Delta DNMT3B variants regulate DNA methylation in a promoter-specific manner

DNA methyltransferase 3B (DNMT3B) is critical in de novo DNA methylation during development and tumorigenesis. We recently reported the identification of a DNMT3B subfamily, DeltaDNMT3B, which contains at least seven variants, resulting from alternative pre-mRNA splicing. DeltaDNMT3Bs are the predominant expression forms of DNMT3B in human lung cancer. A strong correlation was observed between the promoter methylation of RASSF1A gene but not p16 gene (both frequently inactivated by promoter methylation in lung cancer) and expression of DeltaDNMT3B4 in primary lung cancer, suggesting a role of DeltaDNMT3B in regulating promoter-specific methylation of common tumor suppressor genes in tumorigenesis. In this report, we provide first experimental evidence showing a direct involvement of DeltaDNMT3B4 in regulating RASSF1A promoter methylation in human lung cancer cells. Knockdown of DeltaDNMT3B4 expression by small interfering RNA resulted in a rapid demethylation of RASSF1A promoter and reexpression of RASSF1A mRNA but had no effect on p16 promoter in the lung cancer cells. Conversely, normal bronchial epithelial cells with stably transfected DeltaDNMT3B4 gained an increased DNA methylation in RASSF1A promoter but not p16 promoter. We conclude that promoter DNA methylation can be differentially regulated and DeltaDNMT3Bs are involved in regulation of such promoter-specific de novo DNA methylation.     

Expression changes in EZH2, but not in BMI-1, SIRT1, DNMT1 or DNMT3B are associated with DNA methylation changes in prostate cancer

The polycomb proteins BMI-1, EZH2, and SIRT1 are characteristic components of the PRC1, PRC2, and PRC4 repressor complexes, respectively, that modify chromatin. Moreover, EZH2 may influence DNA methylation by direct interaction with DNA methyltransferases. EZH2 expression increases during prostate cancer progression, whereas BMI-1 and SIRT1 are not well investigated. Like EZH2 expression, DNA methylation alterations escalate in higher stage prostate cancers, raising the question whether these epigenetic changes are related. Expression of EZH2, BMI-1, SIRT1, and the DNA methyltransferases DNMT1 and DNMT3B measured by qRT-PCR in 47 primary prostate cancers was compared to APC, ASC, GSTP1, RARB2, and RASSF1A hypermethylation and LINE-1 hypomethylation. SIRT1 and DNMT3B were overexpressed in cancerous over benign tissues, whereas BMI-1 was rather downregulated and DNMT1 significantly diminished. Nevertheless, cancers with higher DNMT1 and BMI-1 expression had worse clinical characteristics, as did those with elevated EZH2. In particular, above median DNMT1 expression predicted a worse prognosis. EZH2 and SIRT1 overexpression were well correlated with increased MKI67. Immunohistochemistry confirmed limited EZH2 and heterogeneous DNMT3B overexpression and explained the decrease in BMI-1 by pronounced heterogeneity among tumor cells. EZH2 overexpression, specifically among all factors investigated, was associated with more frequent hypermethylation, in particular of GSTP1 and RARB2, and also with LINE-1 hypomethylation. Our data reveal complex changes in the composition of polycomb repressor complexes in prostate cancer. Heterogeneously expressed BMI-1 and slightly increased EZH2 may characterize less malignant cancers, whereas more aggressive cases express both at higher levels. SIRT1 appears to be generally increased in prostate cancers. Intriguingly, our data suggest a direct influence of increased EZH2 on altered DNA methylation patterns in prostate cancer.     

Decreased DNA methylation in acute myeloid leukemia patients with DNMT3A mutations and prognostic implications of DNA methylation

We examined 79 acute myeloid leukemia (AML) patients for DNA methylation of 12 tumor suppressor genes (TSG) and 24 homeobox domain (Hox) genes, and additionally for mutations in DNMT3A gene. We observed lower levels of DNA methylation (P<0.0001) as well as smaller numbers of concurrently hypermethylated genes (P<0.0001) in patients with DNMT3A mutations. Our study of the impact of DNA methylation on prognosis in intermediate and high risk AML patients revealed a relation between higher DNA methylation and better patients' outcome. Lower DNA methylation was linked with higher relapse rates and an inferior overall survival.     

