Identity of the human cancer cell DNA hypermethylome revealed by gene expression profiling
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE4763 | GEO | 2007/08/08
SECONDARY ACCESSION(S): PRJNA95579
REPOSITORIES: GEO
Similar Datasets
Project description:CpG island promoter hypermethylation of tumor suppressor genes is a common hallmark of human cancer, and new large-scale epigenomic technologies might be useful in our attempts to define the complete DNA hypermethylome of tumor cells. Here we report a functional search for hypermethylated CpG islands using the colorectal cancer cell line HCT-116, in which two major DNA methyltransferases, DNMT1 and DNMT3b, have been genetically disrupted (DKOcells). Using methylated DNA immunoprecipitation (MeDIP) methodology in conjunction with promoter microarray analyses we found that DKO cells experience a significant loss of hypermethylated CpG islands. Further characterization of these candidate sequences demonstrates CpG island promoter hypermethylation and silencing of genes with potentially important roles in tumorigenesis, such as the Ras guanine-nucleotide release factor RASGRF2, the apoptosis-associated basic helixloop transcription factor BHLHB9, and the homeobox gene HOXD1. Hypermethylation of these genes occurs already in premalignant lesions and accumulates during tumorigenesis. Thus, our results demonstrate the usefulness of DNMT genetic disruption strategies combined with MeDIP in searching for unknown hypermethylated candidate genes in human cancer that might aid our understanding of the biology of the disease and be of potential translational use. Keywords: Human Proximal Promoter Array; DNA Methylation Comparative experiment: methylated DNA immunoprecipitated/INPUT(Total Genomic DNA) from the colon cancer cell line HCT116 wild type vs methylated DNA imonoprecipitated/INPUT from the colon cancer cell line HCT116 DNMT1/3b doubleknockout (DKO)
2010-05-27 | E-GEOD-9267 | biostudies-arrayexpress
Project description:CpG island promoter hypermethylation of tumor suppressor genes is a common hallmark of human cancer, and new large-scale epigenomic technologies might be useful in our attempts to define the complete DNA hypermethylome of tumor cells. Here we report a functional search for hypermethylated CpG islands using the colorectal cancer cell line HCT-116, in which two major DNA methyltransferases, DNMT1 and DNMT3b, have been genetically disrupted (DKOcells). Using methylated DNA immunoprecipitation (MeDIP) methodology in conjunction with promoter microarray analyses we found that DKO cells experience a significant loss of hypermethylated CpG islands. Further characterization of these candidate sequences demonstrates CpG island promoter hypermethylation and silencing of genes with potentially important roles in tumorigenesis, such as the Ras guanine-nucleotide release factor RASGRF2, the apoptosis-associated basic helixloop transcription factor BHLHB9, and the homeobox gene HOXD1. Hypermethylation of these genes occurs already in premalignant lesions and accumulates during tumorigenesis. Thus, our results demonstrate the usefulness of DNMT genetic disruption strategies combined with MeDIP in searching for unknown hypermethylated candidate genes in human cancer that might aid our understanding of the biology of the disease and be of potential translational use. Keywords: Human Proximal Promoter Array; DNA Methylation
2008-01-01 | GSE9267 | GEO
Project description:Altered gene expression is a hallmark of human cancers and arises in part through abnormal epigenetic regulation of gene transcription. The best characterized epigenetic alteration involves tumor suppressor gene inactivation via transcriptional repression associated with aberrant DNA hypermethylation of promoter region CpG islands1. Despite characterization of a growing number of such genes, the majority have yet to be identified. We now describe a genome wide microarray gene expression approach for human colorectal cancer cells, which can efficiently identify hundreds of hypermethylated genes for any cancer type. We compared isogenic cells altered pharmacologically versus genetically to induce genomic demethylation, to pinpoint genes activated by DNA demethylation, but not by inhibition of class I and II histone deacetylases (HDACs). We achieve an 82% success rate in predicting genes with densely hypermethylated CpG islands and complete gene silencing. The genes are similarly hypermethylated in primary tumors and have previously undetected tumor suppressor functions. Our approach provides the first highly efficient, comprehensive, platform for defining the cancer âDNA hypermethylome" Experiment Overall Design: Cell culture and treatment. HCT116 cells and isogenic genetic knockout derivatives were maintained as previously described14. For drug treatments, log phase HCT116 cells were cultured in McCoys 5A media (Invitrogen) containing 10% BCS and 1x penicillin/streptomycin with ïµïM 5aza-deoxycytidine (DAC) (Sigma; stock solution: 1mM in PBS) for 96 hours, replacing media and DAC every 24 hours. Cell treatment with 300nM Trichostatin A (Sigma; stock solution: 1.5mM dissolved in ethanol) was performed for 18 hours. Control cells underwent mock treatment in parallel with addition of equal volume of PBS or ethanol without drugs. Experiment Overall Design: Microarray analysis. Total RNA was harvested from log phase cells using the Qiagen kit according to the manufacturers instructions, including a DNAase step. RNA was quantified using the NanoDrop ND-100 followed by quality assessment with 2100 Bioanalyzer (Agilent Technologies). RNA concentrations for individual samples were greater than 200ng/ul, with 28s/18s ratios greater than 2.2 and RNA integrity numbers of 10 (highest). Sample amplification and labeling procedures were carried out using the Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies) according to the manufacturers instructions. The labeled cRNA was purified using the RNeasy mini kit (Qiagen) and quantified. RNA spike-in controls (Agilent Technologies) were added to RNA samples before amplification. 