NORE1A,a homologue of RASSF1A tumour suppressor gene is inactivated in human cancers

We recently demonstrated that RASSF1A, a new tumour-suppressor gene located at 3p21.3 is frequently inactivated by promoter region hypermethylation in a variety of human cancers including lung, breast, kidney and neuroblastoma. We have identified another member of the RASSF1 gene family by in silico sequence analysis using BLAST searches. NORE1 located at 1q32.1 exists in three isoforms (NORE1Aalpha, NORE1Abeta and NORE1B). Both NORE1A and NORE1B isoforms have separate CpG islands spanning their first exons. NORE1Aalpha Produces a 418 aa protein containing a Ras-association (RA) domain and a diacylglycerol (DAG) binding domain. NORE1Abeta produces a C-terminal truncation of the RA domain. NORE1B also contains the RA domain but not the DAG domain. NORE1 is the human homologue of the mouse Ras effector Nore1. No inactivating somatic mutations were found in lung tumour lines; however, NORE1A promoter region CpG island was hypermethylated in primary tumours and tumour cell lines. NORE1A promoter was methylated in 10/25 breast, 4/40 SCLC, 3/17 NSCLC, 1/6 colorectal and 3/9 kidney tumour cell lines, while NORE1B promoter was unmethylated in the same tumour cell lines. While 24% (6/25) of primary NSCLC underwent NORE1A methylation, methylation in SCLC was a rare event (0/22); (P = 0.0234). NORE1A expression in tumour cell lines was reactivated after treatment with a demethylating agent. There was no correlation between NORE1A and RASSF1A methylation status in NSCLC. Our results demonstrate that NORE1A is inactivated in a subset of human cancers by CpG island promoter hypermethylation, and in lung cancer this hypermethylation may be histological type specific.     

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.     

Examination of a CpG island methylator phenotype and implications of methylation profiles in solid tumors

The CpG island methylator phenotype (CIMP), thoroughly described in colorectal cancer and to a lesser extent in other solid tumors, is important in understanding epigenetics in carcinogenesis and may be clinically useful for classification of neoplastic disease. Therefore, we investigated whether this putative phenotype exists in exposure-related solid tumors, where somatic gene alterations and enhanced clonal growth are selected for by carcinogens, and examined the ability of methylation profiles to classify malignant disease. We studied promoter hypermethylation of 16 tumor suppressor genes and 3 MINT loci (acknowledged classifiers of CIMP) in 344 bladder cancers, 346 head and neck squamous cell carcinomas (HNSCC), 146 non-small-cell lung cancer (NSCLC), and 71 malignant pleural mesotheliomas (MPM). We employed rigorous statistical methods to examine the distribution of promoter methylation and the usefulness of these profiles for disease classification. In bladder cancer, HNSCC, and NSCLC, there was a significant correlation (P < 0.0001) between methylation of the three MINT loci and methylation index, although the distribution of methylated loci varied significantly across these disease. Although there was a significant (P < 0.001) association between gene methylation profile and disease, rates of misclassification of each disease by their methylation profile ranged from 28% to 32%, depending on the classification scheme used. These data suggest that a form of CIMP exists in these solid tumors, although its etiology remains elusive. Whereas the gene profiles of hypermethylation among examined loci could not unequivocally distinguish disease type, the existence of CIMP and the relative preponderance of hypermethylation in these cancers suggest that methylation analysis may be clinically useful as a targeted screening tool.     

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.     

Discovery of novel epigenetic markers in non-Hodgkin's lymphoma

Non-Hodgkin's lymphoma (NHL) is a group of malignancies with heterogeneous genetic and epigenetic alterations. Discovery of molecular markers that better define NHL should improve diagnosis, prognosis and understanding of the biology. We developed a CpG island DNA microarray for discovery of aberrant methylation targets in cancer, and now apply this method to examine NHL cell lines and primary tumors. This methylation profiling revealed differential patterns in six cell lines originating from different subtypes of NHL. We identified 30 hypermethylated genes in these cell lines and independently confirmed 10 of them. Methylation of 6 of these genes was then further examined in 75 primary NHL specimens composed of four subtypes representing different stages of maturation. Each gene (DLC-1, PCDHGB7, CYP27B1, EFNA5, CCND1 and RARbeta2) was frequently hypermethylated in these NHLs (87, 78, 61, 53, 40 and 38%, respectively), but not in benign follicular hyperplasia. Although some genes such as DLC-1 and PCDHGB7 were methylated in the vast majority of NHLs, others were differentially methylated in specific subtypes. The methylation of the candidate tumor suppressor gene DLC-1 was detected in a high proportion of primary tumor and plasma DNA samples by using quantitative methylation-specific PCR analysis. This promoter hypermethylation inversely correlated with DLC-1 gene expression in primary NHL samples. Thus, this CpG island microarray is a powerful discovery tool to identify novel methylated genes for further studies of their relevant molecular pathways in NHLs and identification of potential epigenetic biomarkers of disease.     

