Novel DNA methylation markers with potential prognostic relevance in advanced malignant melanoma identified using COBRA assays.

Aberrant methylation of promoter regions involved in silencing of tumor suppressor genes is a key feature of many human cancers including melanoma. These DNA methylation events occur early in cancer development, increase with progression, and may therefore serve as biomarkers for the detection and staging of cancer. In our study, we used an epigenomic reactivation screening approach including Combined Bisulfite Restriction Analyses (COBRA) assays to identify novel methylation markers in late-stage melanoma. Two human xenograft melanoma models have been used to identify genes methylated in cancer and reactivated upon treatment with a histone deacetylase inhibitor. Gene expression analysis and promoter scanning for DNA methylation by COBRA assays and bisulfite sequencing were used to identify candidate genes. The methylation status of the CpG island promoter region of genes related to melanoma pathophysiology in skin, lymph node, and visceral metastatic metastases in 28 patients (samples n=35) were assessed. These methylation markers have been evaluated in melanoma metastasis tissue and in control samples from normal skin. The screening in in-vitro and in-vivo systems for methylated genes in melanoma samples showed 10 candidate genes. Using COBRA assays, we detected a methylation pattern in the promoter region of 10 genes with two genes (BASP1, CDH11), together with the patient's age and the log-S100B-level at biopsy, constructing a descriptor with a trend to correlate with shorter time to death.

Antiproliferative effects of DNA methyltransferase 3B depletion are not associated with DNA demethylation.

Silencing of genes by hypermethylation contributes to cancer progression and has been shown to occur with increased frequency at specific genomic loci. However, the precise mechanisms underlying the establishment and maintenance of aberrant methylation marks are still elusive. The de novo DNA methyltransferase 3B (DNMT3B) has been suggested to play an important role in the generation of cancer-specific methylation patterns. Previous studies have shown that a reduction of DNMT3B protein levels induces antiproliferative effects in cancer cells that were attributed to the demethylation and reactivation of tumor suppressor genes. However, methylation changes have not been analyzed in detail yet. Using RNA interference we reduced DNMT3B protein levels in colon cancer cell lines. Our results confirm that depletion of DNMT3B specifically reduced the proliferation rate of DNMT3B-overexpressing colon cancer cell lines. However, genome-scale DNA methylation profiling failed to reveal methylation changes at putative DNMT3B target genes, even in the complete absence of DNMT3B. These results show that DNMT3B is dispensable for the maintenance of aberrant DNA methylation patterns in human colon cancer cells and they have important implications for the development of targeted DNA methyltransferase inhibitors as epigenetic cancer drugs.

Epigenetic cancer therapy: Proof of concept and remaining challenges.

Over the past few years several drugs that target epigenetic modifications have shown clinical benefits, thus seemingly validating epigenetic cancer therapy. More recently, however, it has become clear that these drugs are either characterized by low specificity or that their target enzymes have low substrate specificity. As such, clinical proof-of-concept for epigenetic cancer therapies remains to be established. Human cancers are characterized by widespread changes in their genomic DNA methylation and histone modification patterns. Epigenetic cancer therapy aims to restore normal epigenetic modification patterns through the inhibition of epigenetic modifier enzymes. In this review, we provide an overview about the known functional roles of DNA methyltransferases, histone deacetylases, histone methyltransferases, and demethylases in cancer development. The available data identify several examples that warrant further consideration as drug targets. Future research should be directed toward targeted enzyme inhibition and toward exploring interactions between epigenetic pathways to maximize cancer specificity.

The human let-7a-3 locus contains an epigenetically regulated microRNA gene with oncogenic function.