0.75 microgram of samples labeled with Cy3 or Cy5 were mixed with control targets (Agilent Technologies), assembled on Oligo Microarray, hybridized, and processed according to the Agilent microarray protocol. Scanning was performed with the Agilent G2565BA microarray scanner under default settings recommended by Agilent Technologies. Experiment Overall Design: Data analysis. All arrays were subject to quality checks recommended by the manufacturer. Images were visually inspected for artifacts and distributions of signal and background intensity of both red and green channels were examined to identify anomalous arrays. No irregularities were observed, and all arrays were retained and used. All calculations were performed using the R statistical computing platform37 and packages from Bioconductor bioinformatics software project38. The log ratio of red signal to green signal was calculated after background-subtraction and LoEss normalization as implemented in the limma package from Bioconductor39,40. Individual arrays were scaled to have the same inter-quartile range (75th percentile -25th percentile) Log fold changes were averaged over dye-swap replicate microarrays to produce a single set of expression values for each condition.
2008-06-13 | E-GEOD-4763 | biostudies-arrayexpress
Project description:Epigenetic gene regulation is a key determinant of heritable gene expression patterns and is critical for normal cellular function. Dysregulation of epigenetic transcriptional control is a fundamental feature of cancer, particularly manifesting as increased promoter DNA methylation with associated aberrant gene silencing which plays a significant role in tumor progression. We now globally map key chromatin parameters for genes with promoter CpG island DNA hypermethylation in colon cancer cells by combining microarray gene expression analyses with ChIP on chip technology. We first show that the silent state of such genes universally correlates with a broad distribution of a low, but distinct, level of the PcG mediated histone modification, methylation of lysine 27 of histone 3 (H3K27me) and a very low level of the active mark, H3K4me2. This chromatin pattern, and particularly H3K4me2 levels, crisply separates DNA hypermethylated genes from those where histone deacetylation is responsible for transcriptional silencing. Moreover, the chromatin pattern can markedly enhance identification of truly silent and DNA hypermethylated genes. We additionally find that when DNA hypermethylated genes are de-methylated and re-expressed, they adopt a “bivalent” chromatin pattern which is associated with the poised gene expression state of a large group of ES cell genes, and is characterized by an increase in levels of both the H3K27me3 and H3K4me2 marks. Our data have great relevance for the increasing interest in re-expression of DNA hypermethylated genes for the treatment of cancer. Keywords: DNA methylation, chromatin, histone modifications, cancer, epigenetic, ChIP-chip, expression microarray, hypermethylome, DNA-hypermethylation, DAC, TSA, colorectal cancer
2009-01-20 | GSE11173 | GEO
Project description:Clear cell renal cell carcinoma (ccRCC) is the most common adult renal cancer. Although the molecular characteristics of ccRCC are currently being studied, the biologically and clinically relevant ccRCC methylome remains to be elucidated. To explore the RCC hypermethylome we employed massive sequencing of methyl-binding protein enriched DNA and validated the biological relevance of the identified hypermethylated sites by pharmacological inhibition of DNA methylation. We identified four candidate tumor suppressor genes (GREM1, LAD1, NEFH, and NEURL) of which promoter CpG island hypermethylation was strongly predictive for ccRCC survival in two independent series (n=150 and n=185) of ccRCC primary samples. The four markers combined are strongly associated with risk for cancer-related death in the test series (HR 3.64, 95% CI 1.02-13.01) as well as, independently of other clinicopathological characteristics, in the validation series (HR 7.54, 95% CI 2.68-21.19) using Cox proportional hazard models. According to Harrell’s C statistics and the Akaike Information Criterion (AIC) the four marker panel provides the best predictive capacity with the best fit of the model as assessed for the tested markers. These results provide novel insights into the ccRCC hypermethylome and identify a strong methylation marker panel to potentially guide personalized ccRCC patient management. Preliminary to implementation of this marker panel into clinical practice, enrollment of these patients in a Phase-III trial to study adjuvant treatment efficacy is essential. 4 independant clear cell renal cancer cell lines were used before and after treatment with the DNA methylation inhibitor AZA or the HAZA inhibitor TSA.
