Genome-Wide Hypomethylation in Head and Neck Cancer Is More Pronounced in HPV-Negative Tumors and Is Associated with Genomic Instability

Loss of genome-wide methylation is a common feature of cancer, and the degree of hypomethylation has been correlated with genomic instability. Global methylation of repetitive elements possibly arose as a defense mechanism against parasitic DNA elements, including retrotransposons and viral pathogens. Given the alterations of global methylation in both viral infection and cancer, we examined genome-wide methylation levels in head and neck squamous cell carcinoma (HNSCC), a cancer causally associated with human papilloma virus (HPV). We assayed global hypomethylation levels in 26 HNSCC samples, compared with their matched normal adjacent tissue, using Pyrosequencing-based methylation assays for LINE repeats. In addition, we examined cell lines derived from a variety of solid tumors for LINE and SINE (Alu) repeats. The degree of LINE and Alu hypomethylation varied among different cancer cell lines. There was only moderate correlation between LINE and Alu methylation levels, with the range of variation in methylation levels being greater for the LINE elements. LINE hypomethylation was more pronounced in HPV-negative than in HPV-positive tumors. Moreover, genomic instability, as measured by genome-wide loss-of-heterozygosity (LOH) single nucleotide polymorphism (SNP) analysis, was greater in HNSCC samples with more pronounced LINE hypomethylation. Global hypomethylation was variable in HNSCC. Its correlation with both HPV status and degree of LOH as a surrogate for genomic instability may reflect alternative oncogenic pathways in HPV-positive versus HPV-negative tumors

LINE-1 Hypomethylation in Cancer Is Highly Variable and Inversely Correlated with Microsatellite Instability

BACKGROUND: Alterations in DNA methylation in cancer include global hypomethylation and gene-specific hypermethylation. It is not clear whether these two epigenetic errors are mechanistically linked or occur independently. This study was performed to determine the relationship between DNA hypomethylation, hypermethylation and microsatellite instability in cancer. METHODOLOGY/PRINCIPAL FINDINGS: We examined 61 cancer cell lines and 60 colorectal carcinomas and their adjacent tissues using LINE-1 bisulfite-PCR as a surrogate for global demethylation. Colorectal carcinomas with sporadic microsatellite instability (MSI), most of which are due to a CpG island methylation phenotype (CIMP) and associated MLH1 promoter methylation, showed in average no difference in LINE-1 methylation between normal adjacent and cancer tissues. Interestingly, some tumor samples in this group showed increase in LINE-1 methylation. In contrast, MSI-showed a significant decrease in LINE-1 methylation between normal adjacent and cancer tissues (P<0.001). Microarray analysis of repetitive element methylation confirmed this observation and showed a high degree of variability in hypomethylation between samples. Additionally, unsupervised hierarchical clustering identified a group of highly hypomethylated tumors, composed mostly of tumors without microsatellite instability. We extended LINE-1 analysis to cancer cell lines from different tissues and found that 50/61 were hypomethylated compared to peripheral blood lymphocytes and normal colon mucosa. Interestingly, these cancer cell lines also exhibited a large variation in demethylation, which was tissue-specific and thus unlikely to be resultant from a stochastic process. CONCLUSION/SIGNIFICANCE: Global hypomethylation is partially reversed in cancers with microsatellite instability and also shows high variability in cancer, which may reflect alternative progression pathways in cancer

Cyclin A1 promoter hypermethylation in human papillomavirus-associated cervical cancer

BACKGROUND: The aim of this study was to evaluate epigenetic status of cyclin A1 in human papillomavirus-associated cervical cancer. Y. Tokumaru et al., Cancer Res 64, 5982-7 (Sep 1, 2004)demonstrated in head and neck squamous-cell cancer an inverse correlation between cyclin A1 promoter hypermethylation and TP53 mutation. Human papillomavirus-associated cervical cancer, however, is deprived of TP53 function by a different mechanism. Therefore, it was of interest to investigate the epigenetic alterations during multistep cervical cancer development. METHODS: In this study, we performed duplex methylation-specific PCR and reverse transcriptase PCR on several cervical cancer cell lines and microdissected cervical cancers. Furthermore, the incidence of cyclin A1 methylation was studied in 43 samples of white blood cells, 25 normal cervices, and 24, 5 and 30 human papillomavirus-associated premalignant, microinvasive and invasive cervical lesions, respectively. RESULTS: We demonstrated cyclin A1 methylation to be commonly found in cervical cancer, both in vitro and in vivo, with its physiological role being to decrease gene expression. More important, this study demonstrated that not only is cyclin A1 promoter hypermethylation strikingly common in cervical cancer, but is also specific to the invasive phenotype in comparison with other histopathological stages during multistep carcinogenesis. None of the normal cells and low-grade squamous intraepithelial lesions exhibited methylation. In contrast, 36.6%, 60% and 93.3% of high-grade squamous intraepithelial lesions, microinvasive and invasive cancers, respectively, showed methylation. CONCLUSION: This methylation study indicated that cyclin A1 is a potential tumor marker for early diagnosis of invasive cervical cancer

