Decreased expression and aberrant methylation of Gadd45G is associated with tumor progression and poor prognosis in esophageal squamous cell carcinoma

The growth arrest DNA damage-inducible gene (Gadd45) family, which is composed of Gadd45A, Gadd45B, and Gadd45G, is involved in DNA damage response and cell growth arrest. The present study was to detect the role of Gadd45 gene family in esophageal cancer and the relationship of Gadd45G methylation to a series of pathological parameters in a large esophageal squamous cell carcinoma (ESCC) sample, in order to elucidate more information on the role of Gadd45 gene family with regard to the pathogenesis of ESCC. Frequent silencing of Gadd45G but not Gadd45A and Gadd45B were found in esophageal cancer cell lines and the silencing of Gadd45G may be reversed by 5-Aza-dC or TSA treatment in Eca109 cell line. The aberrant proximal promoter methylation of Gadd45G induces silencing of Gadd45G expression in Eca109 cell line. Gadd45A mRNA and protein expression in ESCC tumor tissues was significantly different compared to corresponding normal tissues. Decreased mRNA and protein expression of Gadd45G was observed in ESCC tumor tissues and was associated with Gadd45G proximal promoter methylation. Gadd45A or Gadd45B expression was not correlated with ESCC patients survival, while Gadd45G methylation status and protein expression were independently associated with ESCC patients’ survival. These data indicated that Gadd45G may be a functional tumor suppressor and its inactivation through proximal promoter methylation may play an important role in ESCC carcinogenesis and reactivation of Gadd45G gene may has therapeutic potential and may be used as a prognostic marker for ESCC patients.     

Hypermethylation-modulated down-regulation of CDH1 expression contributes to the progression of esophageal cancer

CDH1, a cell adhesion molecule, which plays a key role in maintaining the epithelial phenotype, is regarded as an invasion-suppressor gene in light of accumulating evidence from in vitro experiments and clinical observations. In an attempt to clarify the mechanism responsible for inactivation of this gene in carcinomas, we investigated the methylation status of the CDH1 gene 5'-CpG islands and its regulatory mechanism in the progression of esophageal squamous cell carcinoma. Real-time methylation-specific polymerase chain reaction (qMSP) and treatment with the demethylating agent 5-aza-2'-deoxycytidine (5-Aza-CdR) were conducted to analyze the methylation status at the CDH1 promoter region in the human esophageal carcinoma cell lines, EC1 and EC9706. A total of 235 invasive esophageal squamous cell carcinomas (ESCC) at stages I-IV and their corresponding normal tissue samples, were included in an immunohistochemistry study and methylation analysis of CDH1. The results demonstrate that in EC1 and EC9706 cells, the CDH1 promoter is methylated and treatment with 5-Aza-CdR restored CDH1 expression. Enhanced CDH1 expression decreased cell migration, invasion ability and increased adhesion ability. Decreased CDH1 expression was detected in 59.6% of ESCC tissues, compared with their adjacent non-neoplastic epithelia, which had a close correlation with the primary tumor status, lymph node status, distant metatasis and clinicopathologic stage. Hypermethylation at the CDH1 promoter was detected in 97.9% of 140 cases of ESCC with low CDH1 expression. The methylation of CDH1 promoters (P=0.929) was closely correlated with the lack of expression of their corresponding proteins. The Cox regression model for survival analysis showed that increases in CDH1 methylation had a greater impact on the prognosis than tumor clinical stage. These findings suggest that CDH1 gene silencing by promoter hypermethylation and the resultant reduction of CDH1 expression may play an important role in the progression of ESCC. CDH1 methylation was a significant predictor of survival in ESCC patients after surgery.     

The role of RASSF1A methylation in cancer

Tumour suppressor gene inactivation is critical to the pathogenesis of cancers; such loss of function may be mediated by irreversible processes such as gene deletion or mutation. Alternatively tumour suppressor genes may be inactivated via epigenetic processes a reversible mechanism that promises to be more amenable to treatment by therapeutic agents. The CpG dinucleotide is under-represented in the genome, but it is found in clusters within the promoters of some genes, and methylation of these CpG islands play a critical role in the control of gene expression. Inhibitors of the DNA methyltransferases DNMT1 and DNMT3b have been used in a clinical setting, these nucleotide analogues lack specificity but the side effects of low dose treatments were minimal and in 2004 Vidaza (5-azacitidine) was licensed for use in myelodysplastic syndrome. Methylation inhibitors are also entering trials in conjunction with another class of epigenetic modifiers, the histone deacetylase inhibitors and this epigenetic double bullet offers hope of improved treatment regimes. Recently there has been a plethora of reports demonstrating epigenetic inactivation of genes that play important roles in development of cancer, including Ras-association domain family of genes. Epigenetic inactivation of RASSF1A (Ras-association domain family 1, isoform A) is one of the most common molecular changes in cancer. Hypermethylation of the RASSF1A promoter CpG island silences expression of the gene in many cancers including lung, breast, prostate, glioma, neuroblastoma and kidney cancer. Several recent studies have illustrated the diagnostic and prognostic potential of RASSF1A methylation. This presents RASSF1A methylation as an attractive biomarker for early cancer detection which, for most cancers, results in improved clinical outcome. DNA methylation analysis is applicable to a range of body fluids including serum, urine, bronchioalveolar lavage and sputum. The ease with which these body fluids can be acquired negates the need for invasive procedures to obtain biopsy material. This review will discuss the feasibility of using RASSF1A methylation as a diagnostic and prognostic marker in cancer management.     