2013-11-22 | E-GEOD-33916 | biostudies-arrayexpress
Project description:Clear cell renal cell carcinoma (ccRCC) is the most common adult renal cancer. Although the molecular characteristics of ccRCC are currently being studied, the biologically and clinically relevant ccRCC methylome remains to be elucidated. To explore the RCC hypermethylome we employed massive sequencing of methyl-binding protein enriched DNA and validated the biological relevance of the identified hypermethylated sites by pharmacological inhibition of DNA methylation. We identified four candidate tumor suppressor genes (GREM1, LAD1, NEFH, and NEURL) of which promoter CpG island hypermethylation was strongly predictive for ccRCC survival in two independent series (n=150 and n=185) of ccRCC primary samples. The four markers combined are strongly associated with risk for cancer-related death in the test series (HR 3.64, 95% CI 1.02-13.01) as well as, independently of other clinicopathological characteristics, in the validation series (HR 7.54, 95% CI 2.68-21.19) using Cox proportional hazard models. According to Harrell’s C statistics and the Akaike Information Criterion (AIC) the four marker panel provides the best predictive capacity with the best fit of the model as assessed for the tested markers. These results provide novel insights into the ccRCC hypermethylome and identify a strong methylation marker panel to potentially guide personalized ccRCC patient management. Preliminary to implementation of this marker panel into clinical practice, enrollment of these patients in a Phase-III trial to study adjuvant treatment efficacy is essential.
2013-11-22 | GSE33916 | GEO
Project description:Aberrant hypermethylation of CpG dinucleotides located in CpG islands within the promoters of key cancer genes is an epigenetic abnormality associated with heritable transcriptional gene silencing and inactivation in cancer. The genes involved include important tumor suppressors affecting key pathways for tumor initiation and progression. These methylated sequences can serve as potentially valuable markers for cancer risk assessment, diagnosis, prognosis, and prediction of therapeutic responses. In addition, many key cancer genes may be targeted by both epigenetic and genetic alterations and, thus epigenetic analysis can help focus the search for mutations, and vice versa. Studies of major cancer types suggest that any individual patient’s tumor may harbor at least 300 or more DNA hypermethylated genes. In TCGA, a pilot project is underway to begin defining these genes for GBM via genomic approaches. The approach in the epigenetic pilot is a two-tiered one which, first, involves pharmacological treatment of both well established human GBM cell lines, and a cell line grown as a neurosphere to enrich for tumor propagating cells, with a DNA methylation inhibitor (5-aza-2’-deoxycytidine, DAC) or a histone deacetylation inhibitor (trichostatin A) followed by an expression transcriptome analysis as previously described (Schuebel et. al.). This has resulted in identification of more than 3,700 total candidate genes. In the second tier, the top candidates are then analyzed on a custom Illumina GoldenGate array with the capacity to monitor methylation at a single CpG dinucleotide in the CpG islands of 1,498 gene promoters for the high throughput analysis of TCGA GBM samples. Keywords: Microarray, Hypermethylome, DNA-hypermethylation, DAC, TSA, Epigenetic, TCGA, The Cancer Genome Atlas, GBM, Glioblastoma, Glioblastoma multiforme, Brain
2009-01-01 | GSE11233 | GEO
Project description:Background: Neuroblastoma is a childhood cancer in which many children still have poor outcomes, emphasising the need to better understand its pathogenesis. Despite recent genome-wide mutation analyses, most neuroblastomas do not contain recognisable driver mutations, suggesting that epigenetic changes could underlie many cases. Methods: To discover genes that become epigenetically deregulated during neuroblastoma tumorigenesis, we compared neuroblastomas to their neural crest precursor cells, using genome-wide DNA methylation analysis; probing CpG island promoter microarrays with methyl CpG-immunoprecipitated DNA. Results: We identified 93 genes that were significantly differently methylated between neuroblastoma cell lines and neural crest cells, of which 26 (28%) were hypermethylated and 67 (72%) were hypomethylated. Concentrating on hypermethylated genes to identify candidate tumour suppressor loci, we found the cell engulfment and adhesion factor gene MEGF10 to be epigenetically repressed by DNA hypermethylation or by H3K27/K9 methylation in neuroblastoma cell lines. MEGF10 showed significantly down-regulated expression in neuroblastoma tumour samples; furthermore patients with the lowest-expressing tumours had reduced relapse-free survival. Knock-down of MEGF10 expression in neuroblastoma cell lines promoted cell growth. Conclusion: Our results suggest that MEGF10 is a clinically relevant, epigenetically-deregulated neuroblastoma tumour suppressor.
