肺癌患者MPDZ基因异常甲基化的研究

目的:探讨肺癌组织以及相应的外周血浆和痰液中MPDZ基因异常甲基化的情况,以及其在肺癌诊断中的作用。方法:采用甲基化特异性PCR(MSP)方法检测49例肺癌组织、20例癌旁正常组织以及相应的外周血浆和痰液中MPDZ基因甲基化发生情况。结果:49例肺癌组织中MPDZ基因甲基化发生率为67%(33/49),癌旁正常组织中的MPDZ基因甲基化发生率为0(0/20)(P=0.00)。MPDZ基因甲基化检出率与临床分期相关(P=0.039),但与患者的年龄、性别、是否吸烟、肿瘤的分化程度以及肿瘤的病理分类无显著相关(P>0.05)。33例癌组织MPDZ基因发生了甲基化的肺癌患者相应血浆的DNA标本中有25例发生了甲基化,甲基化检出率为76%(25/33);痰液标本中有18例发生了甲基化,甲基化检出率为55%(18/33)。16例癌组织MPDZ基因未甲基化的肺癌患者其对应的血浆和痰液标本未检出该基因甲基化。提示痰液和血浆标本中MPDZ基因甲基化能较好地反映肿瘤组织中该基因的甲基化状况。结论:MPDZ基因在肺癌患者的癌组织中、血浆中及痰液中均有较高比例甲基化检出率,提示MPDZ基因甲基化可能在肺癌的诊断中具有一定的价值。     

肺癌驱动基因及其靶向治疗药物

随着肿瘤分子生物学的发展,针对肺癌驱动基因的分子靶向治疗已成为晚期肺癌不可或缺的一部分。目前最成功的例子就是针对表皮生长因子受体（EGFR）和间变性淋巴瘤激酶（ALK）的靶向治疗。现在越来越多的肺癌驱动基因突变已被发现,包括ROS1基因融合、成纤维细胞生长因子1扩增、KRAS、BRAF和PIK3 CA基因突变等。明确这些基因突变的频率及临床意义,对指导肺癌的临床治疗有着重要的意义。     

IGFBP7 is a p53 target gene inactivated in human lung cancer by DNA hypermethylation

Insulin-like growth factor binding protein 7 (IGFBP7) was considered a tumor suppressor gene in lung cancer. However, the mechanism responsible for the downregulation of this gene has not yet been fully understood. In this study, we analyzed the epigenetic inactivation of IGFBP7 expression in human lung cancer. We found that 14 out of 16 lung cancer cell lines showed decreased expression of IGFBP7 compared to control cells by real-time RT-PCR, and 42 out of 90 patients (46.7%) with primary lung tumor exhibited negative staining of IGFBP7 by immunohistochemistry analysis. The IGFBP7 expression could be restored by demethylation agent 5-aza-2′-deoxycytidine (DAC) in 7 cancer cell lines. Methylation status of IGFBP7 was further evaluated by bisulfite sequencing (BS) and methylation-specific-PCR (MSP). It turned out that low expression of IGFBP7 was associated with DNA methylation in lung cancer cell lines and in primary lung tumors (P = 0.019). To explore the regulatory role of p53 on IGFBP7, we transfected a wild type p53 expression vector into lung cancer cell lines H1299, H2228, and H82. Forced expression of p53 increased IGFBP7 expression only in H82 harboring no IGFBP7 methylation, while transfection in combination with DAC induced the expression of IGFBP7 in H1299 and H2228, in which IGFBP7 was methylated. Additionally, treatment with p53 inducer adriamycin (ADR) alone or in combination with DAC increased the expression of IGFBP7 in the 3 cell lines. Our data suggest that IGFBP7 is inactivated in lung cancer by DNA hypermethylation in both lung cancer cell lines and primary lung tumors, and IGFBP7 might be regulated by p53 in lung cancer cells.     

Differential DNA hypermethylation of critical genes mediates the stage-specific tobacco smoke-induced neoplastic progression of lung cancer.

