Convergence of Mutation and Epigenetic Alterations Identifies Common Target Genes in Breast and Colon Cancer
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE10613 | GEO | 2009/09/23
SECONDARY ACCESSION(S): PRJNA107789
REPOSITORIES: GEO
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Project description:The identification and characterization of tumor suppressor genes has enhanced our understanding of the biology of cancer and enabled the development of new diagnostic and therapeutic modalities. Whereas in past decades, a handful of tumor suppressors have been slowly identified using techniques such as linkage analysis, large-scale sequencing of the cancer genome has enabled the rapid identification of a large number of genes that are mutated in cancer. However, determining which of these many genes play key roles in cancer development has proven challenging. Specifically, recent sequencing of human breast and colon cancers has revealed a large number of somatic gene mutations, but virtually all are heterozygous, occur at low frequency, and are tumor-type specific. Keywords: Microarray, Hypermethylome, DNA-hypermethylation, DAC, TSA, Epigenetic, Mutation, Colorectal Cancer, Breast Cancer The candidate hypermethylome genes were identified as described below. These genes were compared to the CAN genes identified by Sjöblom T et al (Science, 2006). Cell culture and treatment. For drug treatments, log phase MCF7, T-47D, MDA-MB231 and MDA-MB-468 cells were cultured in McCoys 5A media (Invitrogen) containing 10% BCS and 1x penicillin/streptomycin with 5M 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. 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. 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 platform and packages from Bioconductor bioinformatics software project. The log ratio of red signal to green signal was calculated and Loess normalization as implemented in the limma package from Bioconductor.
2009-09-23 | E-GEOD-10613 | biostudies-arrayexpress
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:The contribution of aberrant DNA methylation and the downstream effects in tumorogenesis through silencing of tumor suppressor genes (TSGs) and microRNAs has been investigated. Since these epigenetic alterations can be reversed, we investigated the effects of the epigenetic therapy in breast cancer cell lines. We used microarrays to investigate the global microRNA expression profile after demethylation treatment with 5-aza-2’-deoxycytidine (DAC) in breast cancer cell lines and identified distinct classes of early and late systematic stable or transient effects of the treatment. Three selected breast cell lines including MDA-MB231, SKBR3, BT549, HS578T, MCF7 and HB2 (a breast epithelial cell line as control) were subject for miRNA isolation before treatment, after treatment with DAC and at five point follow-ups (1st, 3rd, 5th, 7th and 10th passages) at “drug holiday” condition and hybridized on Affymetrix microarrays.
2012-01-24 | E-GEOD-28969 | biostudies-arrayexpress
Project description:Recently, the H3K4 demethylase, KDM5B, was shown to be amplified and overexpressed in luminal breast cancer, suggesting it might constitute a potential cancer therapy target. Here, we characterize, in breast cancer cells, the molecular effects of a recently developed small-molecule inhibitor of the KDM5 family of proteins, either alone, or in combination with the DNA demethylating agent 5-aza-2’ deoxycytidine (DAC). Alone, the KDM5 inhibitor (KDM5i) increased expression of a small number of genes, but when combined with DAC, the drug enhanced the effects of the latter for increasing expression of hundreds of DAC responsive genes. ChIP-seq studies revealed that KDM5i resulted in the broadening of existing, and creation of thousands of new H3K4me3 domains. When compared to DAC alone, increased promoter and gene body H3K4me3 occupancy at DAC responsive genes was observed in cells treated with the drug combination. Importantly, treatment with either DAC or DAC+KDM5i induced a dramatic increase in H3K27ac at enhancers with an associated significant increase in target gene expression, suggesting a previously unappreciated effect of DAC on transcriptional regulation. Finally, we found that KDM5i could synergize with DAC to reduce the viability of luminal breast cancer cells in in-vitro assays. Our study provides the first look into the molecular effects of novel KDM5i compounds and suggests that combining these with DAC may represent an exciting new approach to epigenetic therapy.
2017-12-08 | GSE97481 | GEO
Project description:The contribution of aberrant DNA methylation and the downstream effects in tumorogenesis through silencing of tumor suppressor genes (TSGs) and microRNAs has been investigated. Since these epigenetic alterations can be reversed, we investigated the effects of the epigenetic therapy in breast cancer cell lines. We used microarrays to investigate the global gene expression profile after demethylation treatment with 5-aza-2’-deoxycytidine (DAC) in breast cancer cell lines and identified distinct classes of early and late systematic stable or transient effects of the treatment. Three selected breast cell lines including MDA-MB231 (a highly aggressive cell line), SKBR3 (a non-aggressive cell line) and HB2 (a breast epithelial cell line as control) were subject for RNA isolation before treatment, after treatment with DAC and at five point follow-ups (1st, 3rd, 5th, 7th and 10th passages) at “drug holiday” condition and hybridized on Affymetrix microarrays.
2012-01-24 | E-GEOD-28968 | 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:Recently, the H3K4 demethylase, KDM5B, was shown to be amplified and overexpressed in luminal breast cancer, suggesting it might constitute a potential cancer therapy target. Here, we characterize, in breast cancer cells, the molecular effects of a recently developed small-molecule inhibitor of the KDM5 family of proteins, either alone, or in combination with the DNA demethylating agent 5-aza-2’ deoxycytidine (DAC). Alone, the KDM5 inhibitor (KDM5i) increased expression of a small number of genes, but when combined with DAC, the drug enhanced the effects of the latter for increasing expression of hundreds of DAC responsive genes. To determine if this combinatorial action was the result of enhanced DNA demethylation, we profiled genome-wide DNA methylation in cells treated with KDM5i, DAC, or both. Methylation levels at approximately 450,000 unique CpG loci were detected using the Infinium Illumina HumanMethylation 450 array. We found that KDM5i did not significantly affect DNA methylation either alone, or in combination with DAC.
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