HOX gene clusters are hotspots of de novo methylation in CpG islands of human lung adenocarcinomas

CpG island methylation results in the silencing of the associated gene and is an important step in tumorigenesis. Following a comprehensive isolation of CpG islands that were methylated in human lung adenocarcinoma, we found that in cancer cells de novo CpG island methylation generally occurred in a sporadic manner. However, some methylated CpG islands appeared to cluster in discrete chromosomal regions. In this study, we have investigated the methylation status of CpG islands located at such chromosomal loci. We have found that many CpG islands at the HOXA and HOXD loci were methylated in human lung adenocarcinoma. The de novo methylation of these CpG islands was also observed in patient's DNA from noncancerous portions of lung tissue. These results indicate the presence of specific chromosomal regions that are susceptible to de novo methylation.     

A survey of methylated candidate tumor suppressor genes in nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is a rare malignancy with unique genetic, viral and environmental characteristic that distinguishes it from other head and neck carcinomas. The clinical management of NPC remains challenging largely due to the lack of early detection strategies for this tumor. In our study, we have sought to identify novel genes involved in the pathogenesis of NPC that might provide insight into this tumor's biology and could potentially be used as biomarkers. To identify these genes, we studied the epigenetics of NPC by characterizing a panel of methylation markers. Eighteen genes were evaluated by quantitative methylation‐specific polymerase chain reaction (PCR) in cell lines as well as in tissue samples including 50 NPC tumors and 28 benign nasopharyngeal biopsies. Significance was evaluated using Fisher's exact test and quantitative values were optimized using cut off values derived from receiver–operator characteristic curves. The methylation status of AIM1, APC, CALCA, deleted in colorectal carcinomas (DCC), DLEC, deleted in liver cancer 1 (DLC1), estrogen receptor alpha (ESR), FHIT, KIF1A and PGP9.5 was significantly associated with NPC compared to controls. The sensitivity of the individual genes ranged from 26 to 66% and the specificity was above 92% for all genes except FHIT. The combination of PGP9.5, KIF1A and DLEC had a sensitivity of 84% and a specificity of 92%. Ectopic expression of DCC and DLC1 lead to decrease in colony formation and invasion properties. Our results indicate that methylation of novel biomarkers in NPC could be used to enhance early detection approaches. Additionally, our functional studies reveal previously unknown tumor suppressor roles in NPC.     

Identification of differentially methylated CpG islands in prostate cancer

Epigenetic change such as DNA methylation is one important mechanism for regulating gene expression as genetic change, such as mutation or loss of heterozygosity. Methylation of cancer-related genes has been shown to play an important role in carcinogenesis and tumor progression. Using methylated CpG island amplification (MCA)/representational difference analysis (RDA), we identified four CpG islands in neurotrophin tyrosine kinase receptor type 2 (NTRK2), Protocadherine Flamingo1 and MFPC (Methylated Fragments in Prostate Cancer) 7 and 8. Bisulfite sequencing revealed that 2 regions of NTRK2 as well as MFPC7 and MFPC8 were aberrantly methylated in prostate cancer cell lines, and COBRA showed that 48 (76.24%), 37 (58.7%) and 14 (22.2%) of 63 prostate cancer tissues were methylated, respectively, for these sites. On the other hand, none of 13 benign prostate samples were methylated, except for 1 (7.7%) with NTRK2. For NTRK2, mRNA expression was negative in prostate cancer cell lines (LNCaP and DU145) but was recovered on a methyltransferase inhibitor (5-Aza-CdR) treatment. The role of NTRK2 within NTRK remains unclear. Our results suggest that these 3 hypermethylated DNA fragments also may be markers of prostate cancer detection.     

Recent progress in the study of methylated tumor suppressor genes in gastric cancer

Gastric cancer is one of the most common malignancies and a leading cause of cancer mortality worldwide.The pathogenesis mechanisms of gastric cancer are still not fully clear.Inactivation of tumor suppressor genes and activation of oncogenes caused by genetic and epigenetic alterations are known to play significant roles in carcinogenesis.Accumulating evidence has shown that epigenetic silencing of the tumor suppressor genes,particularly caused by hypermethylation of CpG islands in promoters,is critical to carcinogenesis and metastasis.Here,we review the recent progress in the study of methylations of tumor suppressor genes involved in the pathogenesis of gastric cancer.We also briefly describe the mechanisms that induce tumor suppressor gene methylation and the status of translating these molecular mechanisms into clinical applications.     

