Analysis of repetitive element DNA methylation by MethyLight

Repetitive elements represent a large portion of the human genome and contain much of the CpG methylation found in normal human postnatal somatic tissues. Loss of DNA methylation in these sequences might account for most of the global hypomethylation that characterizes a large percentage of human cancers that have been studied. There is widespread interest in correlating the genomic 5-methylcytosine content with clinical outcome, dietary history, lifestyle, etc. However, a high-throughput, accurate and easily accessible technique that can be applied even to paraffin-embedded tissue DNA is not yet available. Here, we report the development of quantitative MethyLight assays to determine the levels of methylated and unmethylated repeats, namely, Alu and LINE-1 sequences and the centromeric satellite alpha (Satα) and juxtacentromeric satellite 2 (Sat2) DNA sequences. Methylation levels of Alu, Sat2 and LINE-1 repeats were significantly associated with global DNA methylation, as measured by high performance liquid chromatography, and the combined measurements of Alu and Sat2 methylation were highly correlative with global DNA methylation measurements. These MethyLight assays rely only on real-time PCR and provide surrogate markers for global DNA methylation analysis. We also describe a novel design strategy for the development of methylation-independent MethyLight control reactions based on Alu sequences depleted of CpG dinucleotides by evolutionary deamination on one strand. We show that one such Alu-based reaction provides a greatly improved detection of DNA for normalization in MethyLight applications and is less susceptible to normalization errors caused by cancer-associated aneuploidy and copy number changes

Analysis of the Association between CIMP and BRAFV600E in Colorectal Cancer by DNA Methylation Profiling

A CpG island methylator phenotype (CIMP) is displayed by a distinct subset of colorectal cancers with a high frequency of DNA hypermethylation in a specific group of CpG islands. Recent studies have shown that an activating mutation of BRAF (BRAFV600E) is tightly associated with CIMP, raising the question of whether BRAFV600E plays a causal role in the development of CIMP or whether CIMP provides a favorable environment for the acquisition of BRAFV600E. We employed Illumina GoldenGate DNA methylation technology, which interrogates 1,505 CpG sites in 807 different genes, to further study this association. We first examined whether expression of BRAFV600E causes DNA hypermethylation by stably expressing BRAFV600E in the CIMP-negative, BRAF wild-type COLO 320DM colorectal cancer cell line. We determined 100 CIMP-associated CpG sites and examined changes in DNA methylation in eight stably transfected clones over multiple passages. We found that BRAFV600E is not sufficient to induce CIMP in our system. Secondly, considering the alternative possibility, we identified genes whose DNA hypermethylation was closely linked to BRAFV600E and CIMP in 235 primary colorectal tumors. Interestingly, genes that showed the most significant link include those that mediate various signaling pathways implicated in colorectal tumorigenesis, such as BMP3 and BMP6 (BMP signaling), EPHA3, KIT, and FLT1 (receptor tyrosine kinases) and SMO (Hedgehog signaling). Furthermore, we identified CIMP-dependent DNA hypermethylation of IGFBP7, which has been shown to mediate BRAFV600E-induced cellular senescence and apoptosis. Promoter DNA hypermethylation of IGFBP7 was associated with silencing of the gene. CIMP-specific inactivation of BRAFV600E-induced senescence and apoptosis pathways by IGFBP7 DNA hypermethylation might create a favorable context for the acquisition of BRAFV600E in CIMP+ colorectal cancer. Our data will be useful for future investigations toward understanding CIMP in colorectal cancer and gaining insights into the role of aberrant DNA hypermethylation in colorectal tumorigenesis

