Concurrent DNA hypermethylation of multiple genes in acute myeloid leukemia.

Hypermethylation in cancer often occurs in CpG islands that span the promoter regions of tumor suppressor genes. However, it is not clear if hypermethylation is limited to single target genes or if multiple genes are simultaneously methylated. To understand the extent of aberrant de novo methylation, we have analyzed the methylation pattern of a number of tumor-related genes in leukemia from the same cohort of patients. We used bisulfite genomic sequencing to characterize the methylation pattern of the CpG islands associated with the calcitonin, estrogen receptor, E-cadherin, p15, p16, Rb, GST-Pi, and HIC1 genes in the bone marrow from 9 normal and 20 patients with acute myeloid leukaemia (AML). All of the normal control samples were essentially unmethylated for each of the eight tumor-related genes studied. In contrast, 19 of 20 (95%) of the AML patients had an abnormal methylation pattern in at least one gene, and 15 of 20 (75%) had abnormal methylation patterns in two or more of the target genes. We conclude that there is a general deregulation of CpG island methylation in leukemia and that hypermethylation is not limited to single genes, but a number of genes are methylated concurrently. Moreover, the subset of genes that are commonly methylated in leukemia appear to be cancer type specific.     

Genome-wide identification of OTP gene as a novel methylation marker of breast cancer

Aberrant DNA methylation occurs early and frequently in tumorigenesis. Identification of DNA methylation biomarkers is a field that provides potential for improving the clinical process of breast cancer diagnosis. We utilized a genome-wide technique, methylated DNA isolation assay (MeDIA), in combination with high-resolution CpG microarray analysis to identify hypermethylated genes in breast cancer. Among differentially methylated genes between tumor and adjacent normal tissues, 3 candidate genes (LHX2, WT1 and OTP) were finally selected through a step-wise filtering process and examined for methylation status in normal tissues, primary tumor, and paired adjacent normal-appearing tissues from 39 breast cancer patients. Based on the calculated cut-off values, all genes showed significantly higher frequencies of aberrant hypermethylation in primary tumors (43.6% for LHX2, 89.7% for WT1 and 100% for OTP, p<0.05) while frequencies were intermediate in paired adjacent normal tissues and absent in normal tissues. On further analysis, the methylation level in primary tumors was not significantly correlated with clinicopathological features. Interestingly, DNA methylation of a novel gene OTP was detected in adjacent normal tissues even 6?cm away from primary tumors, suggesting that OTP methylation may qualify as a biomarker for the early detection of breast cancer. In conclusion, we successfully identified a novel gene OTP frequently methylated in breast cancer by genome-wide screening. Our results suggest that the OTP gene may play a crucial role in breast carcinogenesis, although further clinical validation will be needed to evaluate the potential application of OTP in the early detection of breast cancer.     

MIRA-assisted microarray analysis, a new technology for the determination of DNA methylation patterns, identifies frequent methylation of homeodomain-containing genes in lung cancer cells

We present a straightforward and comprehensive approach for DNA methylation analysis in mammalian genomes. The methylated-CpG island recovery assay (MIRA), which is based on the high affinity of the MBD2/MBD3L1 complex for methylated DNA, has been used to detect cell type-dependent differences in DNA methylation on a microarray platform. The procedure has been verified and applied to identify a series of novel candidate lung tumor suppressor genes and potential DNA methylation markers that contain methylated CpG islands. One gene of particular interest was DLEC1, located at a commonly deleted area on chromosome 3p22-p21.3, which was frequently methylated in primary lung cancers and melanomas. Among the identified methylated genes, homeodomain-containing genes were unusually frequent (11 of the top 50 hits) and were targeted on different chromosomes. These genes included LHX2, LHX4, PAX7, HOXB13, LBX1, SIX2, HOXD3, DLX1, HOXD1, ONECUT2, and PAX9. The data show that MIRA-assisted microarray analysis has a low false-positive rate and has the capacity to catalogue methylated CpG islands on a genome-wide basis. The results support the hypothesis that cancer-associated DNA methylation events do not occur randomly throughout the genome but at least some are targeted by specific mechanisms.     

Characterizing DNA methylation patterns in pancreatic cancer genome

We performed a global methylation profiling assay on 1505 CpG sites across 807 genes to characterize DNA methylation patterns in pancreatic cancer genome. We found 289 CpG sites that were differentially methylated in normal pancreas, pancreatic tumors and cancer cell lines. We identified 23 and 35 candidate genes that are regulated by hypermethylation and hypomethylation in pancreatic cancer, respectively. We also identified candidate methylation markers that alter the expression of genes critical to gemcitabine susceptibility in pancreatic cancer. These results indicate that aberrant DNA methylation is a frequent epigenetic event in pancreatic cancer; and by using global methylation profiling assay, it is possible to identify these markers for diagnostic and therapeutic purposes in this disease.     

