Aberrant promoter methylation and gene amplification in colorectal cancer
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ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Odilia Popanda
PROVIDER: E-GEOD-47413 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) is usually incurable. Contrary to genetic mechanisms involved in PDAC pathogenesis, epigenetic alterations are ill defined. Here we determine the contribution of epigenetically silenced genes to the development of PDAC. METHODS: We investigated methylated DNAs from PDACs, chronic pancreatitis and normal pancreatic tissues using Methyl-CpG immunoprecipitation followed by microarray hybridization. Promoter methylation of selected genes was confirmed with the Epityper assay. Expression levels were evaluated by quantitative RT-PCR. WNK2 was further investigated in tissue microarrays, methylation analysis of early pancreatic intraepithelial neoplasia (PanINs), mouse models for PDAC and pancreatitis, re-expression studies after demethylation, and cell growth assays using WNK2 overexpression. RESULTS: A total of 3.8% of 27.800 interrogated CpG islands were hypermethylated in PDAC versus normal and chronic pancreatitis tissues. Hypermethylation was confirmed in 12 out of 13 selected islands and was associated with gene silencing in 4 of them. The most prominently hypermethylated gene, WNK2, was further investigated. Demethylation assays confirmed the link between methylation and expression. WNK2 hypermethylation was higher in pancreatic tumor cells than in surrounding inflamed tissues and was observed in PanIN lesions as well as in a PDAC mouse model. WNK2 mRNA and protein expression were lower in PDAC and chronic pancreatitis compared to normal tissues both in patients and mouse models. Overexpression of WNK2 led to a reduced cell growth and WNK2 expression in tissues correlated negatively with the expression of pERK1/2, a downstream target of WNK2 responsible for cell proliferation. CONCLUSIONS: WNK2 is downregulated by promoter hypermethylation early in PDAC pathogenesis and may support tumor cell growth via the ERK-MAPK pathway. 3 types of pancreatic tissue samples: 5 normals, 2 chronic pancreatitis, 7 tumors (PDAC)
2012-11-01 | E-GEOD-37480 | biostudies-arrayexpress
Project description:To identify new markers for colorectal cancer we scrutinized the methylation status by methyl-CpG immunoprecipitation followed by global methylation profiling on a CpG island microarray, as altered expression could drive genomic and chromosomal instability observed in these tumors. We show for the first time hypermethylation of MMP9, DNMT3A, and LIG4 in CRC which was confirmed in two independent ethnic CRC patients groups. Hypermethylation of the LIG4 promoter is a new mechanism to control ligase IV expression. It may represent a new epigenetic marker for colorectal cancer independent of known markers.
2013-12-31 | GSE47413 | GEO
Project description:Determination of the profile of genes commonly aberrantly methylated in colorectal cancer (CRC) has substantial potential value for diagnostic and therapeutic application. However, our knowledge of the DNA methylation pattern in CRC is currently limited. Therefore, we analyzed the methylation profile of 27,578 CpG sites spanning more than 14,000 genes in CRC and in adjacent normal mucosa using beadchip array-based technology. We identified 621 CpG sites located in promoter region and CpG islands that were significantly hypermethylated in CRC compared to normal mucosa. Genes on chromosome 18 showed promoter hypermethylation most frequently. According to gene ontology analysis, the most common biologically relevant class of genes affected by methylation was the class associated with the cadherin signaling pathway. In comparison with genome-wide expression array, mRNA expression was more like to be down-regulated in the genes demonstrating promoter hypermethylation, although this was not statistically significant. We validated 10 CpG sites that were shown to be hypermethylated in the array studies (ADHFE1, BOLL, SLC6A15, ADAMTS5, TFPI2, EYA4, NPY, TWIST1, LAMA1, GAS7) and 2 CpG sites showing hypomethylaion (MAEL, SFT2D3) in CRC compared to normal mucosa using pyrosequencing. The methylation status measured by pyrosequencing was consistent with the methylation array data. In conclusion, we have shown that methylation profiling based on beadchip arrays is an effective method for screening for aberrantly methylated genes in CRC. In addition, we identified novel methylated genes that are candidate diagnostic or prognostic markers for CRC. We measured the methylation status of the 27,578 CpG sites (Human Methylation27 DNA Analysis BeadChip) in 22 pairs of CRC tissue and adjacent normal mucosa to identify genes that are commonly aberrantly methylated in CRC.