Microsatellite instability and promoter hypermethylation in colorectal cancer in India

Microsatellite instability (MSI) is an important factor in tumor development and is a hypermutable phenotype caused by the loss of DNA mismatch repair activity. It is important to identify tumors with microsatellite instability as the patients have a better prognosis and differ with response to chemotherapy. Limited data are available on the incidence of MSI in Indian colorectal cancers (CRCs). The objectives of this study were to identify the extent of MSI in Indian CRC patients below 50 years and to determine promoter methylation status of hMLH1 and hMSH2 in relation to MSI. A total of 450 patients were diagnosed with CRC, out of which 91 individuals were recruited as per Bethesda guidelines and were tested for instability by the NCI-recommended Bethesda panel (BAT25, BAT26, D2S123, D5S346, and D17S2720) using labeled primers. The fragments were separated and analyzed on a Beckman GeXP sequencer. Promoter methylation status was determined by restriction enzyme digestion and PCR. MSI (high and low) was seen in 48.4 % (44/91) of CRC patients, out of which microsatellite instability-high (MSI-H) was detected in 13.2 % (12/91) and microsatellite instability-low (MSI-L) in 35.2 % (32/91) and the rest were microsatellite stable (MSS), 51.6 % (47/91). Majority of the MSI-H tumors were adenocarcinomas (10/12), in the rectum (8/12), and moderately or poorly differentiated (12/12). Promoter hypermethylation was seen in 75 % of the MSI-H, 56.24 % of MSI-L, and only 23.4 % of MSS individuals. MSI (high and low) was associated with 48.4 % of CRC patients, and a significantly higher proportion of promoter hypermethylation of hMLH1 and hMSH2 genes was associated with instable tumors.     

Aberrant hypermethylation of OGDHL gene promoter in sporadic colorectal cancer

BackgroundAberrant methylation patterns of certain genes including tumor suppressors, a major epigenetic event, contribute mainly to tumorigenesis. Promoter CpG island methylation in Oxoglutarate dehydrogenase like (OGDHL) gene has been reported to reduce gene expression and hence apoptosis induction. This gene has been shown to be involved in colorectal cancer progression. In the present study, we investigated methylation status of OGDHL gene promoter in patients with colorectal cancer and evaluated its potential as a diagnostic biomarker.Methods and materialAfter collecting clinicopathologic data of patients, tumor and matched tumor free margin samples were obtained from 40 individuals; total genomic DNA was extracted and subjected to bisulfite modification. Methylation status of the gene promoter was studied using quantitative methylation-specific PCR method. Finally, its potential as a diagnostic biomarker was evaluated by receiver operating characteristic curve analysis.ResultsThere was not any significant correlation for clinicopathologic features including tumor stage, grade, size, and location with methylation status of OGDHL promoter. However, a significant high methylation level was observed in tumoral tissues compared with nontumoral marginal samples (P < 0.0001). Moreover, receiver operating characteristic curve analysis revealed 97.5% sensitivity and 95%, specificity for OGDHL promoter methylation in a cut off of 27.37% methylation as a biomarker for colorectal cancer.ConclusionThe promoter of OGDHL gene is hypermethylated in colorectal cancer and might be considered as a biomarker for its development.     

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.     

Predicting lung cancer by detecting aberrant promoter methylation in sputum

Despite the promise of using DNA markers for the early detection of cancer, none has proven universally applicable to the most common and lethal forms of human malignancy. Lung carcinoma, the leading cause of tumor-related death, is a key example of a cancer for which mortality could be greatly reduced through the development of sensitive molecular markers detectable at the earliest stages of disease. By increasing the sensitivity of a PCR approach to detect methylated DNA sequences, we now demonstrate that aberrant methylation of the p16 and/or O6-methyl-guanine-DNA methyltransferase promoters can be detected in DNA from sputum in 100% of patients with squamous cell lung carcinoma up to 3 years before clinical diagnosis. Moreover, the prevalence of these markers in sputum from cancer-free, high-risk subjects approximates lifetime risk for lung cancer. The use of aberrant gene methylation as a molecular marker system seems to offer a potentially powerful approach to population-based screening for the detection of lung cancer, and possibly the other common forms of human cancer.     