MicroRNAs (miRNAs) are small noncoding RNAs that repress their target mRNAs by complementary base pairing and induction of the RNA interference pathway. It has been shown that miRNA expression can be regulated by DNA methylation and it has been suggested that altered miRNA gene methylation might contribute to human tumorigenesis. In this study, we show that the human let-7a-3 gene on chromosome 22q13.31 is associated with a CpG island. Let-7a-3 belongs to the archetypal let-7 miRNA gene family and was found to be methylated by the DNA methyltransferases DNMT1 and DNMT3B. The gene was heavily methylated in normal human tissues but hypomethylated in some lung adenocarcinomas. Let-7a-3 hypomethylation facilitated epigenetic reactivation of the gene and elevated expression of let-7a-3 in a human lung cancer cell line resulted in enhanced tumor phenotypes and oncogenic changes in transcription profiles. Our results thus identify let-7a-3 as an epigenetically regulated miRNA gene with oncogenic function and suggest that aberrant miRNA gene methylation might contribute to the human cancer epigenome.

Monitoring methylation changes in cancer.

Methylation of cytosines at their carbon-5 position plays an important role both during development and in tumorgenesis. The methylation occurs almost exclusively in CpG dinucleotides. While the bulk of human genomic DNA is depleted in CpG sites, there are CpG-rich stretches, so-called CpG islands, which are located in promoter regions of more than 70% of all known human genes. In normal cells, CpG islands are unmethylated, reflecting an transcriptionally active state of the respective gene. Epigenetic silencing of tumor suppressor genes by hypermethylation of CpG islands is a very early and stable characteristic of tumorigenesis. The detection of DNA methylation is based on a treatment of genomic DNA with sodium bisulfite, which converts only unmethylated cytosines to uracil, while methylated cytosines stay unaltered. This sequence conversion can be detected in the same way as a single nucleotide polymorphism. Even though different approaches have been established for analysing DNA methylation, so far detection methods that are capable of surveying the methylation status of multiple gene promoters have been restricted to a limited number of cytosines. The use of oligonucleotide microarrays permits the parallel analysis of the methylation status of individual cytosines on a genome-wide and gene-specific level. On the one hand, a hybridization-based setup is described employing microarrays that contain oligonucleotide probes of 17-25 bases in length reflecting the methylated as well as the unmethylated status of each CpG site. After hybridization of sodium bisulfite treated and fluorescently labeled targets, methylation status of individual CpG dinucleotides can be computed based on resulting signal intensities. Secondly, a microarray-based approach for detecting methylation-specific sequence polymorphisms via an on-chip enzymatic primer extension is described.

Allelic silencing at the tumor-suppressor locus 13q14.3 suggests an epigenetic tumor-suppressor mechanism.

Genomic material from chromosome band 13q14.3 distal to the retinoblastoma locus is recurrently lost in a variety of human neoplasms, indicating an as-yet-unidentified tumor-suppressor mechanism. No pathogenic mutations have been found in the minimally deleted region until now. However, in B cell chronic lymphocytic leukemia tumors with loss of one copy of the critical region, respective candidate tumor-suppressor genes are down-regulated by a factor >2, which would be expected by a normal gene-dosage effect. This finding points to an epigenetic pathomechanism. We find that the two copies of the critical region replicate asynchronously, suggesting differential chromatin packaging of the two copies of 13q14.3. Although we also detect monoallelic silencing of genes localized in the critical region, monoallelic expression originates from either the maternal or paternal copy, excluding an imprinting mechanism. DNA methylation analyses revealed one CpG island of the region to be methylated. DNA demethylation of this CpG island and global histone hyperacetylation induced biallelic expression, whereas replication timing was not affected. We propose that differential replication timing represents an early epigenetic mark that distinguishes the two copies of 13q14.3, resulting in differential chromatin packaging and monoallelic expression. Accordingly, deletion of the single active copy of 13q14.3 results in significant down-regulation of the candidate genes and loss of function, providing a model for the interaction of genetic lesions and epigenetic silencing at 13q14.3 in B cell chronic lymphocytic leukemia.

Tissue-specific effects of in vitro fertilization procedures on genomic cytosine methylation levels in overgrown and normal sized bovine fetuses.