Promoter hypermethylation and reduced expression of RASSF1A are frequent molecular alterations of endometrial carcinoma.

Alterations in the regulation of the RAS-MAPK pathway are frequent in endometrial carcinoma. RASSF1A is a tumor-suppressor gene that can regulate this pathway negatively. RASSF1A has been found to be inactivated by promoter methylation in some human tumors. The aim of the study was to assess the immunohistochemical expression of RASSF1A in normal endometrium and endometrial carcinoma, and to correlate its expression with K-RAS mutations, presence of microsatellite instability, RASSF1A promoter methylation, and clinicopathological data. RASSF1A immunostaining was evaluated in one tissue microarray constructed from 80 paraffin-embedded samples of normal endometrium, and two tissue microarrays constructed with a total of 157 endometrial carcinomas (one constructed with 95 endometrial carcinomas previously evaluated for K-RAS mutations, and microsatellite instability, and another one containing 62 endometrial carcinomas that were also subjected to RASSF1A promoter methylation analysis). RASSF1A immunostaining was correlated with cell proliferation (Ki67), Cyclin D1 expression and clinicopathological data. Promoter methylation of RASSF1A was assessed by methylation-specific PCR. RASSF1A immunostaining was variable during the menstrual cycle in normal endometrium. RASSF1A expression was significantly reduced in 48% of endometrial carcinomas, particularly in tumors exhibiting microsatellite instability. RASSF1A-promoter methylation was very frequent in endometrial carcinoma (74%), and was frequently associated with reduced expression of RASSF1A. RASSF1A-promoter hypermethylation was common in advanced-stage endometrial carcinoma. The results suggest that reduced expression of RASSF1A may play a role in endometrial carcinogenesis by controlling cell proliferation and apoptosis through the MAPK-signaling pathway.     

髓母细胞瘤与幕上原始神经外胚叶肿瘤中RASSF1A基因的甲基化差异

目的通过研究髓母细胞瘤与幕上原始神经外胚叶肿瘤（SPNET）中RASSF1A基因的甲基化改变,探讨颅内原始神经外胚叶肿瘤（PNET）的不同亚型中该基因的表遗传学差异及其意义。方法收集25例原发髓母细胞瘤,9例原发SPNET,3株髓母细胞瘤细胞系和2株SPNET细胞系。采用甲基化特异性聚合酶链反应（MSP）检测RASSF1A基因启动子区的甲基化状态。应用去甲基化试剂5-aza-2’deoxycytidine处理存在基因表达缺失的细胞系,探讨基因表达与甲基化之间的关系。结果100％（25／25）的原发髓母细胞瘤、6／9的原发SPNET及全部PNET细胞系中均检测到RASSFIA基因的甲基化。相反,该基因甲基化在全部正常组织（包括2例小脑,5例大脑）中均未检测到。并且,RASSF1A在SPNET中的甲基化率明显低于髓母细胞瘤（Fisher精确检验,P=0．014）。在经去甲基化试剂处理的PNET细胞中,该基因表达得以恢复,证明甲基化与该基因沉默相关。结论RASSF1A甲基化是肿瘤特异性的,RASSF1A甲基化与PNET的发生有一定关联,不同亚型的PNET之间RASSF1A基因的不同甲基化状态提示髓母细胞瘤和SPNET是表遗传学上存在差异的两类肿瘤。     

RASSF1A, BLU, NORE1A, PTEN and MGMT expression and promoter methylation in gliomas and glioma cell lines and evidence of deregulated expression of de novo DNMTs

Methylation of CpG islands in gene promoters can lead to gene silencing. Together with deletion or mutation, it may cause a loss of function of tumor suppressor genes. RASSF1A (3p21.3), NORE1A (1q32.1) and BLU (3p21.3) have been shown to be downregulated by methylation in cancer, and PTEN (10q23.3) and MGMT (10q26.1) are located in areas commonly deleted in astrocytomas. MGMT methylation predicts a better response and a longer overall survival in patients with glioblastomas treated with temozolomide. We analyzed 53 astrocytoma samples and 10 high-grade glioma cell lines. Gene expression was assessed by RT-PCR. Bisulfite sequencing, MSP and a melting curve analysis-based real-time PCR were performed to detect promoter methylation. Treatments with 5'-aza-2'-deoxicitidine were applied to restore gene expression in cell lines. Ninety-two percent of tumor samples were methylated for RASSF1A , 30%–57% for BLU and 47% for MGMT , suggesting promoter methylation of these genes to be a common event in glioma tumorigenesis. Only 4% of the tumors revealed a methylated promoter for NORE1A . No association between methylation and loss of expression could be established for PTEN . We identified de novo DNMTs overexpression in a subset of tumors which may explain the methylation phenotype of individual gliomas.     

