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Genome-wide methylation and expression profiling identifies promoter characteristics affecting demethylation-induced gene up-regulation in melanoma.

ABSTRACT: Abberant DNA methylation at CpG dinucleotides represents a common mechanism of transcriptional silencing in cancer. Since CpG methylation is a reversible event, tumor supressor genes that have undergone silencing through this mechanism represent promising targets for epigenetically active anti-cancer therapy. The cytosine analog 5-aza-2'-deoxycytidine (decitabine) induces genomic hypomethylation by inhibiting DNA methyltransferase, and is an example of an epigenetic agent that is thought to act by up-regulating silenced genes.It is unclear why decitabine causes some silenced loci to re-express, while others remain inactive. By applying data-mining techniques to large-scale datasets, we attempted to elucidate the qualities of promoter regions that define susceptibility to the drug's action. Our experimental data, derived from melanoma cell strains, consist of genome-wide gene expression data before and after treatment with decitabine, as well as genome-wide data on un-treated promoter methylation status, and validation of specific genes by bisulfite sequencing.We show that the combination of promoter CpG content and methylation level informs the ability of decitabine treatment to up-regulate gene expression. Promoters with high methylation levels and intermediate CpG content appear most susceptible to up-regulation by decitabine, whereas few of those highly methylated promoters with high CpG content are up-regulated. For promoters with low methylation levels, those with high CpG content are more likely to be up-regulated, whereas those with low CpG content are underrepresented among up-regulated genes.Clinically, elucidating the patterns of action of decitabine could aid in predicting the likelihood of up-regulating epigenetically silenced tumor suppressor genes and others from pathways involved with tumor biology. As a first step toward an eventual translational application, we build a classifier to predict gene up-regulation based on promoter methylation and CpG content, which achieves a performance of 0.77 AUC.

SUBMITTER: Rubinstein JC

PROVIDER: S-EPMC2843643 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature

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Genome-wide methylation and expression profiling identifies promoter characteristics affecting demethylation-induced gene up-regulation in melanoma.

Rubinstein Jill C JC Tran Nam N Ma Shuangge S Halaban Ruth R Krauthammer Michael M

BMC medical genomics 20100209

<h4>Background</h4>Abberant DNA methylation at CpG dinucleotides represents a common mechanism of transcriptional silencing in cancer. Since CpG methylation is a reversible event, tumor supressor genes that have undergone silencing through this mechanism represent promising targets for epigenetically active anti-cancer therapy. The cytosine analog 5-aza-2'-deoxycytidine (decitabine) induces genomic hypomethylation by inhibiting DNA methyltransferase, and is an example of an epigenetic agent that ...[more]

PMID: 20144234

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Project description:BackgroundPromoter hypermethylation coupled with loss of heterozygosity at the same locus results in loss of gene function in many tumor cells. The "rules" governing which genes are methylated during the pathogenesis of individual cancers, how specific methylation profiles are initially established, or what determines tumor type-specific methylation are unknown. However, DNA methylation markers that are highly specific and sensitive for common tumors would be useful for the early detection of cancer, and those required for the malignant phenotype would identify pathways important as therapeutic targets.Methods and findingsIn an effort to identify new cancer-specific methylation markers, we employed a high-throughput global expression profiling approach in lung cancer cells. We identified 132 genes that have 5' CpG islands, are induced from undetectable levels by 5-aza-2'-deoxycytidine in multiple non-small cell lung cancer cell lines, and are expressed in immortalized human bronchial epithelial cells. As expected, these genes were also expressed in normal lung, but often not in companion primary lung cancers. Methylation analysis of a subset (45/132) of these promoter regions in primary lung cancer (n = 20) and adjacent nonmalignant tissue (n = 20) showed that 31 genes had acquired methylation in the tumors, but did not show methylation in normal lung or peripheral blood cells. We studied the eight most frequently and specifically methylated genes from our lung cancer dataset in breast cancer (n = 37), colon cancer (n = 24), and prostate cancer (n = 24) along with counterpart nonmalignant tissues. We found that seven loci were frequently methylated in both breast and lung cancers, with four showing extensive methylation in all four epithelial tumors.ConclusionsBy using a systematic biological screen we identified multiple genes that are methylated with high penetrance in primary lung, breast, colon, and prostate cancers. The cross-tumor methylation pattern we observed for these novel markers suggests that we have identified a partial promoter hypermethylation signature for these common malignancies. These data suggest that while tumors in different tissues vary substantially with respect to gene expression, there may be commonalities in their promoter methylation profiles that represent targets for early detection screening or therapeutic intervention.