CpG island arrays: an application toward deciphering epigenetic signatures of breast cancer.

CpG island hypermethylation is a frequent epigenetic event in cancer. We have recently developed an array-based method, called differential methylation hybridization (DMH), allowing for a genome-wide screening of CpG island hypermethylation in breast cancer cell lines (T. H-M. Huang et al., Hum. Mol. Genet., 8: 459-470, 1999). In the present study, DMH was applied to screen 28 paired primary breast tumor and normal samples and to determine whether patterns of specific epigenetic alterations correlate with pathological parameters in the patients analyzed. Amplicons, representing a pool of methylated CpG DNA derived from these samples, were used as hybridization probes in an array panel containing 1104 CpG island tags. Close to 9% of these tags exhibited extensive hypermethylation in the majority of breast tumors relative to their normal controls, whereas others had little or no detectable changes. Pattern analysis in a subset of CpG island tags revealed that CpG island hypermethylation is associated with histological grades of breast tumors. Poorly differentiated tumors appeared to exhibit more hypermethylated CpG islands than their moderately or well-differentiated counterparts (P = 0.041). This early finding lays the groundwork for a population-based DMH study and demonstrates the need to develop a database for examining large-scale methylation data and for associating specific epigenetic signatures with clinical parameters in breast cancer.     

CpG methylation in the Fhit regulatory region: relation to Fhit expression in murine tumors

To determine if: (1) 5' CpG island methylation is related to Fhit inactivation; (2) there are tumor or carcinogen-specific methylation patterns, we examined 35 CpG sites in the promoter, exon and intron 1 of the mouse Fhit gene. In primary tumors of lung, urinary bladder and tongue, induced by different carcinogens, 15-35% of sites were methylated, with specific methylation patterns associated with each cancer type, suggesting cancer- or tissue-specific methylation patterns. The methylation patterns were associated with reduced Fhit expression, as determined by immunohistochemical analyses. Methylation of rat Fhit 5' CpGs in mammary adenocarcinomas, detected by methylation specific PCR amplification, also correlated with reduced gene expression. Thus, there was an overall association between promoter/exon 1 methylation and decreased Fhit expression. In contrast, in cancer-derived cell lines 70-95% of the CpG sites were methylated. This is the first detailed study of the relationship between Fhit 5' CpG island methylation and Fhit expression in murine tumors, our main models for preclinical cancer studies, and provides evidence that loss of Fhit expression and methylation are correlated in these mouse models and these models will be useful to examine the complex relationships among gene expression, methylation patterns and organ specificity.     

A systematic profile of DNA methylation in human cancer cell lines

Human cancer cell lines are commonly used in basic cancer research to understand the behavior of primary tumors. Aberrations in the DNA methylation patterns are nowadays recognized as a hallmark of the cancer cell. However, no comprehensive study defines the DNA methylation environment present in the established cancer cell lines used in everyday laboratory-based research. To address this matter, we have analyzed 70 widely used human cancer cell lines of 12 different tumor types for CpG island promoter hypermethylation of 15 tumor suppressor genes, global 5-methylcytosine genomic content, chemical response to the demethylating agent 5-aza-2'-deoxycytidine, and their genetic haplotype for methyl-group metabolism genes. Several conclusions arise from our study: (a) a specific profile of CpG island hypermethylation exists for each tumor type, allowing its classification within hierarchical clusters according to the originating tissue; (b) cancer cell lines generally have higher levels of CpG island hypermethylation than primary tumors, because of the contribution of particular CpG islands and tumor types; and (c) there are no major differences between cell lines in their 5-methylcytosine DNA content, efficacy of 5-aza-2'-deoxycytidine treatment, and distribution of allelotypes of methyl-group metabolism genes. Our data provide a basis for a better use of human cancer cell lines in basic and translational research with respect to their DNA methylation environment.     

食管癌细胞系中MT-3基因CpG岛甲基化的意义

目的研究食管癌细胞系中MT-3基因CpG岛甲基化与其mRNA表达的关系。方法选取4种食管癌细胞系OE21，OE19，OE33和TE7，经重亚硫酸钠处理后，采用限制性酶切图谱分析(COBRA)和逆转录聚合酶链反应(RT-PCR)技术，在DNA甲基转移酶抑制剂5-氮2^ -脱氧胞苷(5-aza-CdR)处理前后，对其MT-3基因CpG岛的甲基化情况及mRNA的表达情况进行对比研究。结果4种食管癌细胞系的MT-3基因均存在不同程度的CpG岛超甲基化。经5-aza-CdR处理后，超甲基化状态解除，mRNA的表达水平也较处理前明显提高(P=0.002)。结论食管癌细胞系中MT-3基因CpG岛的超甲基化可抑制其mRNA表达。当其超甲基化解除后，MT-3基因的mRNA表达水平也会相应升高。