Chronic nonsteroidal anti-inflammatory drug (NSAID) use suppresses multiple CpG islands hyper methylation (CIHM) of tumor suppressor genes in the human gastric mucosa

There have been reports showing a protective role of nonsteroidal anti‐inflammatory drugs (NSAIDs) against gastrointestinal cancers. CpG island hyper methylation (CIHM) of tumor suppressor genes is a major event in carcinogenesis. We investigated the CIHM status of non‐cancerous gastric mucosa in chronic NSAID users and non‐users and assessed the effect of NSAIDs on CIHM. Gastric mucosa samples were obtained from 51 chronic NSAID users and 180 non‐users. CIHM of p14(ARF), p16(INK4a), death‐associated protein kinase (DAP‐kinase), and E‐cadherin (CDH1) genes were determined by methylation‐specific PCR. CIHM high was defined as two or more CpG islands methylated. CIHM of p14, p16, CDH1, and CIHM high were lower in chronic NSAID users than in non‐users (p14: non‐users vs users = 32.2%vs 9.8%, P = 0.003; p16: non‐users vs users = 35.0%vs 15.7%, P = 0.02; CDH1: non‐users vs users = 36.1%vs 9.8%, P = 0.0009; CIHM high: non‐users vs users = 44.4%vs 17.6%, P = 0.0009). NSAID use was also associated with decreased number of CIHM by anova (R = –0.32, P < 0.0001). Multivariate logistic regression analysis with adjustment for sex, age, Helicobacter pylori infection, and NSAID use revealed that NSAID use was inversely correlated with all four CIHM and CIHM high as an independent factor (p14: odds ratio [OR] = 0.17, 95% confidence interval [CI] = 0.06–0.48; p16: OR = 0.32, 95% CI = 0.14–0.75; DAP‐kinase: OR = 0.45, 95% CI = 0.22–0.92; CDH1: OR = 0.18, 95% CI = 0.06–0.48; CIHM high: OR = 0.21, 95% CI = 0.09–0.49). No association was found between CIHM status and the duration or dose of NSAIDs. Chronic NSAID use suppresses CIHM in human gastric mucosa. NSAIDs may have a suppressive role against methylation‐related gastric carcinogenesis. (Cancer Sci 2009; 100: 1192–1197)     

Frequently methylated tumor suppressor genes in head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is a very aggressive cancer. In advanced stages, the patient has poor chances of receiving effective treatment, and survival rates are low. To facilitate timely diagnosis and improve treatment, elucidation of early detection markers is crucial. DNA methylation markers are particularly advantageous because DNA methylation is an early event in tumorigenesis, and the epigenetic modification, 5-methylcytosine, is a stable mark. A genome-wide screen using Restriction Landmark Genomic Scanning found a set of genes that are most commonly methylated in head and neck cancers. Five candidate genes: septin 9 (SEPT9), sodium-coupled monocarboxylate transporter 1 (SLC5A8), functional smad-suppressing element on chromosome 18 (FUSSEL18), early B-cell factor 3 (EBF3), and iroquois homeobox 1 (IRX1) were methylated in 27% to 67% of the HNSCC patient samples tested. Furthermore, approximately 50% of the methylated tumor samples shared methylation between two of the five genes (most commonly between EBF3 and IRX1), and 15% shared methylation between three of the five genes. Expression analysis revealed candidate gene down-regulation in 25% to 93% of the HNSCC samples, and 5-aza-2'-deoxycytidine treatment was able to restore expression in at least 2 of 5 HNSCC cell lines for all of the genes tested. Overexpression of the three most frequently down-regulated candidates, SLC5A8, IRX1, and EBF3, validated their tumor suppressor potential by growth curve analysis and colony formation assay. Interestingly, all of the candidates identified may be involved in the transforming growth factor beta signaling pathway, which is often disrupted in HNSCC.     