Phenotype-specific CpG island methylation events in a murine model of prostate cancer

Aberrant DNA methylation plays a significant role in nearly all human cancers and may contribute to disease progression to advanced phenotypes. Study of advanced prostate cancer phenotypes in the human disease is hampered by limited availability of tissues. We therefore took advantage of the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) model to study whether three different phenotypes of TRAMP tumors (PRIM, late-stage primary tumors; AIP, androgen-independent primary tumors; and MET, metastases) displayed specific patterns of CpG island hypermethylation using Restriction Landmark Genomic Scanning. Each tumor phenotype displayed numerous hypermethylation events, with the most homogeneous methylation pattern in AIP and the most heterogeneous pattern in MET. Several loci displayed a phenotype-specific methylation pattern; the most striking pattern being loci methylated at high frequency in PRIM and AIP but rarely in MET. Examination of the mRNA expression of three genes, BC058385, Goosecoid, and Neurexin 2, which exhibited nonpromoter methylation, revealed increased expression associated with downstream methylation. Only methylated samples showed mRNA expression, in which tumor phenotype was a key factor determining the level of expression. The CpG island in the human orthologue of BC058385 was methylated in human AIP but not in primary androgen-stimulated prostate cancer or benign prostate. The clinical data show a proof-of-principle that the TRAMP model can be used to identify targets of aberrant CpG island methylation relevant to human disease. In conclusion, phenotype-specific hypermethylation events were associated with the overexpression of different genes and may provide new markers of prostate tumorigenesis.     

微阵列定量检测急性白血病患者E-cadherin基因启动子区的甲基化

目的利用微阵列定量检测急性白血病患者E-cadherin基因启动子区CpG岛的甲基化改变,初步探讨基因芯片在肿瘤甲基化检测中的作用。方法用亚硫酸氢盐处理基因组DNA,并以此为模板进行PCR扩增。目的序列中未甲基化的CpG位点被翻转成TpG,而甲基化的CpG位点保持不变。设计5组探针构建一种检测急性白血病E-cadherin基因甲基化改变的微阵列。通过绘制标准曲线,定量检测样本的甲基化改变。结果利用微阵列定量检测了5例急性白血病患者的样本E-cadherin基因的甲基化改变,5个检测区域不同程度发生了甲基化,且发生高甲基化的区域一致。检测结果经过基因测序验证。结论基因芯片能够作为一种定量检测基因甲基化改变的有效工具,可用于白血病甲基化的定量研究。其较基因测序省时省力,且能实现定量和高通量检测。     

Aberrant methylation and silencing of ARHI,an imprinted tumor suppressor gene in which the function is lost in breast cancers

ARHI is a maternally imprinted tumor suppressor gene that maps to a site on chromosome 1p31 where loss of heterozygosity has been observed in 40% of human breast and ovarian cancers. ARHI is expressed in normal ovarian and breast epithelial cells, but ARHI expression is lost in a majority of ovarian and breast cancers. Expression of ARHI from the paternal allele can be down-regulated by multiple mechanisms in addition to loss of heterozygosity. This article explores the role of DNA methylation in silencing ARHI expression. There are three CpG islands in the ARHI gene. CpG islands I and II are located in the promoter region, whereas CpG island III is located in the coding region. Consistent with imprinting, we have found that all three CpG islands were partially methylated in normal human breast epithelial cells. Additional confirmation of imprinting has been obtained by studying DNA methylation and ARHI expression in murine A9 cells that carry either the maternal or the paternal copy of human chromosome 1. All three CpG islands were methylated, and ARHI was not expressed in A9 cells that contained the maternal allele. Conversely, CpG islands were not methylated and ARHI was expressed in A9 cells that contained the paternal allele of human chromosome 1. Aberrant methylation was found in several breast cancer cell lines that exhibited decreased ARHI expression. Hypermethylation was detected in 67% (6 of 9) of breast cancer cell lines at CpG island I, 33% (3 of 9) at CpG island II, and 56% (5 of 9) at CpG island III. Hypomethylation was observed in 44% (4 of 9) of breast cancer cell lines at CpG island II. When methylation of CpG islands was studied in 20 surgical specimens, hypermethylation was not observed in CpG island I, but 3 of 20 cases exhibited hypermethylation in CpG island II (15%), and 4 of 20 cases had hypermethylation in CpG island III (20%). Treatment with 5-aza-2'-deoxycytidine, a methyltransferase inhibitor, could reverse aberrant hypermethylation of CpG island I, II and III and partially restore ARHI expression in some, but not all of the cell lines. Treatment with 5-aza-2'-deoxycytidine partially reactivated ARHI expression in cell lines with hypermethylation of CpG islands I and II but not in cell lines with partial methylation or hypomethylation of these CpG islands. To test the impact of CpG island methylation on ARHI promoter activity more directly, constructs were prepared with the ARHI promoter linked to a luciferase reporter and transfected into SKBr3 and human embryo kidney 293 cells. Methylation of the entire construct destroyed promoter activity. Selective methylation of CpG island II alone or in combination with CpG island I also abolished ARHI promoter activity. Methylation of CpG I alone partially inhibited promoter activity of ARHI. Thus, hypermethylation of CpG island II in the promoter region of ARHI is associated with the complete loss of ARHI expression in breast cancer cells. Other epigenetic modifications such as hypermethylation in CpG island III may also contribute to the loss of ARHI expression.     

