Melanie Ehrlich

DNA methylation in cancer: too much, but also too little

Cancer-associated DNA hypomethylation is as prevalent as cancer-linked hypermethylation, but these two types of epigenetic abnormalities usually seem to affect different DNA sequences. Much more of the genome is generally subject to undermethylation rather than overmethylation. Genomic hypermethylation in cancer has been observed most often in CpG islands in gene regions. In contrast, very frequent hypomethylation is seen in both highly and moderately repeated DNA sequences in cancer, including heterochromatic DNA repeats, dispersed retrotransposons, and endogenous retroviral elements. Also, unique sequences, including transcription control sequences, are often subject to cancer-associated undermethylation. The high frequency of cancer-linked DNA hypomethylation, the nature of the affected sequences, and the absence of associations with DNA hypermethylation are consistent with an independent role for DNA undermethylation in cancer formation or tumor progression. Increased karyotypic instability and activation of tumor-promoting genes by cis or trans effects, that might include altered heterochromatin-euchromatin interactions, may be important consequences of DNA hypomethylation which favor oncogenesis. The relationship of DNA hypomethylation to tumorigenesis is important to be considered in the light of cancer therapies involving decreasing DNA methylation. Inducing DNA hypomethylation may have short-term anticancer effects, but might also help speed tumor progression from cancer cells surviving the DNA demethylation chemotherapy.

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.

DNA hypomethylation in cancer cells

DNA hypomethylation was the initial epigenetic abnormality recognized in human tumors. However, for several decades after its independent discovery by two laboratories in 1983, it was often ignored as an unwelcome complication, with almost all of the attention on the hypermethylation of promoters of genes that are silenced in cancers (e.g., tumor-suppressor genes). Because it was subsequently shown that global hypomethylation of DNA in cancer was most closely associated with repeated DNA elements, cancer linked-DNA hypomethylation continued to receive rather little attention. DNA hypomethylation in cancer can no longer be considered an oddity, because recent high-resolution genome-wide studies confirm that DNA hypomethylation is the almost constant companion to hypermethylation of the genome in cancer, just usually (but not always) in different sequences. Methylation changes at individual CpG dyads in cancer can have a high degree of dependence not only on the regional context, but also on neighboring sites. DNA demethylation during carcinogenesis may involve hemimethylated dyads as intermediates, followed by spreading of the loss of methylation on both strands. In this review, active demethylation of DNA and the relationship of cancer-associated DNA hypomethylation to cancer stem cells are discussed. Evidence is accumulating for the biological significance and clinical relevance of DNA hypomethylation in cancer, and for cancer-linked demethylation and de novo methylation being highly dynamic processes.

Hypomethylation of pericentromeric DNA in breast adenocarcinomas

Drug‐induced DNA demethylation in normal human cells and inherited localized hypomethylation in mitogen‐stimulated lymphocytes from patients with a rare recessive disease (ICF: i―mmunodeficiency, c―entromeric region instability, f―acial anomalies) are associated with karyotypic instability. This chromosomal recombination is targeted to heterochromatin in the vicinity of the centromere (pericentromeric region) of human chromosome 1. Pericentromeric rearrangements in this chromosome as well as overall genomic hypomethylation are frequently observed in many kinds of cancer, including breast adenocarcinoma. We found that almost half of 25 examined breast adenocarcinomas exhibited hypomethylation in satellite 2 DNA, which is located in the long region of heterochromatin adjacent to the centromere of chromosome 1 and is normally highly methylated. One of the 19 examined non‐malignant breast tissues displaying fibrocystic changes was similarly hypomethylated in this satellite DNA. We also looked at an opposing type of methylation alteration in these cancers, namely, hypermethylation in a tumor‐suppressor gene region that is frequently hypermethylated in breast cancers. We found that increased methylation in the E‐cadherin promoter region and decreased methylation in satellite 2 DNA were often present in the same breast cancers. While hypermethylation in certain tumor‐suppressor gene regions may favor tumorigenesis by repressing transcription, demethylation of other DNA sequences may predispose to cancer‐promoting chromosomal re‐arrangements. Int. J. Cancer 77:833–838, 1998.