CpG island methylator phenotype involving tumor suppressor genes located on chromosome 3p in non-small cell lung cancer

CpG island methylator phenotype (CIMP) involving methylation abnormalities of tumor suppressor gene (TSG) on short arm of chromosome 3 (chromosome 3p) has not been so far epigenetically elucidated in non-small cell lung cancer (NSCLC). Using methylation-specific PCR (MSP) method, we examined methylation profiles for eight TSGs harbored in chromosome 3p in 60 NSCLC tissues and 60 paired normal tissues as well as 11 normal blood samples. CIMP positive is referred to having four or more than four synchronously methylated genes per sample. Consequently, 59 of 60 (98.3%) NSCLC presented promoter methylation of at least one gene while only one malignant tumor showed no methylation of any of eight genes. The frequency of promoter methylation for eight genes explored ranged from 12% for hMLH1 to 67% for RASSF1A given that of VHL (none) was not considered. Interestingly, CIMP+ was found in 56.7% (34/60) of NSCLC, and in 6.7% (4/60) of paired normal tissues and 0% (0/11) of normal blood samples, respectively; CIMP- was present in 43.3% (26/60) of NSCLC, 93.3% (56/60) of paired normal tissues, and 100% (11/11) of normal blood samples, respectively. The data suggest that CIMP status was significantly associated with NSCLC, paired normal tissues and normal blood samples (P<0.001). In addition, there appeared to be a significant association between CIMP status and survival prognosis of NSCLC (P=0.0166). In the present study, for the first time, we shed light on the presence of chromosome 3p-specific CIMP, which might play an important role in tumorigenesis of NSCLC.     

Role of tumor suppressor genes in transplacental lung carcinogenesis

Most human cancers involve multiple genetic changes, including activation of oncogenes such as Ki-ras-2 (Kras2) and inactivation of any one of a number of tumor suppressor genes such as p53 and members of the retinoblastoma (Rb) regulatory axis. As part of an ongoing project to determine how in utero exposure to chemical carcinogens affects the molecular pathogenesis of murine lung tumors, the p53 and p16^Cdkn2a genes were analyzed by using paraffin-embedded lung tissues from mice treated transplacentally with 3-methylcholanthrene. Single-strand conformation polymorphism analysis of exons 5–8 of the p53 gene, as well as their flanking introns, demonstrated an absence of mutations at this gene locus. However, a genetic polymorphism was identified at nt 708 in intron 4 of the DBA/2 strain of mice 5 bp downstream of a 3′ branching-point splice signal. Analysis of exons 1 and 2 of the Cdkn2a gene by single-strand conformation polymorphism and sequence analyses revealed mutations in exon 2 in 7% of the tumors examined. Tumor 23-1 exhibited a CAC→TAC transition at nt 301 (His⁷⁴→Tyr⁷⁴), and tumor 36-1 exhibited a GGG→GAG transition at nucleotide 350 (Gly⁹⁰→Glu ⁹⁰). Northern blot analysis of 14 of the larger tumors showed a marked decrease in the levels of Rb RNA expression. Immunohistochemical analysis revealed a spectrum of pRb expression, with the smaller adenomas showing moderate numbers of nuclei with heterogeneous staining for pRb in contrast with a highly reduced or near-complete absence of expression in the nuclei of larger tumors with features of adenocarcinomas. The low incidence of mutations at tumor suppressor loci suggested that inactivation of tumor suppressor genes was a late event in murine lung tumor pathogenesis. The identification of both mutations at the Cdkn2a gene locus and reduced levels of Rb expression combined with previous studies demonstrating a high incidence of mutated Kras2 alleles in these tumors implies that alterations of the Rb regulatory axis, in combination with mutation of Kras2, may be the preferred pathway for the pathogenesis of pulmonary tumors in transplacentally exposed mice. Mol. Carcinog. 21:177–184, 1998. © 1998 Wiley-Liss, Inc.     