2016-11-30 | GSE71958 | GEO
Project description:Colorectal cancer (CRC) is one of the most common forms of cancer and the second leading cause of cancer-related deaths in the western world.
CRC mortality is related to stage of disease with a five year survival for early-stage disease of 77.0% and 50.8% for late stage disease. Methods for early detection of primary as well as recurrent CRC are therefore important to increase patient survival. Tumour biomarkers from blood, stool, or urine could aid the early diagnostics of CRC, but despite extensive research such markers have only provided limited clinical value.
Sporadic CRC develops as a result of the accumulation of genetic and epigenetic alterations. Epigenetic alterations include DNA hypermethylation, which through transcriptional silencing of tumour suppressor genes is associated with cancer development and cancer progression. The search for gene promoter regions hypermethylated in cancer has been ongoing for nearly two decades, and a number of genes have been shown to be preferentially hypermethylated in CRC. Therefore, hypermethylated DNA in plasma has been suggested as a marker for tumour-stage and survival in CRC patients. The only approved biomarker for the detection of CRC recurrence is the protein carcinoembryonic antigen (CEA). CEA is limited by its low sensitivity and therefore not recommended as a diagnostic biomarker. Hypermethylation of CRC specific genes as part of a molecular biomarker panel measured in blood could prove to be a recurrence marker in CRC patients, with elevated sensitivity and specificity.
The aims of this project are to examine if hypermethylation of specific genes measured from cell-free DNA in plasma of CRC patients can be used to detect primary CRC, to detect CRC recurrence and to be a biomarker for CRC prognosis.
Development of a reliable sensitive and specific biomarker for CRC will immensely improve the diagnostics and handling of CRC patients.
| 2227102 | ecrin-mdr-crc
Project description:To identify novel hypermethylated genes in colorectal cancer (CRC) and to test their potential application in CRC early diagnosis, we performed a genome-wide screening of 57,723 CpG dinucleotides covering 4,010 genes in paired DNA samples extracted from 3 fresh frozen CRC tissues and their matching non-tumor adjacent tissues from a cohort of 3 CRC patients undergoing curative surgery using MIRA-based microarray. We also validated candidate hypermethylated genes screened by MIRA-based microarray in independent CRC samples using combined bisulfite restriction analysis. A total of 297 CpG dinucleotides in CRC covering 211 genes were found to be hypermethylated in CRC tissues. From these 211 candidate methylated genes, seven novel methylated genes were picked up for validation and three genes were confirmed to be methylated in cancer samples but not in non-cancer samples.We also compared the methylation levels of these three novel hypermethylated genes with those of Vimentin and SEPT9, well-known hypermethylated genes in CRC, and found that methylated PHOX2B, FGF12 and GAD2 were better than methylated Vimentin and SEPT9 in differentiating CRC cancer tissue from normal tissue. Significant enrichment analysis of GO terms of the hypermethylated genes showed that a high proportion of hypermethylated genes in tumor tissues are involved in regulation of transcription. Paired experiments, colorectal cancer tissue vs. adjacent non-cancer tissue. Biological replicates: 3 cancer replicates, 3 paired non-cancer replicates.
2011-11-14 | E-GEOD-33545 | biostudies-arrayexpress