Hypomethylation of a LINE-1 Promoter Activates an Alternate Transcript of the MET Oncogene in Bladders with Cancer

It was recently shown that a large portion of the human transcriptome can originate from within repetitive elements, leading to ectopic expression of protein-coding genes. However the mechanism of transcriptional activation of repetitive elements has not been definitively elucidated. For the first time, we directly demonstrate that hypomethylation of retrotransposons can cause altered gene expression in humans. We also reveal that active LINE-1s switch from a tetranucleosome to dinucleosome structure, acquiring H2A.Z- and nucleosome-free regions upstream of TSSs, previously shown only at active single-copy genes. Hypomethylation of a specific LINE-1 promoter was also found to induce an alternate transcript of the MET oncogene in bladder tumors and across the entire urothelium of tumor-bearing bladders. These data show that, in addition to contributing to chromosomal instability, hypomethylation of LINE-1s can alter the functional transcriptome and plays a role not only in human disease but also in disease predisposition

Birthweight, Maternal Weight Trajectories and Global DNA Methylation of LINE-1 Repetitive Elements

Low birthweight, premature birth, intrauterine growth retardation, and maternal malnutrition have been related to an increased risk of cardiovascular disease, type 2 diabetes mellitus, obesity, and neuropsychiatric disorders later in life. Conversely, high birthweight has been linked to future risk of cancer. Global DNA methylation estimated by the methylation of repetitive sequences in the genome is an indicator of susceptibility to chronic diseases. We used data and biospecimens from an epigenetic birth cohort to explore the association between trajectories of fetal and maternal weight and LINE-1 methylation in 319 mother-child dyads. Newborns with low or high birthweight had significantly lower LINE-1 methylation levels in their cord blood compared to normal weight infants after adjusting for gestational age, sex of the child, maternal age at delivery, and maternal smoking during pregnancy (p = 0.007 and p = 0.036, respectively), but the magnitude of the difference was small. Infants born prematurely also had lower LINE-1 methylation levels in cord blood compared to term infants, and this difference, though small, was statistically significant (p = 0.004). We did not find important associations between maternal prepregnancy BMI or gestational weight gain and global methylation of the cord blood or fetal placental tissue. In conclusion, we found significant differences in cord blood LINE-1 methylation among newborns with low and high birthweight as well as among prematurely born infants. Future studies may elucidate whether chromosomal instabilities or other functional consequences of these changes contribute to the increased risk of chronic diseases among individuals with these characteristics

Hypomethylation of Intragenic LINE-1 Represses Transcription in Cancer Cells through AGO2

In human cancers, the methylation of long interspersed nuclear element -1 (LINE-1 or L1) retrotransposons is reduced. This occurs within the context of genome wide hypomethylation, and although it is common, its role is poorly understood. L1s are widely distributed both inside and outside of genes, intragenic and intergenic, respectively. Interestingly, the insertion of active full-length L1 sequences into host gene introns disrupts gene expression. Here, we evaluated if intragenic L1 hypomethylation influences their host gene expression in cancer. First, we extracted data from L1base (http://l1base.molgen.mpg.de), a database containing putatively active L1 insertions, and compared intragenic and intergenic L1 characters. We found that intragenic L1 sequences have been conserved across evolutionary time with respect to transcriptional activity and CpG dinucleotide sites for mammalian DNA methylation. Then, we compared regulated mRNA levels of cells from two different experiments available from Gene Expression Omnibus (GEO), a database repository of high throughput gene expression data, (http://www.ncbi.nlm.nih.gov/geo) by chi-square. The odds ratio of down-regulated genes between demethylated normal bronchial epithelium and lung cancer was high (p<1E−27; OR = 3.14; 95% CI = 2.54–3.88), suggesting cancer genome wide hypomethylation down-regulating gene expression. Comprehensive analysis between L1 locations and gene expression showed that expression of genes containing L1s had a significantly higher likelihood to be repressed in cancer and hypomethylated normal cells. In contrast, many mRNAs derived from genes containing L1s are elevated in Argonaute 2 (AGO2 or EIF2C2)-depleted cells. Hypomethylated L1s increase L1 mRNA levels. Finally, we found that AGO2 targets intronic L1 pre-mRNA complexes and represses cancer genes. These findings represent one of the mechanisms of cancer genome wide hypomethylation altering gene expression. Hypomethylated intragenic L1s are a nuclear siRNA mediated cis-regulatory element that can repress genes. This epigenetic regulation of retrotransposons likely influences many aspects of genomic biology