Promoter DNA methylation status of six genes in samples derived from 27 bronchial epithelial cells and matching blood samples from 22 former/current smokers and five nonsmokers as well as 49 primary non-small cell lung cancer samples with corresponding blood controls was determined using methylation-specific PCR (MSP). Lung tumor tissues showed a significantly higher frequency of promoter DNA methylation in p16, MGMT, and DAPK (P < 0.05; Fisher's exact test). p16 promoter DNA methylation in tumors was observed at consistently higher levels when compared with all the other samples analyzed (P = 0.001; Fisher's exact test). ECAD and DAPK exhibited statistically insignificant differences in their levels of DNA methylation among the tumors and bronchial epithelial cells from the smokers. Interestingly, similar levels of methylation were observed in bronchial epithelial cells and corresponding blood from smokers for all four genes (ECAD, p16, MGMT, and DAPK) that showed smoking/lung cancer-associated methylation changes. In summary, our data suggest that targeted DNA methylation silencing of ECAD and DAPK occurs in the early stages and that of p16 and MGMT in the later stages of lung cancer progression. We also provide preliminary evidence that peripheral lymphocytes could potentially be used as a surrogate for bronchial epithelial cells to detect altered DNA methylation in smokers.     

Promoter hypermethylation of hallmark cancer genes in atypical adenomatous hyperplasia of the lung.

PURPOSE: According to current models of tumorigenesis, the progression of phenotypic changes culminating in overtly malignant carcinoma is driven by genetic and epigenetic alterations. The recognition of an early form of glandular neoplasia termed atypical adenomatous hyperplasia (AAH), a precursor lesion from which lung adenocarcinomas arise, provides an opportunity for characterizing early epigenetic alterations involved in lung tumorigenesis. EXPERIMENTAL DESIGN: We evaluated AAHs, adjacent normal lung tissue, and synchronous lung adenocarcinomas for promoter hypermethylation of genes implicated in lung tumorigenesis (p16, TIMP3, DAPK, MGMT, RARbeta, RASSF1A, and hTERT). RESULTS: For individual genes and the number of genes methylated, we observed a significant increase in the frequency of promoter hypermethylation in the histologic progression from normal to AAH, with low-grade or high-grade atypia, and finally to adenocarcinoma (P(trend) 相似文献   

Promoter hypermethylation of multiple genes in sputum precedes lung cancer incidence in a high-risk cohort

A sensitive screening approach for lung cancer could markedly reduce the high mortality rate for this disease. Previous studies have shown that methylation of gene promoters is present in exfoliated cells within sputum prior to lung cancer diagnosis. The purpose of the current study is to conduct a nested case-control study of incident lung cancer cases from an extremely high-risk cohort for evaluating promoter methylation of 14 genes in sputum. Controls (n = 92) were cohort members matched to cases (n = 98) by gender, age, and month of enrollment. The comparison of proximal sputum collected within 18 months to >18 months prior to diagnosis showed that the prevalence for methylation of gene promoters increased as the time to lung cancer diagnosis decreased. Six of 14 genes were associated with a >50% increased lung cancer risk. The concomitant methylation of three or more of these six genes was associated with a 6.5-fold increased risk and a sensitivity and specificity of 64%. This is the first study to prospectively examine a large panel of genes for their ability to predict lung cancer and shows the promise of gene promoter hypermethylation in sputum as a molecular marker for identifying people at high risk for cancer incidence.     