DNA Methylation Profiles of Ovarian Epithelial Carcinoma Tumors and Cell Lines

BACKGROUND:Epithelial ovarian carcinoma is a significant cause of cancer mortality in women worldwide and in the United States. Epithelial ovarian cancer comprises several histological subtypes, each with distinct clinical and molecular characteristics. The natural history of this heterogeneous disease, including the cell types of origin, is poorly understood. This study applied recently developed methods for high-throughput DNA methylation profiling to characterize ovarian cancer cell lines and tumors, including representatives of three major histologies. METHODOLOGY/PRINCIPAL FINDINGS:We obtained DNA methylation profiles of 1,505 CpG sites (808 genes) in 27 primary epithelial ovarian tumors and 15 ovarian cancer cell lines. We found that the DNA methylation profiles of ovarian cancer cell lines were markedly different from those of primary ovarian tumors. Aggregate DNA methylation levels of the assayed CpG sites tended to be higher in ovarian cancer cell lines relative to ovarian tumors. Within the primary tumors, those of the same histological type were more alike in their methylation profiles than those of different subtypes. Supervised analyses identified 90 CpG sites (68 genes) that exhibited 'subtype-specific' DNA methylation patterns (FDR<1%) among the tumors. In ovarian cancer cell lines, we estimated that for at least 27% of analyzed autosomal CpG sites, increases in methylation were accompanied by decreases in transcription of the associated gene. SIGNIFICANCE:The significant difference in DNA methylation profiles between ovarian cancer cell lines and tumors underscores the need to be cautious in using cell lines as tumor models for molecular studies of ovarian cancer and other cancers. Similarly, the distinct methylation profiles of the different histological types of ovarian tumors reinforces the need to treat the different histologies of ovarian cancer as different diseases, both clinically and in biomarker studies. These data provide a useful resource for future studies, including those of potential tumor progenitor cells, which may help illuminate the etiology and natural history of these cancers

Identification of a panel of sensitive and specific DNA methylation markers for squamous cell lung cancer

<p>Abstract</p> <p>Background</p> <p>Lung cancer is the leading cause of cancer death in men and women in the United States and Western Europe. Over 160,000 Americans die of this disease every year. The five-year survival rate is 15% – significantly lower than that of other major cancers. Early detection is a key factor in increasing lung cancer patient survival. DNA hypermethylation is recognized as an important mechanism for tumor suppressor gene inactivation in cancer and could yield powerful biomarkers for early detection of lung cancer. Here we focused on developing DNA methylation markers for squamous cell carcinoma of the lung. Using the sensitive, high-throughput DNA methylation analysis technique MethyLight, we examined the methylation profile of 42 loci in a collection of 45 squamous cell lung cancer samples and adjacent non-tumor lung tissues from the same patients.</p> <p>Results</p> <p>We identified 22 loci showing significantly higher DNA methylation levels in tumor tissue than adjacent non-tumor lung. Of these, eight showed highly significant hypermethylation in tumor tissue (p < 0.0001): GDNF, MTHFR, OPCML, TNFRSF25, TCF21, PAX8, PTPRN2 and PITX2. Used in combination on our specimen collection, this eight-locus panel showed 95.6% sensitivity and specificity.</p> <p>Conclusion</p> <p>We have identified 22 DNA methylation markers for squamous cell lung cancer, several of which have not previously been reported to be methylated in any type of human cancer. The top eight markers show great promise as a sensitive and specific DNA methylation marker panel for squamous cell lung cancer.</p

Epigenetic Subgroups of Esophageal and Gastric Adenocarcinoma with Differential GATA5 DNA Methylation Associated with Clinical and Lifestyle Factors