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Project description:Extensive changes in DNA methylation are common in cancer and may contribute to oncogenesis through transcriptional silencing of tumor suppressor genes. Genome-scale studies have yielded important insights into these changes, but have focused on CpG islands or gene promoters. We used whole-genome bisulfite sequencing (Bisulfite-Seq) to comprehensively profile a primary human colorectal tumor and adjacent-normal colon tissue at single-basepair resolution. Regions of focal hypermethylation in the tumor were located primarily at CpG islands and were concentrated within regions of long-range (>100 kb) hypomethylation. These hypomethylated domains covered nearly half the genome and coincided with late replication and attachment to the nuclear lamina in human cell lines. The confluence of hypermethylation and hypomethylation within these domains was confirmed in 25 diverse colorectal tumors with matched adjacent tissue. We propose that widespread DNA methylation changes in cancer are linked to silencing programs orchestrated by the 3D organization of chromatin within the nucleus. dbGAP study: phs000385 Primary tissue samples sequenced using 76-bp bisulfite sequencing (WGBS or Methyl-seq) using the Illumina GAII platform. Two independent libraries were constructed for each sample, and these libraries were combined into a single data file for each sample.
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Project description:We sought to understand the mechanisms of cancer dissemination and to improve treatments for late-stage cancer patients. Laser tissue microdissection of 10 to 25 x 106 µm2 epithelium from sections of freshly-frozen colorectal cancers (no=6), colorectal cancer liver metastases (no=12) and normal colonic mucosa (no=3) was performed to extract DNA. Methylated DNA was captured using a methylCpG binding domain (MBD) polypeptide and deep sequenced. We detected hyper and hypomethylation in both colorectal cancers and metastases compared to normal tissue.
2020-01-03 | E-MTAB-8232 | biostudies-arrayexpress
Project description:To globally define methylation-âproneâ and -âprotectedâ CpG islands in leukemia, we analyzed the methylation status of 23,000 CpG islands of the human genome in two acute leukemia cell lines as well as normal blood monocytes using our previously described methyl-CpG immunoprecipitation (MCIp) technique (Gebhard et al. 2006; Schilling and Rehli 2007). This method enriches for highly CpG methylated DNA that can be directly applied to fluorescent labeling and oligonucleotide microarray hybridization without an additional amplification step. Keywords: MCIp-on-Chip; comparative genomic hybridization CpG-methylated genomic DNA was enriched using methyl-CpG immunoprecipitation (MCIp). On each microarray, the enriched material from leukemia cell lines was compared to the enriched material from normal blood monocytes to identify aberrantly methylated regions. Three biological replicates were analysed for each cell line.
2010-06-05 | E-GEOD-17455 | biostudies-arrayexpress
Project description:Using MCIp, we enriched highly methylated DNA from a normal mucosa control and from three OPSCC patient subgroups regarding the HPV status: group A, DNA-RNA- (Tu1-Tu5); group B, DNA+RNA- (Tu6-Tu10); group C, DNA+RNA+ (Tu11-Tu15). 3 groups of tumor biopsies, 5 samples each regarding HPV status: DNA-RNA-, DNA+RNA-, DNA+RNA+ and a control sample (normal mucosa from healthy individual): 15 tumor samples/ 1 control sample
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Project description:MGMT promoter methylation status is currently the only established molecular prognosticator in IDH wild-type glioblastoma multiforme (GBM). Therefore, we aimed to discover novel therapy-associated epigenetic biomarkers. After enrichment for hypermethylated fractions using methyl-CpG-immunoprecipitation (MCIp), we performed global DNA methylation profiling for 14 long-term (LTS; >36 months) and 15 short-term (STS; 6â??10 months) surviving GBM patients. Even after exclusion of the G-CIMP phenotype, we observed marked differences between the LTS and STS methylome. A total of 1,247 probes in 706 genes were hypermethylated in LTS and 463 probes in 305 genes were found to be hypermethylated in STS patients (p values < 0.05, log2 fold change ± 0.5). We identified 13 differentially methylated regions (DMRs) with a minimum of four differentially methylated probes per gene. Indeed, we were able to validate a subset of these DMRs through a second, independent method (MassARRAY) in our LTS/STS training set (ADCY1, GPC3, LOC283731/ISLR2). These DMRs were further assessed for their prognostic capability in an independent validation cohort (n = 62) of non-G-CIMP GBMs from the TCGA. Hypermethylation of multiple CpGs mapping to the promoter region of LOC283731 correlated with improved patient outcome (p < 0.03). The prognostic performance of LOC283731 promoter hypermethylation was confirmed in a third independent study cohort (n = 89), and was independent of gender, performance (KPS) and MGMT status (p < 0.0485, HR = 0.63). Intriguingly, the prediction was most pronounced in younger GBM patients (<60 years). In conclusion, we provide compelling evidence that promoter methylation status of this novel gene is a prognostic biomarker in IDH1 wild-type/non-G-CIMP GBMs. Global DNA methylation profiling in 14 long-term and 15 short-term surviving glioblastoma patients. DNA of 7 normal brain samples were pooled to create a control DNA for two-color analysis.
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