DNA Hypomethylation and Ovarian Cancer Biology

Hypomethylation of some portions of the genome and hypermethylation of others are very frequent in human cancer. The hypomethylation often involves satellite 2 (Sat2) DNA in the juxtacentromeric (centromere-adjacent) region of chromosome 1. In this study, we analyzed methylation in centromeric and juxtacentromeric satellite DNA in 115 ovarian cancers, 26 non-neoplastic ovarian specimens, and various normal somatic tissue standards. We found that hypomethylation of both types of satellite DNA in ovarian samples increased significantly from non-neoplastic toward cancer tissue. Furthermore, strong hypomethylation was significantly more prevalent in tumors of advanced stage or high grade. Importantly, extensive hypomethylation of Sat2 DNA in chromosome 1 was a highly significant marker of poor prognosis (relative risk for relapse, 4.1, and death, 9.4) and more informative than tumor grade or stage. Also, comparing methylation of satellite DNA and 15 5′ gene regions, which are often hypermethylated in cancer or implicated in ovarian carcinogenesis, we generally found no positive or negative association between methylation changes in satellite DNA and in the gene regions. However, hypermethylation at two loci, CDH13 (at 16q24) and RNR1 (at 13p12), was correlated strongly with lower levels of Sat2 hypomethylation. The CDH13/Sat2 epigenetic correlation was seen also in breast cancers. We conclude that satellite DNA hypomethylation is an important issue in ovarian carcinogenesis as demonstrated by: (a) an increase from non-neoplastic tissue toward ovarian cancer; (b) an increase within the ovarian cancer group toward advanced grade and stage; and (c) the finding that strong hypomethylation was an independent marker of poor prognosis.

Expression of various genes is controlled by DNA methylation during mammalian development

Despite thousands of articles about 5‐methylcytosine (m5C) residues in vertebrate DNA, there is still controversy concerning the role of genomic m5C in normal vertebrate development. Inverse correlations between expression and methylation are seen for many gene regulatory regions [Heard et al., 1997 ; Attwood et al., 2002 ; Plass and Soloway, 2002 ] although much vertebrate DNA methylation is in repeated sequences [Ehrlich et al., 1982 ]. At the heart of this debate is whether vertebrate DNA methylation has mainly a protective role in limiting expression of foreign DNA elements and endogenous transposons [Walsh and Bestor, 1999 ] or also is important in the regulation of the expression of diverse vertebrate genes involved in differentiation [Attwood et al., 2002 ]. Enough thorough studies have now been reported to show that many tissue‐ or development‐specific changes in methylation at vertebrate promoters, enhancers, or insulators regulate expression and are not simply inconsequential byproducts of expression differences. One line of evidence comes from mutants with inherited alterations in genes encoding DNA methyltransferases and from rodents or humans with somatically acquired changes in DNA methylation that illustrate the disease‐producing effects of abnormal methylation. Another type of evidence derives from studies of in vivo correlations between tissue‐specific changes in DNA methylation and gene expression coupled with experiments demonstrating cause‐and‐effect associations between DNA hyper‐ or hypomethylation and gene expression. In this review, I summarize some of the strong evidence from both types of studies. Taken together, these studies demonstrate that DNA methylation in mammals modulates expression of many genes during development, causing major changes in or important fine‐tuning of expression. Also, I discuss previously established and newly hypothesized mechanisms for this epigenetic control. J. Cell. Biochem. 88: 899–910, 2003.

DNA demethylation and pericentromeric rearrangements of chromosome 1

Rearrangements in the vicinity of the centromere of chromosome 1 are over-represented in many types of human cancer and are a characteristic feature of a rare genetic disease called ICF (immunodeficiency, centromeric heterochromatin instability, and facial anomalies). Evidence is presented that implicates DNA hypomethylation in the formation of these pericentromeric chromosomal anomalies. The DNA methylation inhibitors 5-azadeoxycytidine and 5-azacytidine, but not other tested genotoxins, induced the preferential formation of pericentromeric rearrangements of chromosome 1 at a very high frequency in a pro-B-cell line (FLEB14) and at a lower frequency in a mature B-cell line (AHH-1). These abnormal chromosomes appear identical to the diagnostic chromosomal aberrations in the ICF syndrome. A major component of the pericentromeric DNA in chromosome 1, satellite 2, was shown to be hypomethylated in an ICF B-cell line, although DNA from this cell line did not display detectable overall hypomethylation. It is hypothesized that demethylation in certain DNA regions, including in pericentromeric satellite DNA, helps lead to pericentromeric chromosomal rearrangements in lymphocytes from ICF patients and in normal lymphoblastoid cells incubated in vitro with DNA demethylating agents.