HOXA11 hypermethylation is associated with progression of non-small cell lung cancer

This study was aimed at understanding the functional significance of HOXA11 hypermethylation in non-small cell lung cancer (NSCLC). HOXA11 hypermethylation was characterized in six lung cancer cell lines, and its clinical significance was analyzed using formalin-fixed paraffin-embedded tissues from 317 NSCLC patients, and Ki-67 expression was analyzed using immunohistochemistry. The promoter region of HOXA11 was highly methylated in six lung cancer cell lines, but not in normal bronchial epithelial cells. The loss of expression was restored by treatment of the cells with a demethylating agent, 5-aza-2''-deoxycytidine (5-Aza-dC). Transient transfection of HOXA11 into H23 lung cancer cells resulted in the inhibition of cell migration and proliferation. HOXA11 hypermethylation was found in 218 (69%) of 317 primary NSCLCs. HOXA11 hypermethylation was found at a higher prevalence in squamous cell carcinoma than in adenocarcinoma (74% vs. 63%, respectively). HOXA11 hypermethylation was associated with Ki-67 proliferation index (P = 0.03) and pT stage (P = 0.002), but not with patient survival. Patients with pT2 and pT3 stages were 1.85 times (95% confidence interval [CI] = 1.04-3.29; P = 0.04) and 5.47 times (95% CI = 1.18-25.50; P = 0.01), respectively, more likely to show HOXA11 hypermethylation than those with pT1 stage, after adjusting for age, sex, and histology. In conclusion, the present study suggests that HOXA11 hypermethylation may contribute to the progression of NSCLC by promoting cell proliferation or migration.     

肺癌患者外周血血浆中p16基因异常甲基化的检测

目的 分析肺癌患者外周血血浆中p16基因启动子区异常甲基化发生情况,探讨血浆中p16基因异常改变作为肺癌临床辅助诊断分子生物学标志物的可能性。方法 利用半巢式甲基化特异性PCR技术,检测了137例肺癌患者血浆和112例相对应肿瘤组织DNA p16基因的异常甲基化情况。结果 在血浆和肿瘤组织中的p16基因甲基化检出率分别为75．2％和80．4％;其中鳞癌、腺癌、腺鳞癌和小细胞肺癌患者血浆标本的阳性率分别为77．9％,65．1％,75．1％和91．7％。血浆DNAp16基因甲基化仅在肿瘤组织存在同样甲基化的病例中被检出。血浆和肿瘤组织中,p16基因的甲基化异常改变与肿瘤的分期、分型无明显相关性。结论 分析血浆DNA的p16基因异常甲基化有可能成为辅助肺癌诊断的有效方法之一。     

Gene-promoter hypermethylation as a biomarker in lung cancer

Silencing of genes by aberrant promoter hypermethylation is now recognized as a crucial component in cancer initiation and progression. Highly sensitive assays have been developed to assess gene-promoter methylation in biological fluids. The detection of methylated genes in sputum could lead to the development of a screening test to non-invasively identify early cancer in high-risk people.     

miR-152 is a tumor suppressor microRNA that is silenced by DNA hypermethylation in endometrial cancer

The etiology and development of human cancers that remain little understood might be enlightened by defining tumor suppressor microRNAs (TS-miRNA). In this study, we identified TS-miRNAs silenced by aberrant DNA hypermethylation in endometrial cancer. Functional screening of 327 synthetic miRNAs in an endometrial cancer cell proliferation assay identified 103 miRNAs that inhibited cell growth. We then determined the sequence, DNA methylation status, and expression levels of these miRNAs in endometrial cancer cell lines and primary tumors. These determinations led to the identification of miR-152 as a candidate TS-miRNA gene in endometrial cancer. Epigenetic silencing documented in miR-152 was consistent with its location at 17q21.32 in intron 1 of the COPZ2 gene, which is also silenced often in endometrial cancer by DNA hypermethylation, and also with evidence that miR-152 targets the DNA methyltransferase DNMT1. Notably, restoration of miR-152 expression in endometrial cancer cell lines was sufficient to inhibit tumor cell growth in vitro and in vivo. We identified E2F3, MET, and Rictor as novel candidate targets of miR-152, suggesting how its epigenetic silencing can drive endometrial carcinogenesis. Our findings define a central role for miR-152 in endometrial cancer, and they also suggest its use in new therapeutic strategies to treat this cancer.     