Multiple tumor suppressor genes are increasingly methylated with age in non-neoplastic gastric epithelia

A number of tumor suppressor and tumor-related genes are silenced by promoter hypermethylation in gastric cancer. Hypermethylation is not restricted to cancer cells, but is also present in non-neoplastic cells during aging. Such age-related methylation in non-neoplastic gastric epithelia is postulated to constitute a field defect that increases the risk for development of gastric cancer. To quantitatively evaluate age-related methylation in non-neoplastic gastric epithelia, we used a fiber-type DNA microarray on which methylated and unmethylated sequence probes were mounted. After bisulfite modification, a part of the promoter CpG island of four tumor suppressor genes, lysyl oxidase (LOX), p16, RUNX3 and tazarotene-induced gene 1 (TIG1), were amplified by PCR using Cy5 end labeled primers. Methylation rates (MRs) were calculated as the ratio of the fluorescence intensity of a methylated sequence probe to the total fluorescence intensity of methylated and unmethylated probes. Non-neoplastic gastric mucosa was obtained from 24 non-cancer-bearing stomachs at autopsy. MRs ranged from 0.0% to 77.2% (mean, 15.8%) for LOX, 0.0% to 45.8% (mean, 10.0%) for p16, 0.0% to 83.8% (mean, 9.0%) for RUNX3, and 0.0% to 46.1% (mean, 6.6%) for TIG1, and significantly correlated with aging (P < 0.01). The regression curves were: y = 0.013x(2) - 0.6184x + 4.0512, R(2) = 0.5728 (P < 0.001) for LOX; y = 0.0107x(2) - 0.6055x + 5.2943, R(2) = 0.7891 (P < 0.00001) for p16; y = 0.0182x(2) - 1.2234x + 11.566, R(2) = 0.5595 (P < 0.001) for RUNX3; and y = 0.0068 x(2) - 0.3586 x + 2.4306, R(2) = 0.4670 (P < 0.01) for TIG1. Thus, our present results are consistent with the notion that age-related methylation is associated with cancer susceptibility in the elderly. Quantitative analysis of DNA methylation using DNA microarrays is a promising method for risk assessment in the development of gastric cancer.     

Hypermethylation of the large tumor suppressor genes in Japanese lung cancer

Large tumor suppressor (LATS) 1 and 2 are tumor suppressor genes implicated in the regulation of the cell cycle. The methylation statuses of the promoter regions of these genes were studied in Japanese lung cancers. The methylation statuses of the promoter regions of LATS1 and LATS2 were investigated by methylation-specific PCR. The findings were compared to clinicopathological features of lung cancer. Methylation-specific PCR showed that the LATS1 promoter region was hypermethylated in 95 out of 119 (79.8%) lung cancers. The methylation status of LATS1 was significantly associated with squamous histology (p=0.0267) and smoking status (never smoker vs. smoker; p=0.0399). LATS1-ummethylated patients harbored more EGFR mutations (p=0.0143). The LATS2 promoter region was hypermethylated in 160 out of 203 (78.8%) lung cancers. However, the methylation status had no association with the clinicopathological characteristics of the lung cancers cases. Both the LATS1 and LATS2 methylation statuses did not correlate with survival of lung cancer patients. Thus, the EGFR methylation status of the LATS genes has limited value in Japanese lung cancers.     

3株肺癌细胞中9个肿瘤相关基因的启动子甲基化状态和部分基因表达的相关性

目的对3株肺癌细胞中9个肿瘤相关基因的启动子甲基化状态和部分基因表达的相关性进行评估.方法通过甲基化特异性多聚酶链反应法(MSP)结合DNA测序来确定3株肺腺癌细胞株(A549,SH77和SPE-A-1)中9个基因的启动子CpG岛的甲基化状态:APC,CDH13,DAPK1,MGMT,MYOD1,p16INK4a,p73,RAR-β和WT1基因.同时采用半定量RT-PCR的方法来量度下列5个基因的表达:CDH13,MGMT,MYOD1,RAR-β和WT1基因.结果在9个受检基因之中,下列4个基因在三株细胞中均呈同样的甲基化谱式:仅有去甲基化:APC和DAPK1,以及兼有甲基化和去甲基化:p73和p16INK4a.而其余5个基因则表现为不同的甲基化模式.尽管,这5个基因的表达谱式大体上符合其启动子CpG岛甲基化的表达静息的原则,个别的例外亦是有的.这提示着与DNA甲基化状态无关的机制可能也会参与该基因的表达调控.结论肺癌细胞株中的肿瘤相关基因的启动子CpG岛的过甲基化可与该基因的表达状态有较高的相关性.