Tissue-specific differences in DNA methylation in various mammals.

The only naturally occurring modified base in vertebrate DNA is 5-methylcytosine. Using a precise high-performance liquid chromatographic analysis of DNA enzymatically digested to deoxynucleosides, we have shown that rats, mice and four types of monkey display tissue-specific as well as species-specific differences in the extent of methylation of their cytosine residues. Several similarities in the patterns of tissue-specific DNA methylation in these mammals and in the previously studied human samples were observed. Compared to most other types of DNA examined, brain and thymus DNAs were hypermethylated which suggests that this hypermethylation is a determinant or a necessary byproduct of mammalian differentiation. In all of the studied rodents and primates, the highly repeated DNA sequence fraction was more methylated than the moderately repetitive or single copy fractions. The tissue-specific differences in overall DNA methylation showed no correlation with what is known about average cell turnover rates nor with the percentage of the genome that is transcribed. Liver regeneration in the rat following partial hepatectomy did not detectably alter 5-methylcytosine levels in liver DNA. A considerable increase in the extent of methylation of total liver DNA was observed during normal development of the rat. The latter phenomenon may be due to a major change in the cellular composition of the liver.

Frequent Hypomethylation in Wilms Tumors of Pericentromeric DNA in Chromosomes 1 and 16

Rearrangements in the pericentromeric heterochromatin of chromosome 1 or 16 are often found in many types of cancers, including Wilms tumors, and have been suggested to contribute to oncogenesis or tumor progression. The oncogenic potential of these rearrangements has been ascribed to the resulting chromosome arm imbalances affecting the dosage of tumor suppressor genes or protooncogenes. Because DNA hypomethylation has been linked to rearrangements in the pericentromeric regions of chromosome 1 and 16 in two types of non-cancer cell populations, we examined methylation of normally highly methylated satellite DNA sequences in these regions in Wilms tumors. Hypomethylation was found to be frequent in juxtacentromeric (satellite 2) sequences and, especially, in centromeric (satellite alpha) sequences of chromosome 1. Hypomethylation of satellite 2 DNA of chromosome 16 showed a high degree of concordance with that of satellite 2 DNA of chromosome 1. We discuss the relationship of this satellite DNA hypomethylation in Wilms tumors to chromosome aberrations, as determined by assays for loss of heterozygosity.

Hypomethylation and hypermethylation of DNA in Wilms tumors

We quantitatively analysed hypermethylation at CpG islands in the 5′ ends of 12 genes and one non-CpG island 5′ region (MTHFR) in 31 Wilms tumors. We also determined their global genomic 5-methylcytosine content. Compared with various normal postnatal tissues, ∼40–90% of these pediatric kidney cancers were hypermethylated in four of the genes, MCJ, RASSF1A, TNFRSF12 and CALCA as determined by a quantitative bisulfite-based assay (MethyLight). Interestingly, the non-CpG island 5′ region of MTHFR was less methylated in most tumors relative to the normal tissues. By chromatographic analysis of DNA digested to deoxynucleosides, about 60% of the Wilms tumors were found to be deficient in their overall levels of DNA methylation. We also analysed expression of the three known functional DNA methyltransferase genes. No relationship was observed between global genomic 5-methylcytosine levels and relative amounts of RNA for DNA methyltransferases DNMT1, DNMT3A, and DNMT3B. Importantly, no association was seen between CpG island hypermethylation and global DNA hypomethylation in these cancers. Therefore, the overall genomic hypomethylation frequently observed in cancers is probably not just a response or a prelude to hypermethylation elsewhere in the genome. This suggests that the DNA hypomethylation contributes independently to oncogenesis or tumor progression.