Project description:BACKGROUND:Measuring the DNA methylome may offer the opportunity to identify novel disease biomarkers and insights into disease mechanisms. Although aberrant DNA methylation has been investigated in many human cancers and precancerous lesions, the DNA methylation landscape of gastric cardiac intestinal metaplasia (IM) remains unknown. Therefore, we aimed to investigate the genome-wide DNA methylation landscape and to search for potential epigenetic biomarkers of gastric cardiac IM. METHODS:Histopathologic profiling was performed on a total of 118 gastric cardiac biopsies from cancer-free individuals. Genome-wide DNA methylation analysis was performed on 11 gastric cardiac mucosal biopsies (IM?=?7; normal?=?4) using Illumina 850K microarrays. Transcriptional relevance of any candidate epigenetic biomarker was validated by qRT-PCR. RESULTS:The detection rate of gastric cardiac IM was 23% (27/118) in cancer-free individuals. Genome-wide DNA methylation profiling showed a global decrease in methylation in IM compared with normal tissues (median methylation?=?0.64 and 0.70 for gastric cardiac IM and normal tissues, respectively). Differential methylation analysis between gastric cardiac IM and normal tissues identified 38,237 differentially methylated probes (DMPs) with a majority of sites showing hypermethylation in IM compared with normal tissues (56.3% vs. 43.7%). Subsequent analysis revealed a significant enrichment of hypermethylated DMPs in promoter and CpG islands (p?<?0.001 for both, Pearson ?2 test). For DMPs located in promoter CpG islands showing extreme hypermethylation, the candidate gene with the largest number of DMPs (n?=?7) was mapped to HOXA5. Accordingly, mRNA expression of HOXA5 was significantly reduced in IM compared to normal tissue. CONCLUSIONS:Our results suggest the implication of alterations in DNA methylation in gastric cardiac IM and highlight that HOXA5 hypermethylation may be a promising epigenetic biomarker, emphasizing the role of aberrant HOXA5 expression in the pathogenesis of gastric cardiac IM.

Project description:Background: DNA methylation is an important component of epigenetic modifications that influences the transcriptional machinery and is aberrant in many human diseases. In particular, dysregulation of promoter methylation in cancer has already been shown to be associated with repression of tumor suppressors and activation of oncogenes. In addition, detection of altered promoter methylation status is suitable for the design of diagnostic or prognostic tests. In this study we present a new methodological approach for the robust identification of promoter methylation markers and the first genome-wide study for the detection of methylation markers in melanoma. Methods and Findings: Genome-wide promoter methylation and gene expression of ten early passage melanoma cell strains are compared to newborn and adult normal melanocytes. For the identification of markers we applied linear mixed effect models (LME) in combination with a series of filters based on the localization of promoter methylation relative to the transcription start site, overall promoter CpG content, and differential gene expression. The aim of this methodology is to identify markers whose promoter differential methylation is likely to be functionally related to differential expression. We identified 76 markers, 68 hyper- and 8-hypo-methylated in melanomas (LME P<0.05). Promoter methylation profiles and differential expression of five of these markers were successfully validated. In addition, promoter demethylation following Aza treatment consistently restored expression of markers hyper-methylated in melanoma. Conclusions: The proposed methodology allows the identification of robust markers and can be applied to other experimental scenarios where promoter methylation is evaluated. More importantly, the list of markers represents the first systematic effort in the identification of methylation markers in melanoma. Many of the identified markers were not previously known to be regulated by promoter methylation and/or associated with this or other types of cancer.

Dataset Information

Genome-wide methylation and expression profiling identifies promoter characteristics affecting demethylation-induced gene up-regulation in melanoma.

Publications

Genome-wide methylation and expression profiling identifies promoter characteristics affecting demethylation-induced gene up-regulation in melanoma.

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