assessing treatment options for breast cancer.     

Epigenetic silencing of PEG3 gene expression in human glioma cell lines

Genomic imprinting, the phenomenon in which alleles of genes are expressed differentially depending on their parental origins, has important consequences for mammalian development, and disturbance of normal imprinting leads to abnormal embryogenesis and some inherited diseases and is also associated with various cancers. In the context of screening for novel imprinted genes on human chromosome 19q13.4 with mouse A9 hybrids, we identified a maternal allele-specific methylated CpG island in exon 1 of paternally expressed imprinted gene 3 (PEG3), a gene that exhibits paternal allele-specific expression. Because PEG3 expression is downregulated in some gliomas and glioma cell lines, despite high-level expression in normal brain tissues, we investigated whether the loss of PEG3 expression is related to epigenetic modifications involving DNA methylation. We found monoallelic expression of PEG3 in all normal brain tissues examined and five of nine glioma cell lines that had both unmethylated and methylated alleles; the remaining four glioma cell lines exhibited gain of imprinting with hypermethylated alleles. In addition, treatment of glioma cell lines with the DNA demethylating agent 5-aza-2'-deoxycytidine reversed the silencing of PEG3 biallelically. In this article, we report that the epigenetic silencing of PEG3 expression in glioma cell lines depends on aberrant DNA methylation of an exonic CpG island, suggesting that PEG3 contributes to glioma carcinogenesis in certain cases.     

Characterizing DNA methylation patterns in pancreatic cancer genome

We performed a global methylation profiling assay on 1505 CpG sites across 807 genes to characterize DNA methylation patterns in pancreatic cancer genome. We found 289 CpG sites that were differentially methylated in normal pancreas, pancreatic tumors and cancer cell lines. We identified 23 and 35 candidate genes that are regulated by hypermethylation and hypomethylation in pancreatic cancer, respectively. We also identified candidate methylation markers that alter the expression of genes critical to gemcitabine susceptibility in pancreatic cancer. These results indicate that aberrant DNA methylation is a frequent epigenetic event in pancreatic cancer; and by using global methylation profiling assay, it is possible to identify these markers for diagnostic and therapeutic purposes in this disease.     

Epigenetic silencing of the kinase tumor suppressor WNK2 is tumor-type and tumor-grade specific

Both genetic and epigenetic mechanisms contribute to meningioma development by altering gene expression and protein function. To determine the relative contribution of each mechanism to meningioma development, we used an integrative approach measuring copy number and DNA methylation changes genomewide. We found that genetic alterations affected 1.9%, 7.4%, and 13.3% of the 691 loci studied, whereas epigenetic mechanisms affected 5.4%, 9.9%, and 10.3% of these loci in grade I, II, and III meningiomas, respectively. Genetic and epigenetic mechanisms rarely involved the same locus in any given tumor. The predilection for epigenetic rather than genetic silencing was exemplified at the 5′ CpG island of WNK2, a serine-threonine kinase gene on chromosome 9q22.31. WNK2 is known to negatively regulate epidermal growth factor receptor signaling via inhibition of MEK1 (mitogen-activated protein kinase kinase 1), and point mutations have been reported in WNK1, WNK2, WNK3, and WNK4. In meningiomas, WNK2 was aberrantly methylated in 83% and 71% of grade II and III meningiomas, respectively, but rarely in a total of 209 tumors from 13 other tumor types. Aberrant methylation of the CpG island was associated with decreased expression in primary tumors. WNK2 could be reactivated with a methylation inhibitor in IOMM-Lee, a meningioma cell line with a densely methylated WNK2 CpG island and lack of WNK2 expression. Expression of exogenous WNK2 inhibited colony formation, implicating it as a potential cell growth suppressor. These findings indicate that epigenetic mechanisms are common across meningiomas of all grades and that for specific genes such as WNK2, epigenetic alteration may be the dominant, grade-specific mechanism of gene inactivation.     