Epigenetic perturbations are assumed to be responsible for phenotypic abnormalities of fetuses and offspring originating from in vitro embryo techniques. We studied 29 viable Day-80 bovine fetuses to assess the effects of two in vitro fertilization protocols (IVF1 and IVF2) on fetal phenotype and genomic cytosine methylation levels in liver, skeletal muscle, and brain. The IVF1 protocol employed 0.01 U/ml of FSH and LH in oocyte maturation medium and 5% estrous cow serum (ECS) in embryo culture medium, whereas the IVF2 protocol employed 0.2 U/ml of FSH and no LH for oocyte maturation and 10% ECS for embryo culture. Comparisons with in vivo-fertilized controls (n=14) indicated an apparently normal phenotype for IVF1 fetuses (n=5), but IVF2 fetuses (n=10) were significantly heavier (19.9%) and longer (4.7%), with increased heart (25.2%) and liver (27.9%) weights, and thus displayed an overgrowth phenotype. A clinicochemical screen of 18 plasma parameters revealed significantly increased levels of insulin-like growth factor 1 (40.8%) and creatinine (37.5%) in IVF2, but not in IVF1, fetuses. Quantification of genomic 5-methylcytosine (5mC) by capillary electrophoresis indicated that both IVF1 and IVF2 fetuses differed from controls. We observed significant DNA hypomethylation in liver and muscle of IVF1 fetuses (-16.1% and -9.3%, respectively) and significant hypermethylation in liver of IVF2 fetuses (+11.2%). The 5mC level of cerebral DNA was not affected by IVF protocol. Our data indicate that bovine IVF procedures can affect fetal genomic 5mC levels in a protocol- and tissue-specific manner and show that hepatic hypermethylation is associated with fetal overgrowth and its correlated endocrine changes.

Reactivation of epigenetically silenced genes by DNA methyltransferase inhibitors: basic concepts and clinical applications.

Hypermethylation of tumor suppressor genes is one of the most consistent hallmarks of human cancers. This epigenetic alteration has been associated with gene silencing and thus represents an important pathway for generating loss-of-function mutations. In this review, we survey the available literature on systematic, genome-wide approaches aimed at the identification of epigenetically silenced loci. These studies uncovered a variety of diverse genes, but a common signature for epigenetic reactivation has not been identified. Nevertheless, DNA methyltransferase inhibitors have shown significant clinical benefits, mostly in the therapy of leukemias. Recent analyses revealed substantial drug-induced methylation changes that can now be used as endpoints for the further refinement of clinical treatment schedules. Further optimization of epigenetic cancer therapies should be feasible through the use of novel DNA methyltransferase inhibitors with improved specificity. Rational design of epigenetic inhibitors might provide the foundation for a broader use of these drugs in the treatment of cancer.

Characterization of DNA demethylation effects induced by 5-Aza-2'-deoxycytidine in patients with myelodysplastic syndrome.

Azanucleoside drugs such as 5-azacytidine (Vidaza) and 5-aza-2'-deoxycytidine (decitabine, Dacogen) function as DNA methyltransferase inhibitors in vitro and represent promising new drugs for the treatment of myelodysplastic syndrome (MDS) and acute myeloid leukemia. In this study, we aimed to determine the effect of decitabine on the genomic methylation level in MDS patients. Comparison of different assays established micellar electrokinetic chromatography as a reliable method for the analysis of genomic methylation levels. When used for the determination of DNA methylation levels in bone marrow DNA from MDS patients during various time points of decitabine treatment, the results revealed a significant (up to 70%) demethylation in five of seven patients. Interestingly, genome-wide demethylation appeared after karyotype normalization, which suggests demethylation of nonclonal cells. Drug-induced demethylation dynamics were also confirmed by bisulfite sequencing of pericentromeric satellite elements. Our results are the first to show a genome-wide demethylating activity of decitabine in tumor material. In addition, our data uncovers novel targets of decitabine-mediated demethylation that are important for the refinement of treatment schedules with demethylating drugs.

Array-based analysis of genomic DNA methylation patterns of the tumour suppressor gene p16INK4A promoter in colon carcinoma cell lines.