Relationship of the aberrant DNA hypermethylation of cancer-related genes with carcinogenesis of endometrial cancer

Epigenetic abnormalities including the aberrant DNA hypermethylation of the promoter CpG islands play a key role in the mechanism of gene inactivation in cell carcinogenesis. To identify the genes associated with aberrant DNA hypermethylation in endometrial carcinogenesis, we studied the hypermethylation of the promoter regions of five genes: hMLH1, APC, E-cadherin, RAR-beta and p16. The frequencies of aberrant hypermethylation were 40.4% (21/52) in hMLH1, 22% (11/50) in APC, 14% (7/50) in E-cadherin, and 2.3% (1/44) in RAR-beta in endometrial cancer specimens. No aberrant DNA methylation was found in p16. In atypical endometrial hyperplasia, the frequencies of aberrant methylation were 14.3% (2/14) in hMLH1 and 7.3% (1/14) in APC, whereas normal endometrial cells showed no aberrant hypermethylation of any of the five genes. The high frequencies of the aberrant DNA hypermethylation of hMLH1, APC and E-cadherin suggest that the methylation of the DNA mismatch repair and Wnt signal-related genes may be associated with endometrial carcinogenesis.     

Chromate exposure induces DNA hypermethylation of the mismatch repair gene MLH1 in lung cancer

Hexavalent chromium is recognized as a human carcinogen. Our previous studies revealed that lung cancer (LC) in chromate-exposed workers (chromate LC) had molecular features of frequent microsatellite instability (MSI), repression of MLH1 level, and aberrant DNA methylation of several tumor-suppressor genes, including MLH1. In the present study, we quantitatively investigated MLH1-promoter methylation status using bisulfite pyrosequencing of paired tumorous/nontumorous tissues from chromate and nonchromate LCs to determine the effect of chromate exposure on MLH1-promoter methylation. The methylation level of MLH1 promoter was significantly higher in chromate LC tumors (P < .001) than nonchromate LC tumors and, among chromate LC, significantly higher in tumorous tissue than nontumorous tissue (P = .004). Moreover, the methylation level of MLH1 promoter in normal lung tissue tended to be higher in chromate LC than nonchromate LC (P = .062). In addition, LC with reduced levels of MLH1 showed significantly higher methylation levels of MLH1 promoter than LC exhibiting normal MLH1 levels (P = .019). Moreover, immunohistochemical analyses determined that levels of SUV39H1, an H3K9me2-related methyltransferase, were higher in chromate LC than nonchromate LC (P = .076). Furthermore, we evaluated three DNA double-strand break-repair genes (MRE11, RAD50, and DNA-PKcs) as possible targets of MSI by fragment-length polymorphism analysis, revealing the mutation frequency of RAD50 as significantly higher in chromate LC than nonchromate LC (P = .047). These results suggest that chromate exposure might induce MLH1 hypermethylation in LC as a mechanism of chromate-induced carcinogenesis.     

Analysis of RASSF1A promoter hypermethylation in serum DNA of non-small cell lung cancer

OBJECTIVE: To detect the hypermethylation status of RASSF1A promoter in serum DNA of non-small cell lung cancer (NSCLC) patient and evaluate its correlation with clinicopathological parameters. METHODS: Serum DNA was extracted from the peripheral blood of 75 NSCLC patients and another 35 patients with benign pulmonary disease and 15 healthy donors. The methylation status of RASSF1A promoter was determined using methylation-specific PCR (MSP), and the correlation of methylation profiles with clinicopathological parameters was statistically analyzed. RESULTS: Aberrant methylation of RASSF1A was detected in 23 of 75 (30.7%) cancer patients, but in none of patients with benign pulmonary disease or in healthy donors (P <0.001). RASSF1A hypermethylation status was found to be correlated with late stage and poor differentiation (P < 0.05), but not with gender, age or histopathology in NSCLC patients. CONCLUSION: Hypermethylated RASSF1A promoter is frequently found in the serum DNA of non-small cell lung cancer patient, and RASSF1A may become a promising novel biomarker for diagnosis and prognosis prediction in lung cancer.     