维吾尔族妇女宫颈病变与CALCA基因启动子甲基化的关系

目的： 研究维吾尔族妇女宫颈病变与降钙素相关肽α(calcitonin-related polypeptide alpha,CALCA)基因启动子区CpG位点甲基化的关系,进一步揭示宫颈癌组织中该基因表达调控的表观遗传学机制。方法：收集维吾尔族妇女宫颈病变患者手术切除或活检组织标本50例,其中宫颈鳞癌(cervical squamous cell carcinoma,CSCC)25例、宫颈内上皮内瘤样变(cervical intraepithelial neoplasia,CIN)9例及慢性宫颈炎16例,应用Sequenom MassARRAY 甲基化DNA定量分析平台,对组织DNA的CALCA基因启动子区CpG位点进行甲基化水平的定量分析。结果：宫颈炎、CIN和CSCC组织中CALCA基因启动子区CpG片段甲基化率分别为0.22、0.26和0.33,经统计学分析,CSCC与CIN以及CSCC与宫颈炎之间均有显著差异(P<0.05)。从单一CpG位点角度分析,CSCC与宫颈炎组织CpG-2、CpG-3、CpG-9.10.11和CpG-12等6个CpG位点甲基化率均有显著差异(P<0.05)。结论：CALCA基因启动子甲基化与维吾尔族妇女宫颈癌发生密切相关,其中特定CpG位点甲基化水平变化可能成为宫颈癌早期诊断的分子标志物。     

Methylated CpG site count of dapper homolog 1 (DACT1) promoter prediction the poor survival of gastric cancer

Objective: To elucidate the clinical significance of the methylated status of CpG sites of dapper homolog 1 (DACT1) promoter in the survival prediction in gastric cancer (GC). Methods: The large scale GC patients (n=459) were analyzed for the quantitatively methylated status of CpG sites of DACT1 DNA promoter with the bisulphite sequencing PCR (BSP). With gene sequencing analysis, the methylated statuses of 12 cytosine-phosphate-guanine (CpG) sites in DACT1 promoter were detected to supply detailed information for the precisely prognostic prediction. Associations between molecular, clinicopathologic, and survival data were analyzed. Results: With the MSP detection, different methylated levels of DACT1 promoter were identified in the 25 GC tissues, while none of 25 normal gastric mucosal tissues were found to be methylated. DACT1 promoter methylation was found in 28.32% in 459 patients. GC patients with 4 or more methylated CpG sites of DACT1 promoter was significantly associated with the poorer survival (P=0.19). The methylated statuses of CpG -515, CpG -435, and CpG -430 sites were also identified to provide the elaborate survival discrimination for 459 GC patients, respectively (P=0.049, =0.006, and =0.037). In addition, we demonstrated that the methylated CpG site count had smallest AIC and BIC values than other three methylated status of CpG sites for prediction the survival of 459 GC patients. Conclusions: The methylated CpG site count of DACT1 promoter had the significant applicability for clinical evaluation the prognosis of GC.     

DNA methylation in the promoter region of the p16 (CDKN2/MTS-1/INK4A) gene in human breast tumours

The p16 (CDKN2/MTS-1/INK4A) gene is one of several tumour-suppressor genes that have been shown to be inactivated by DNA methylation in various human cancers including breast tumours. We have used bisulphite genomic sequencing to examine the detailed sequence specificity of DNA methylation in the CpG island promoter/exon 1 region in the p16 gene in DNA from a series of human breast cancer specimens and normal human breast tissue (from reductive mammaplasty). The p16 region examined was unmethylated in the four normal human breast specimens and in four out of nine breast tumours. In the other five independent breast tumour specimens, a uniform pattern of DNA methylation was observed. Of the nine major sites of DNA methylation in the amplified region from these tumour DNAs, four were in non-CG sequences. This unusual concentration of non-CG methylation sites was not a general phenomenon present throughout the genome of these tumour cells because the methylated CpG island regions of interspersed L1 repeats had a pattern of (almost exclusively) CG methylation similar to that found in normal breast tissue DNA and in DNA from tumours with unmethylated p16 genes. These data suggest that DNA methylation of the p16 gene in some breast tumours could be the result of an active process that generates a discrete methylation pattern and, hence, could ultimately be amenable to theraputic manipulation. © 1999 Cancer Research Campaign     

Altered mucosal DNA methylation in parallel with highly active Helicobacter pylori-related gastritis

Background

Chronic inflammation triggered by Helicobacter pylori causes altered DNA methylation in stomach mucosae, which is deeply involved in gastric carcinogenesis. This study aimed to elucidate the correlation between altered mucosal DNA methylation levels and activity of H. pylori-related gastritis, because inflammatory activity shows particular correlations with the development of diffuse-type cancer.