Aberrant DNA methylation at CpG dinucleotides can result in epigenetic silencing of tumour suppressor genes and represents one of the earliest events in tumourigenesis. To date, however, high-throughput tools that are capable of surveying the methylation status of multiple gene promoters have been restricted to a limited number of cytosines. Here, we present an oligonucleotide microarray that permits the parallel analysis of the methylation status of individual cytosines, thus combining high throughput and high resolution. The approach was used to study the CpG island in the promoter region of the tumour suppressor gene p16(INK4A). In total, 876 oligonucleotide probes of 21 nt in length were used to inspect the methylation status of 53 CpG dinucleotides, producing correct signals in colorectal cancer cell lines as well as control samples with a defined methylation status. The information was validated by established alternative methods. The overall methylation pattern was consistent for each cell line, while different between them. At the level of individual cytosines, however, significant variations between individual cells of the same type were found, but also consistencies across the panel of cancer cell lines were observed.

Tissue-specific elevated genomic cytosine methylation levels are associated with an overgrowth phenotype of bovine fetuses derived by in vitro techniques.

Epigenetic perturbations are assumed to be responsible for abnormalities observed in fetuses and offspring derived by in vitro techniques. We have designed an experiment with bovine Day 80 fetuses generated by somatic cell nuclear transfer (SCNT), in vitro fertilization (IVF), and artificial insemination (AI) to determine the relationship between fetal phenotype and genome-wide 5-methylcytosine (5mC) content. When compared with AI controls, SCNT and IVF fetuses displayed significantly increased body weight (61% and 28%), liver weight (100% and 36%), and thorax circumference (20% and 11%). A reduced crown-rump length:thorax circumference ratio (1.175 +/- 0.017 in SCNT and 1.292 +/- 0.018 in IVF vs. 1.390 +/- 0.018 in AI, P < 0.001 and P < 0.002) was the external hallmark of this disproportionate overgrowth phenotype. The SCNT fetuses showed significant hypermethylation of liver DNA in comparison with AI controls (3.46% +/- 0.08% vs. 3.17% +/- 0.09% 5mC, P < 0.03), and the cytosine methylation levels for IVF fetuses (3.34% +/- 0.09%) were, as observed for phenotypic parameters, intermediate to the other groups. Regressions of fetal body and liver weight and thorax circumference on 5mC content of liver DNA were positive (P < 0.073-0.079). Furthermore, a significant negative regression (P < 0.021) of the crown-rump length:thorax circumference ratio on liver 5mC was observed. The 5mC content of placental cotyledon DNA was 46% lower than in liver DNA (P < 0.0001) but did not differ among groups. These data are in striking contrast with the recently reported hypomethylation of DNA from SCNT fetuses and indicate that hypermethylation of fetal tissue, but not placenta, is linked to the overgrowth phenotype in bovine SCNT and IVF fetuses.

Comparative analysis of DNA methylation patterns in transgenic Drosophila overexpressing mouse DNA methyltransferases.

DNA methyltransferases (Dnmts) mediate the epigenetic modification of eukaryotic genomes. Mammalian DNA methylation patterns are established and maintained by co-operative interactions among the Dnmt proteins Dnmt1, Dnmt3a and Dnmt3b. Owing to their simultaneous presence in mammalian cells, the activities of individual Dnmt have not yet been determined. This includes a fourth putative Dnmt, namely Dnmt2, which has failed to reveal any activity in previous assays. We have now established transgenic Drosophila strains that allow for individual overexpression of all known mouse Dnmts. Quantitative analysis of genomic cytosine methylation levels demonstrated a robust Dnmt activity for the de novo methyltransferases Dnmt3a and Dnmt3b. In addition, we also detected a weak but significant activity for Dnmt2. Subsequent methylation tract analysis by genomic bisulphite sequencing revealed that Dnmt3 enzymes preferentially methylated CpG dinucleotides in a processive manner, whereas Dnmt2 methylated isolated cytosine residues in a non-CpG dinucleotide context. Our results allow a direct comparison of the activities of mammalian Dnmts and suggest a significant functional specialization of these enzymes.