Detection of p16 hypermethylation in circulating plasma DNA of non-small cell lung cancer patients

We detected tumor-associated aberrant hypermethylation of the p16 gene in plasma DNA from 105 non-small cell lung cancer (NSCLC) patients (65 squamous cell carcinoma (SCC) and 40 adenocarcinoma (ADC)) and 92 matched tumor DNA samples, using a modified semi-nested methylation-specific PCR (MSP). This technique increased the sensibility of detecting p16 hypermethylation from DNA samples in varying stages. p16 hypermethylation was present in 73.3% (77/105) of the plasma samples, and 79.3% (73/92) of the tumor samples. Among those cases with methylated p16 sequence in tumor samples, 87.7% (64/73) also demonstrated this epigenetic alteration in the corresponding plasma DNA. Only patients whose tumor cells had hypermethylated p16 gene exhibited aberrant methylation in their plasma samples. Regarding different clinical stages of SCC and ADC, the frequencies of p16 hypermethylation in plasma DNA were nearly the same as those in corresponding tumors, except for stage I ADC. Our study indicated that aberrant methylation of p16 may be an excellent biomarker for early diagnosis and follow-up of NSCLC patients, and MSP is a reliable method for these purposes.     

痰标本p16和MGMT基因甲基化对肺癌的诊断价值

目的：分析肺癌患者痰标本中p16和MGMT基因启动子区甲基化的改变情况,评价该指标作为肺癌辅助诊断分子标志物的价值。方法：运用甲基化特异性PCR技术,检测77例原发性肺癌患者痰标本和部分对应肿瘤组织(53例),以及30例正常对照者痰标本中p16和MGMT基因启动子区域的甲基化改变。结果：49例(63．6%)肺癌患者痰标本中检测到了pi6基因异常甲基化,34例(44．2%)检测到了MGMT基因异常甲基化,77例患者痰标本中2个基因中至少有1个基因出现甲基化为64例(83．1%),对肿瘤的检出灵敏度较高。长期吸烟史是影响肺鳞状细胞癌痰标本p16(P=0．001)基因启动子区甲基化的因素。随TNM分期增高,肺鳞状细胞癌患者痰标本中p16基因甲基化比例增高(P=0．021)；随TNM分期增高,肺腺癌患者痰标本MGMT基因甲基化比例增高(P=0．023)。对照组正常人痰液标本未发现p16和MGMT基因启动子区甲基化。结论：痰标本中p16和MGMT基因甲基化是临床肺癌辅助诊断的有效生物标志物之一。     

DNA hypermethylation status of multiple genes in prostate adenocarcinomas.

Multiple genetic mutations and epigenetic methylation are believed to be involved in prostate carcinogenesis, but it is not known whether these events are independent or correlated in some fashion. We therefore studied 32 prostate adenocarcinomas not only for deletions and / or mutations of multiple suspect genes, but also for aberrant DNA methylation using methylation-specific PCR (MSP). Of those genes examined, p16(INK4a), O(6)-MGMT, and GST-P were found to be the most frequently methylated (66%, 25% and 75% of cases, respectively), while methylations of p14(ARF), RB1, p21(Waf1), and p27(Kip1) were far less common (3%, 6%, 6% and 6% of cases, respectively). Methylation of O(6)-MGMT and GST-P genes was defective in about 19% of the cases and there were occasional simultaneous deletions and methylations of p14(ARF) and p16(INK4a) genes (13% and 3% of cases, respectively). In p16(INK4a), methylation occurred in the promoter region in 9% of samples and in exon 2 in 66% of tumors. Hypermethylation of O(6)-MGMT with concurrent p53 and ras gene mutations were found in 6% and 13% of specimens, respectively; among those tumors with high Gleason scores were 2 carcinomas showing hypermethylated O(6)-MGMT with G-to-A transitions in K-ras. Our results demonstrate that multiple genes of a subset common in prostate carcinomas are methylated and not infrequently show concurrent deletions. Further, there is a suggestion that specific combinations of hypermethylation and mutation correlate to tumor malignancy.