Abstract
BackgroundHypermethylation of promoter CpG islands with associated loss of gene expression, and hypomethylation of CpG-rich repetitive elements that may destabilize the genome are common events in most, if not all, epithelial cancers.MethodsThe methylation of 6,502 CpG-rich sequences spanning the genome was analyzed in 137 ovarian samples (ten normal, 23 low malignant potential, 18 stage I, 16 stage II, 54 stage III, and 16 stage IV) ranging from normal tissue through to stage IV cancer using a sequence-validated human CpG island microarray. The microarray contained 5' promoter-associated CpG islands as well as CpG-rich satellite and Alu repetitive elements.ResultsResults showed a progressive de-evolution of normal CpG methylation patterns with disease progression; 659 CpG islands showed significant loss or gain of methylation. Satellite and Alu sequences were primarily associated with loss of methylation, while promoter CpG islands composed the majority of sequences with gains in methylation. Since the majority of ovarian tumors are late stage when diagnosed, we tested whether DNA methylation profiles could differentiate between normal and low malignant potential (LMP) compared to stage III ovarian samples. We developed a class predictor consisting of three CpG-rich sequences that was 100% sensitive and 89% specific when used to predict an independent set of normal and LMP samples versus stage III samples. Bisulfite sequencing confirmed the NKX-2-3 promoter CpG island was hypermethylated with disease progression. In addition, 5-aza-2'-deoxycytidine treatment of the ES2 and OVCAR ovarian cancer cell lines re-expressed NKX-2-3. Finally, we merged our CpG methylation results with previously published ovarian expression microarray data and identified correlated expression changes.ConclusionOur results show that changes in CpG methylation are cumulative with ovarian cancer progression in a sequence-type dependent manner, and that CpG island microarrays can rapidly discover novel genes affected by CpG methylation in clinical samples of ovarian cancer.
Highlights
Hypermethylation of promoter CpG islands with associated loss of gene expression, and hypomethylation of CpG-rich repetitive elements that may destabilize the genome are common events in most, if not all, epithelial cancers
DNA methylation differences in benign and malignant ovarian tissue 137 samples of benign and malignant ovarian tissues from various stages and histopathologies were analyzed to develop a broad view of DNA methylation changes with progression of ovarian cancer (Additional File 2)
To examine the relationship between tumor stage and changes in DNA methylation, samples were grouped by stage, and analysis of variance (ANOVA) was performed. 2,042 CpG-rich clones with significant differences in methylation by stage at p = 0.01 (Benjamini Hochberg False Discovery Rate (FDR) adjusted) were identified, and the list reduced to 659 clones by selection for at least a 1.5fold difference in methylation between any two stages (Additional File 3)
Summary
Hypermethylation of promoter CpG islands with associated loss of gene expression, and hypomethylation of CpG-rich repetitive elements that may destabilize the genome are common events in most, if not all, epithelial cancers. Ovarian cancer remains the most deadly gynecologic malignancy. In contrast to other gynecologic malignancies, 81% of ovarian cancers are late stage III or IV at the time of diagnosis, implying upper abdominal or distant metastases[1]. Because of late stage at diagnosis, the 5 year survival of ovarian cancer for all stages is only 45%, and the 5 year survival for patients with stage III or IV disease is only 30%[1]. Normal tissue and tissue samples of low malignant potential were collected for comparison to tumor. Tissues were snap frozen at the time of surgery in liquid nitrogen and high molecular weight tumor DNA extracted with Trizol reagent by following the manufacturer's instructions (Life Technologies, Inc., Gaithersburg, Maryland). The DOT quantitative test was used to estimate the DNA concentration[17]
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