Correlation of Global and Gene-Specific DNA Methylation in Maternal-Infant Pairs

The inheritance of DNA methylation patterns is a popular theory to explain the influence of parental genetic and environmental factors on the phenotype of their offspring but few studies have examined this relationship in humans. Using 120 paired maternal-umbilical cord blood samples randomly selected from a prospective birth cohort in Bangladesh, we quantified DNA methylation by pyrosequencing seven CpG positions in the promoter region of p16, four CpG positions in the promoter region of p53, LINE-1 and Alu. Positive correlations were observed between maternal and umbilical cord blood at p16, LINE-1, and Alu but not p53. Multiple linear regression models observed a significant association between maternal and umbilical cord blood at LINE-1 and Alu (LINE-1: β = 0.63, p<0.0001; Alu: β = 0.28, p = 0.009). After adjusting for multiple comparisons, maternal methylation of p16 at position 4 significantly predicted methylation at the same position in umbilical cord blood (β = 0.43, p = <0.0001). These models explained 48%, 5% and 16% of the observed variability in umbilical cord %5mC for LINE-1, Alu and p16 at position 4, respectively. These results suggest that DNA methylation in maternal blood was correlated with her offspring at LINE-1, Alu, and p16 but not p53. Additional studies are needed to confirm whether these observed associations were due to the inheritance of epigenetic events or the shared environment between mother and fetus. Future studies should also use a multi-generational family-based design that would quantify both maternal and paternal contributions to DNA methylation in offspring across more than one generation

Global Methylation Patterns in Idiopathic Pulmonary Fibrosis

BACKGROUND: Idiopathic Pulmonary Fibrosis (IPF) is characterized by profound changes in the lung phenotype including excessive extracellular matrix deposition, myofibroblast foci, alveolar epithelial cell hyperplasia and extensive remodeling. The role of epigenetic changes in determining the lung phenotype in IPF is unknown. In this study we determine whether IPF lungs exhibit an altered global methylation profile.\ud \ud METHODOLOGY/PRINCIPAL FINDINGS: Immunoprecipitated methylated DNA from 12 IPF lungs, 10 lung adenocarcinomas and 10 normal histology lungs was hybridized to Agilent human CpG Islands Microarrays and data analysis was performed using BRB-Array Tools and DAVID Bioinformatics Resources software packages. Array results were validated using the EpiTYPER MassARRAY platform for 3 CpG islands. 625 CpG islands were differentially methylated between IPF and control lungs with an estimated False Discovery Rate less than 5%. The genes associated with the differentially methylated CpG islands are involved in regulation of apoptosis, morphogenesis and cellular biosynthetic processes. The expression of three genes (STK17B, STK3 and HIST1H2AH) with hypomethylated promoters was increased in IPF lungs. Comparison of IPF methylation patterns to lung cancer or control samples, revealed that IPF lungs display an intermediate methylation profile, partly similar to lung cancer and partly similar to control with 402 differentially methylated CpG islands overlapping between IPF and cancer. Despite their similarity to cancer, IPF lungs did not exhibit hypomethylation of long interspersed nuclear element 1 (LINE-1) retrotransposon while lung cancer samples did, suggesting that the global hypomethylation observed in cancer was not typical of IPF.\ud \ud CONCLUSIONS/SIGNIFICANCE: Our results provide evidence that epigenetic changes in IPF are widespread and potentially important. The partial similarity to cancer may signify similar pathogenetic mechanisms while the differences constitute IPF or cancer specific changes. Elucidating the role of these specific changes will potentially allow better understanding of the pathogenesis of IPF.\ud \u

Long interspersed nuclear element-1 hypomethylation in cancer: biology and clinical applications

Epigenetic changes in long interspersed nuclear element-1s (LINE-1s or L1s) occur early during the process of carcinogenesis. A lower methylation level (hypomethylation) of LINE-1 is common in most cancers, and the methylation level is further decreased in more advanced cancers. Consequently, several previous studies have suggested the use of LINE-1 hypomethylation levels in cancer screening, risk assessment, tumor staging, and prognostic prediction. Epigenomic changes are complex, and global hypomethylation influences LINE-1s in a generalized fashion. However, the methylation levels of some loci are dependent on their locations. The consequences of LINE-1 hypomethylation are genomic instability and alteration of gene expression. There are several mechanisms that promote both of these consequences in cis. Therefore, the methylation levels of different sets of LINE-1s may represent certain phenotypes. Furthermore, the methylation levels of specific sets of LINE-1s may indicate carcinogenesis-dependent hypomethylation. LINE-1 methylation pattern analysis can classify LINE-1s into one of three classes based on the number of methylated CpG dinucleotides. These classes include hypermethylation, partial methylation, and hypomethylation. The number of partial and hypermethylated loci, but not hypomethylated LINE-1s, is different among normal cell types. Consequently, the number of hypomethylated loci is a more promising marker than methylation level in the detection of cancer DNA. Further genome-wide studies to measure the methylation level of each LINE-1 locus may improve PCR-based methylation analysis to allow for a more specific and sensitive detection of cancer DNA or for an analysis of certain cancer phenotypes