Use of DNA from human stools to detect aberrant CpG island methylation of genes implicated in colorectal cancer.

Hypermethylation of cytosine residues in the CpG islands of tumor suppressor genes is a key mechanism of colorectal carcinogenesis. Detection and quantification of CpG island methylation in human DNA isolated from stools might provide a novel strategy for the detection and investigation of colorectal neoplasia. To explore the feasibility of this approach, colorectal biopsies and fecal samples were obtained from 32 patients attending for colonoscopy or surgery, who were found to have adenomatous polyps, colorectal cancer, or no evidence of neoplasia. A further 18 fecal samples were obtained from healthy volunteers, with no bowel symptoms. Isolated DNA was modified with sodium bisulfite and analyzed by methylation-specific PCR and combined bisulfite restriction analysis for CpG island methylation of ESR1, MGMT, HPP1, p16(INK4a), APC, and MLH1. CpG island methylation was readily detectable in both mucosal and fecal DNA with methylation-specific PCR. Using combined bisulfite restriction analysis, it was established that, in volunteers from whom biopsies were available, the levels of methylation at two CpG sites within ESR1 assayed using fecal DNA were significantly correlated with methylation in DNA from colorectal mucosa. Thus, noninvasive techniques can be used to obtain quantitative information about the level of CpG island methylation in human colorectal mucosa. The methods described here could be applied to a much expanded range of genes and may be valuable both for screening purposes and to provide greater insight into the functional consequences of epigenetic changes in the colorectal mucosa of free-living individuals.     

An association of DNMT3b protein expression with P16INK4a promoter hypermethylation in non-smoking female lung cancer with human papillomavirus infection

Our recent report indicated that HPV infection may be associated with an increased frequency of p16INK4a promoter hypermethylation to cause p16 inactivation. In this study, we further speculated that the HPV infection may be linked with the expression of DNA methyltransferase (DNMT) protein in lung cancer patients and it was observed that an association of p16INK4a promoter hypermethylation with HPV infection existed, but only in female cases (P<0.0001). Interestingly, DNMT3b protein expression was significantly correlated with p16INK4a promoter hypermethylation (P=0.023) and HPV 16/18 infections (P<0.001), respectively. Moreover, the correlation between p16INK4a promoter hypermethylation and DNMT3b protein expression was exclusively seen in female cases (P=0.035). These results strongly suggested that the involvement of HPV infection in nonsmoking female lung tumorigenesis may be mediated, at least to a certain extent, through the increase of DNMT3b protein expression to cause p16INK4a promoter hypermethylation.     

Genome-wide analysis of aberrant DNA methylation for identification of potential biomarkers in colorectal cancer patients

Background: Colorectal cancer is one of the leading causes of mortality worldwide. Genome wide analysisstudies have identified sequence mutations causing loss-of-function that are associated with disease occurrence andseverity. Epigenetic modifications, such DNA methylation, have also been implicated in many cancers but haveyet to be examined in the East Asian population of colorectal cancer patients. Methods: Biopsies of tumors andmatched non-cancerous tissue types were obtained and genomic DNA was isolated and subjected to the bisulphiteconversion method for comparative DNA methylation analysis on the Illumina Infinium HumanMethylation27BeadChip. Results: Totals of 258 and 74 genes were found to be hyper- and hypo-methylated as compared tothe individual’s matched control tissue. Interestingly, three genes that exhibited hypermethylation in theirpromoter regions, CMTM2, ECRG4, and SH3GL3, were shown to be significantly associated with colorectalcancer in previous studies. Using heatmap cluster analysis, eight hypermethylated and 10 hypomethylated geneswere identified as significantly differentially methylated genes in the tumour tissues. Conclusions: Genome-widemethylation profiling facilitates rapid and simultaneous analysis of cancerous cells which may help to identifymethylation markers with high sensitivity and specificity for diagnosis and prognosis. Our results show thepromise of the microarray technology in identification of potential methylation biomarkers for colorectal cancers.     