BACKGROUND: Adenocarcinomas located near the gastroesophageal junction have unclear etiology and are difficult to classify. We used DNA methylation analysis to identify subtype-specific markers and new subgroups of gastroesophageal adenocarcinomas, and studied their association with epidemiological risk factors and clinical outcomes. METHODOLOGY/PRINCIPAL FINDINGS: We used logistic regression models and unsupervised hierarchical cluster analysis of 74 DNA methylation markers on 45 tumor samples (44 patients) of esophageal and gastric adenocarcinomas obtained from a population-based case-control study to uncover epigenetic markers and cluster groups of gastroesophageal adenocarcinomas. No distinct epigenetic differences were evident between subtypes of gastric and esophageal cancers. However, we identified two gastroesophageal adenocarcinoma subclusters based on DNA methylation profiles. Group membership was best predicted by GATA5 DNA methylation status. We analyzed the associations between these two epigenetic groups and exposure using logistic regression, and the associations with survival time using Cox regression in a larger set of 317 tumor samples (278 patients). There were more males with esophageal and gastric cardia cancers in Cluster Group 1 characterized by higher GATA5 DNA methylation values (all p<0.05). This group also showed associations of borderline statistical significance with having ever smoked (p-value = 0.07), high body mass index (p-value = 0.06), and symptoms of gastroesophageal reflux (p-value = 0.07). Subjects in cluster Group 1 showed better survival than those in Group 2 after adjusting for tumor differentiation grade, but this was not found to be independent of tumor stage. CONCLUSIONS/SIGNIFICANCE: DNA methylation profiling can be used in population-based studies to identify epigenetic subclasses of gastroesophageal adenocarcinomas and class-specific DNA methylation markers that can be linked to epidemiological data and clinical outcome. Two new epigenetic subgroups of gastroesophageal adenocarcinomas were identified that differ to some extent in their survival rates, risk factors of exposure, and GATA5 DNA methylation

Genome-scale screen for DNA methylation-based detection markers for ovarian cancer.

The identification of sensitive biomarkers for the detection of ovarian cancer is of high clinical relevance for early detection and/or monitoring of disease recurrence. We developed a systematic multi-step biomarker discovery and verification strategy to identify candidate DNA methylation markers for the blood-based detection of ovarian cancer

DNA Methylation in the Human Cerebral Cortex Is Dynamically Regulated throughout the Life Span and Involves Differentiated Neurons

The role of DNA cytosine methylation, an epigenetic regulator of chromatin structure and function, during normal and pathological brain development and aging remains unclear. Here, we examined by MethyLight PCR the DNA methylation status at 50 loci, encompassing primarily 5′ CpG islands of genes related to CNS growth and development, in temporal neocortex of 125 subjects ranging in age from 17 weeks of gestation to 104 years old. Two psychiatric disease cohorts—defined by chronic neurodegeneration (Alzheimer's) or lack thereof (schizophrenia)—were included. A robust and progressive rise in DNA methylation levels across the lifespan was observed for 8/50 loci (GABRA2, GAD1, HOXA1, NEUROD1, NEUROD2, PGR, STK11, SYK) typically in conjunction with declining levels of the corresponding mRNAs. Another 16 loci were defined by a sharp rise in DNA methylation levels within the first few months or years after birth. Disease-associated changes were limited to 2/50 loci in the Alzheimer's cohort, which appeared to reflect an acceleration of the age-related change in normal brain. Additionally, methylation studies on sorted nuclei provided evidence for bidirectional methylation events in cortical neurons during the transition from childhood to advanced age, as reflected by significant increases at 3, and a decrease at 1 of 10 loci. Furthermore, the DNMT3a de novo DNA methyl-transferase was expressed across all ages, including a subset of neurons residing in layers III and V of the mature cortex. Therefore, DNA methylation is dynamically regulated in the human cerebral cortex throughout the lifespan, involves differentiated neurons, and affects a substantial portion of genes predominantly by an age-related increase

DNA Methylation Changes in Atypical Adenomatous Hyperplasia, Adenocarcinoma In Situ, and Lung Adenocarcinoma