Methods

Methylation levels in stomach mucosae of 78 healthy volunteers were determined by real-time methylation-specific PCR or bisulfite pyrosequencing. Examined loci were the promoter CpG islands of six genes (FLNc, HAND1, THBD, p41ARC, HRASLS, and LOX) and the CpG sites of non-coding repetitive elements (Alu and Satα) that are reportedly altered by H. pylori infection. Activity of H. pylori-related gastritis was evaluated using two serum markers: H. pylori antibody titer and pepsinogen II.

Results

Methylation levels of the six CpG islands were consistently increased, and those of the two repetitive elements were consistently decreased in a stepwise manner with the activity of gastric inflammation as represented by serum marker levels. Each serum marker level was well correlated with the overall DNA methylation status of stomach mucosa, and these two serologic markers were additive in the detection of the mucosa with severely altered DNA methylation.

Conclusions

Alteration in mucosal DNA methylation level was closely correlated with activity of H. pylori-related gastritis as evaluated by serum markers. The observed correlation between altered DNA methylation levels and activity of H. pylori-related gastritis appears to be one of the relevant molecular mechanisms underlying the development of diffuse-type cancer.     

Combined DNA methylation and gene expression profiling in gastrointestinal stromal tumors reveals hypomethylation of SPP1 as an independent prognostic factor

Gastrointestinal stromal tumors (GISTs) have distinct gene expression patterns according to localization, genotype and aggressiveness. DNA methylation at CpG dinucleotides is an important mechanism for regulation of gene expression. We performed targeted DNA methylation analysis of 1.505 CpG loci in 807 cancer‐related genes in a cohort of 76 GISTs, combined with genome‐wide mRNA expression analysis in 22 GISTs, to identify signatures associated with clinicopathological parameters and prognosis. Principal component analysis revealed distinct DNA methylation patterns associated with anatomical localization, genotype, mitotic counts and clinical follow‐up. Methylation of a single CpG dinucleotide in the non‐CpG island promoter of SPP1 was significantly correlated with shorter disease‐free survival. Hypomethylation of this CpG was an independent prognostic parameter in a multivariate analysis compared to anatomical localization, genotype, tumor size and mitotic counts in a cohort of 141 GISTs with clinical follow‐up. The epigenetic regulation of SPP1 was confirmed in vitro, and the functional impact of SPP1 protein on tumorigenesis‐related signaling pathways was demonstrated. In summary, SPP1 promoter methylation is a novel and independent prognostic parameter in GISTs, and might be helpful in estimating the aggressiveness of GISTs from the intermediate‐risk category.     

Genome‐wide DNA methylation patterns in pancreatic ductal adenocarcinoma reveal epigenetic deregulation of SLIT‐ROBO,ITGA2 and MET signaling

The importance of epigenetic modifications such as DNA methylation in tumorigenesis is increasingly being appreciated. To define the genome‐wide pattern of DNA methylation in pancreatic ductal adenocarcinomas (PDAC), we captured the methylation profiles of 167 untreated resected PDACs and compared them to a panel of 29 adjacent nontransformed pancreata using high‐density arrays. A total of 11,634 CpG sites associated with 3,522 genes were significantly differentially methylated (DM) in PDAC and were capable of segregating PDAC from non‐malignant pancreas, regardless of tumor cellularity. As expected, PDAC hypermethylation was most prevalent in the 5′ region of genes (including the proximal promoter, 5′UTR and CpG islands). Approximately 33% DM genes showed significant inverse correlation with mRNA expression levels. Pathway analysis revealed an enrichment of aberrantly methylated genes involved in key molecular mechanisms important to PDAC: TGF‐β, WNT, integrin signaling, cell adhesion, stellate cell activation and axon guidance. Given the recent discovery that SLIT‐ROBO mutations play a clinically important role in PDAC, the role of epigenetic perturbation of axon guidance was pursued in more detail. Bisulfite amplicon deep sequencing and qRT‐PCR expression analyses confirmed recurrent perturbation of axon guidance pathway genes SLIT2, SLIT3, ROBO1, ROBO3, ITGA2 and MET and suggests epigenetic suppression of SLIT‐ROBO signaling and up‐regulation of MET and ITGA2 expression. Hypomethylation of MET and ITGA2 correlated with high gene expression, which was associated with poor survival. These data suggest that aberrant methylation plays an important role in pancreatic carcinogenesis affecting core signaling pathways with potential implications for the disease pathophysiology and therapy.