Association of aberrant DNA methylation with clinicopathological features in breast cancer

Aberrant DNA methylation is responsible for the epigenetic silencing of genes associated with tumourigenesis and progression of cancer. In this study, we assessed the methylation status of eight genes in 49 snap-frozen primary breast tumours. Epigenetic alterations of 8 genes were analysed with methylation-specific polymerase chain reaction (MS-PCR) (DCR1, DAPK1, RASSF1A and DCR2) or methylation-sensitive high-resolution melting analysis (MS-HRM) (APC, MGMT, GSTP1 and PTEN). MS-HRM performance was validated by bisulfite pyrosequencing regarding the methylation levels of MGMT. Promoter methylation was observed in APC 54.34%, 40.4% DCR1, 37.5% DAPK1, 33.3% RASSF1A, 22.44% MGMT, 16.6% GSTP1, 6% PTEN and 0% DCR2 promoters, respectively. Interestingly, 37 out of 49 cases (75.5%) displayed aberrant promoter methylation in at least one gene. An association of MGMT promoter methylation with age and tumour grade was recorded. Moreover, a correlation with advanced T-category was elicited for GSTP1, RASSF1 and DAPK1 promoter methylation. Finally, concurrent methylation of several genes showed a marginal statistical relationship with N-category. We conclude that APC, DCR1, DAPK1 and RASSF1A promoter methylation represents a common event in breast cancer tumourigenesis. Our results suggest that GSTP1, RASSF1, DAPK1 and MGMT may be implicated in the acquisition of a more aggressive phenotype in breast cancer.     

DNA methylation markers in colorectal cancer

Colorectal cancer (CRC) arises as a consequence of the accumulation of genetic and epigenetic alterations in colonic epithelial cells during neoplastic transformation. Epigenetic modifications, particularly DNA methylation in selected gene promoters, are recognized as common molecular alterations in human tumors. Substantial efforts have been made to determine the cause and role of aberrant DNA methylation (“epigenomic instability”) in colon carcinogenesis. In the colon, aberrant DNA methylation arises in tumor-adjacent, normal-appearing mucosa. Aberrant methylation also contributes to later stages of colon carcinogenesis through simultaneous methylation in key specific genes that alter specific oncogenic pathways. Hypermethylation of several gene clusters has been termed CpG island methylator phenotype and appears to define a subgroup of colon cancer distinctly characterized by pathological, clinical, and molecular features. DNA methylation of multiple promoters may serve as a biomarker for early detection in stool and blood DNA and as a tool for monitoring patients with CRC. DNA methylation patterns may also be predictors of metastatic or aggressive CRC. Therefore, the aim of this review is to understand DNA methylation as a driving force in colorectal neoplasia and its emerging value as a molecular marker in the clinic.     

GPx3 promoter hypermethylation is a frequent event in human cancer and is associated with tumorigenesis and chemotherapy response

Glutathione peroxidase 3 (GPx3), a plasma antioxidant enzyme, maintains genomic integrity by inactivating reactive oxygen species (ROS), known DNA-damaging agents and mediators of cancer chemotherapy response. In this study, we demonstrate that loss of GPx3 expression by promoter hypermethylation is frequently observed in a wide spectrum of human malignancies. Furthermore, GPx3 methylation correlates with head and neck cancer (HNC) chemoresistance and may serve as a potential prognostic indicator for HNC patients treated with cisplatin-based chemotherapy. Our findings support the hypothesis that defects in the antioxidant system may contribute to tumorigenesis of a wide spectrum of human malignancies. GPx3 methylation may have implications in chemotherapy response and clinical outcome of HNC patients.     

胃和结直肠腺癌患者血清RASSF1A基因启动子异常甲基化检测及其临床意义

目的 研究胃和结直肠腺癌患者血清RASSF1A基因启动子区域的甲基化状态及其临床意义.方法采用甲基化特异性聚合酶链反应(MSP)技术,检测47例胃腺癌患者、45例结直肠腺癌患者、60例胃肠道良性病变患者及30例健康志愿者血清RASSF1A基因启动子区域的甲基化状态,并同时检测25例进行手术的胃腺癌、结直肠腺癌患者的肿瘤...