Abstract
Epigenetic alterations are common and can now be addressed in a parallel fashion. We investigated the methylation in bladder cancer with respect to location in genome, consistency, variation in metachronous tumors, impact on transcripts, chromosomal location, and usefulness as urinary markers. A microarray assay was utilized to analyze methylation in 56 samples. Independent validation was conducted in 63 samples by a PCR-based method and bisulfite sequencing. The methylation levels in 174 urine specimens were quantified. Transcript levels were analyzed using expression microarrays and pathways were analyzed using dedicated software. Global methylation patterns were established within and outside CpG islands. We validated methylation of the eight tumor markers genes ZNF154 (P < 0.0001), HOXA9 (P < 0.0001), POU4F2 (P < 0.0001), EOMES (P = 0.0005), ACOT11 (P = 0.0001), PCDHGA12 (P = 0.0001), CA3 (P = 0.0002), and PTGDR (P = 0.0110), the candidate marker of disease progression TBX4 (P < 0.04), and other genes with stage-specific methylation. The methylation of metachronous tumors was stable and targeted to certain pathways. The correlation to expression was not stringent. Chromosome 21 showed most differential methylation (P < 0.0001) and specifically hypomethylation of keratins, which together with keratin-like proteins were epigenetically regulated. In DNA from voided urine, we detected differential methylation of ZNF154 (P < 0.0001), POU4F2 (P < 0.0001), HOXA9 (P < 0.0001), and EOMES (P < 0.0001), achieving 84% sensitivity and 96% specificity. We initiated a detailed mapping of the methylome in metachronous bladder cancer. Novel genes with tumor, chromosome, as well as pathway-specific differential methylation in bladder cancer were identified. The methylated genes were promising cancer markers for early detection of bladder cancer.
Highlights
Epigenetics is the study of mitotically and/or meiotically heritable changes in gene function that cannot be explained by changes in DNA sequence [1]
We initiated a detailed mapping of the methylome in metachronous bladder cancer
Transcriptional inactivation by CpG island promoter hypermethylation is a well-established mechanism for gene silencing in cancer including bladder cancer [8,9,10,11,12,13,14,15,16,17,18], and aberrant methylation is associated with stage, grade of the tumors as well as recurrence rate and progression [19,20,21,22,23,24]
Summary
Epigenetics is the study of mitotically and/or meiotically heritable changes in gene function that cannot be explained by changes in DNA sequence [1]. CpG dinucleotides outside CpG islands are generally hypermethylated in normal cells and undergo a substantial loss of DNA methylation in cancers. CpG sites within CpG islands are usually in an unmethylated state permissive to transcription in normal cells but become hypermethylated at certain promoters in cancers. Transcriptional inactivation by CpG island promoter hypermethylation is a well-established mechanism for gene silencing in cancer including bladder cancer [8,9,10,11,12,13,14,15,16,17,18], and aberrant methylation is associated with stage, grade of the tumors as well as recurrence rate and progression [19,20,21,22,23,24]
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