BACKGROUND:Aberrant DNA methylation is common in lung adenocarcinoma, but its timing in the phases of tumor development is largely unknown. Delineating when abnormal DNA methylation arises may provide insight into the natural history of lung adenocarcinoma and the role that DNA methylation alterations play in tumor formation. METHODOLOGY/PRINCIPAL FINDINGS:We used MethyLight, a sensitive real-time PCR-based quantitative method, to analyze DNA methylation levels at 15 CpG islands that are frequently methylated in lung adenocarcinoma and that we had flagged as potential markers for non-invasive detection. We also used two repeat probes as indicators of global DNA hypomethylation. We examined DNA methylation in 249 tissue samples from 93 subjects, spanning the putative spectrum of peripheral lung adenocarcinoma development: histologically normal adjacent non-tumor lung, atypical adenomatous hyperplasia (AAH), adenocarcinoma in situ (AIS, formerly known as bronchioloalveolar carcinoma), and invasive lung adenocarcinoma. Comparison of DNA methylation levels between the lesion types suggests that DNA hypermethylation of distinct loci occurs at different time points during the development of lung adenocarcinoma. DNA methylation at CDKN2A ex2 and PTPRN2 is already significantly elevated in AAH, while CpG islands at 2C35, EYA4, HOXA1, HOXA11, NEUROD1, NEUROD2 and TMEFF2 are significantly hypermethylated in AIS. In contrast, hypermethylation at CDH13, CDX2, OPCML, RASSF1, SFRP1 and TWIST1 and global DNA hypomethylation appear to be present predominantly in invasive cancer. CONCLUSIONS/SIGNIFICANCE:The gradual increase in DNA methylation seen for numerous loci in progressively more transformed lesions supports the model in which AAH and AIS are sequential stages in the development of lung adenocarcinoma. The demarcation of DNA methylation changes characteristic for AAH, AIS and adenocarcinoma begins to lay out a possible roadmap for aberrant DNA methylation events in tumor development. In addition, it identifies which DNA methylation changes might be used as molecular markers for the detection of preinvasive lesions

The Sulfotransferase SULT1C2 Is Epigenetically Activated and Transcriptionally Induced by Tobacco Exposure and Is Associated with Patient Outcome in Lung Adenocarcinoma

Lung cancer is the leading cause of cancer-related death. Tobacco exposure is associated with 80&ndash;90% of lung cancer cases. The SULT1C2 sulfotransferase modifies xenobiotic compounds to enhance secretion but can also render these compounds carcinogenic. To determine if SULT1C2 contributes to tobacco-related carcinogenesis in the lung, we analyzed the expression and epigenetic state of SULT1C2 in human lung adenocarcinoma (LUAD) samples and in LUAD cell lines exposed to cigarette smoke condensate (CSC). SULT1C2 expression was significantly positively correlated to overall LUAD patient survival in smokers, was elevated in LUAD tumors compared to adjacent non-tumor lung, and was significantly correlated with levels of patient exposure to tobacco smoke. SULT1C2 promoter DNA methylation was inversely correlated with expression in LUAD, and hypomethylation of the SULT1C2 promoter was observed in Asian patients, as compared to Caucasians. In vitro analysis of LUAD cell lines indicates that CSC stimulates expression of SULT1C2 in a dose-dependent and cell-line-specific manner. In vitro methylation of the SULT1C2 promoter significantly decreased transcriptional activity of a reporter plasmid, and SULT1C2 expression was activated by the DNA demethylating agent 5-Aza-2&prime;-deoxycytidine in a cell line in which the SULT1C2 promoter was hypermethylated. An aryl hydrocarbon receptor (AHR) binding site was detected spanning critical methylation sites upstream of SULT1C2. CSC exposure significantly increased AHR binding to this predicted binding site in the SULT1C2 promoter in multiple lung cell lines. Our data suggest that CSC exposure leads to activation of the AHR transcription factor, increased binding to the SULT1C2 promoter, and upregulation of SULT1C2 expression and that this process is inhibited by DNA methylation at the SULT1C2 locus. Additionally, our results suggest that the level of SULT1C2 promoter methylation and gene expression in normal lung varies depending on the race of the patient, which could in part reflect the molecular mechanisms of racial disparities seen in lung cellular responses to cigarette smoke exposure