Identification of genes uniquely involved in frequent microsatellite instability colon carcinogenesis by expression profiling combined with epigenetic scanning

Gene silencing through CpG island hypermethylation has been associated with genesis or progression of frequent microsatellite instability (MSI-H) cancers. To identify novel methylation sites unique to MSI-H colon cancers in an unbiased fashion, we conducted a global expression profiling-based methylation target search. We identified 81 genes selectively down-regulated in MSI-H cancers using cDNA microarray analysis of 41 primary colon cancers. Forty six of these 81 genes contained CpG islands overlapping their 5'untranslated regions. Initial screening of six genes in 57 primary colon cancers detected the following gene with MSI-H cancer-specific hypermethylation: RAB32, a ras family member and A-kinase-anchoring protein, was methylated in 14 of 25 (56%) MSI-H cancers but in none of 32 non-MSI-H cancers or 23 normal colonic specimens. RAB32 hypermethylation correlated with RAB32 mRNA down-regulation and with hMLH1 hypermethylation. In addition, the protein-tyrosine phosphatase receptor type O gene, PTPRO, was frequently methylated in right-sided tumors. This methylation screening strategy should identify additional genes inactivated by epigenetic silencing in colorectal and other cancers.     

The study of aberrant methylation in cancer via restriction landmark genomic scanning

Restriction landmark genomic scanning (RLGS) has been used to study DNA methylation in cancer for nearly a decade. The strong bias of RLGS for assessing the methylation state of CpG islands genome wide makes this an attractive technique to study both hypo- and hypermethylation of regions of the genome likely to harbor genes. RLGS has been used successfully to identify regions of hypomethylation, candidate tumor suppressor genes, correlations between hypermethylation events and clinical factors, and quantification of hypermethylation in a multitude of malignancies. This review will examine the major uses of RLGS in the study of aberrant methylation in cancer and discuss the significance of some of the findings.     

DNA methylation biomarkers for lung cancer

Changes in DNA methylation patterns are an important characteristic of human cancer including lung cancer. In particular, hypermethylation of CpG islands is a signature of malignant progression. Methylated CpG islands are promising diagnostic markers for the early detection of cancer. However, the full extent and sequence context of DNA hypermethylation in lung cancer has remained unknown. We have used the methylated CpG island recovery assay and high-resolution microarray analysis to find hypermethylated CpG islands in squamous cell carcinomas (SCC) and adenocarcinomas of the lung. Each tumor contained several hundred hypermethylated CpG islands. In an initial microarray screen, 36 CpG islands were methylated in five of five (=100%) of the SCC tumors tested and 52 CpG islands were methylated in at least 75% of the adenocarcinomas tested (n = 8). Using sodium-bisulfite-based approaches, 12 CpG islands (associated with the BARHL2, EVX2, IRX2, MEIS1, MSX1, NR2E1, OC2, OSR1, OTX1, PAX6, TFAP2A, and ZNF577 genes) were confirmed to be methylated in 85% to 100% of the squamous cell carcinomas and 11 CpG islands (associated with the CHAD, DLX4, GRIK2, KCNG3, NR2E1, OSR1, OTX1, OTX2, PROX1, RUNX1, and VAX1 genes) were methylated in >80% of the adenocarcinomas. From the list of genes that were methylated in lung adenocarcinomas, we identified the gene FAT4 and found that this gene was methylated in 39% of the tumors. FAT4 is the closest mammalian homologue of the Drosophila tumor suppressor Fat which is an important component of the Hippo growth control pathway. Many of these newly discovered methylated CpG islands hold promise for becoming biomarkers for the early detection of lung cancer.