食管鳞癌分泌素-3A1基因启动子甲基化异常

[目的]研究具有抗癌活性的细胞因子分泌素-3A1基因启动子超甲基化所至该基因表达抑制在我国食管鳞癌发生中的作用。[方法] 用RT-PCR 方法检测分泌素-3A1基因在12个食管鳞癌细胞系的表达,并用甲基化特异PCR技术（methylation specific PCR, MSP）检测上述细胞系及37例来自我国河南的原发性食管鳞癌标本分泌素-3A1基因启动子甲基化状态,通过5-aza-dc处理使培养细胞去甲基化,RT-PCR技术检测处理后该基因在Kyse30的重新表达。[结果] 该基因仅在Kyse410、Kyes520和TE8等3个细胞系有表达,5-aza-dc处理可使Kyse30重新表达分泌素-3A1。此外,在18/37（48%）例原发性食管鳞癌细胞有分泌素-3A1基因启动子区域的超甲基化。[结论] 启动子超甲基化造成分泌素-3A1基因在上述食管鳞癌细胞系表达抑制,原发性食管鳞癌细胞分泌素-3A1基因启动子超甲基化提示该基因表达抑制可能与食管鳞癌发生有关。     

肾细胞癌RASSF1A基因启动子区甲基化状况与表达水平研究

目的检测19例肾细胞癌患者癌组织及癌旁组织Ras相关区域家族蛋白1A（RASSFlA）基因启动子区甲基化情况并检测其mRNA表达水平，探索两者之间的联系。方法收集肾细胞癌患者癌组织及相应癌旁组织19份，分别提取其基因组DNA，以甲基化特异性PCR（Methylation special PCR，MSP）法检测RASSFlA基因启动子区甲基化情况。并以QPCR法检测了RASSFlA基因的mRNA表达水平。结果19例中有lO例肾细胞癌患者癌组织存在高甲基化，mRNA表达水平表达降低，二者之间存在显著负相关性（r=-0．8734，P〈0．01）。结论肾细胞癌中RASSFlA基因启动子区存在高甲基化，并抑制该基因的表达。     

RASSF1A及其在肿瘤发生中的作用

RASSF1A（Ras-assotiation domain family 1 A）基因是一个新型抑癌基因。目前的研究已经在许多肿瘤中发现了这个基因的失活。虽然这个基因的失活可因基因缺失或突变引起，但最常见的原因还是该基因的启动子区甲基化紊乱。这种表形遗传学改变被证实是肿瘤形成的一个早期事件，是肿瘤形成的主要因素之一。RASSF1A有多个结构域，被认为是一种潜在的Ras癌蛋白效应分子，能与活化的Ras结合，调节凋亡及细胞周期信号通路，在细胞的凋亡、增殖、分化及维持细胞的稳定中发挥多种生物学效应，与肿瘤的发生发展密切相关。     

High frequency of promoter hypermethylation of RASSF1A in tumorous and non-tumourous tissue of breast cancer

AIM: To determine the presence of RASSF1A promoter methylation in tumorous and non-tumorous tissues of breast cancer. METHODS: Methylation-specific PCR was used to detect RASSF1A methylation in DNA extracted from tumorous and paired non-tumorous tissues of 40 breast cancer patients. The associations of RASSF1A hypermethylation with clinicopathological characteristics in tumorous and non-tumorous breast tissues were analysed. RESULTS: RASSF1A promoter hypermethylation was detected in 38 of the 40 breast cancer tissues (95%) and 37 of the paired non-tumorous tissues (92.5%). When compared with the non-tumorous tissues, aberrant methylation was detected to be higher in 24 of the tumorous tissues (60%). The latter was found to be associated with lower histological grade tumours (p=0.048). CONCLUSION: RASSF1A promoter hypermethylation occurred at a high frequency in breast cancer tumorous and non-tumorous tissues; the majority of tumours have a higher level of methylation status when compared with non-tumorous tissues. This supports the notion that RASSF1A methylation is an early and premalignant alteration.     

RASSF1A and NORE1A methylation and BRAFV600E mutations in thyroid tumors

We analyzed RASSF1A and NORE1A methylation and BRAF mutation in 89 thyroid tumors, 42 non-neoplastic thyroid tissues and three thyroid tumor cell lines using polymerase chain reaction (PCR), methylation-specific PCR, Western blotting and DNA sequencing in order to study thyroid tumor pathogenesis and progression. RASSF1A promoter methylation was present in all three thyroid cell lines and in 27/78 (35%) of benign and malignant thyroid tumors. We showed for the first time that there was generally good agreement between RASSF1A methylation status and RASSF1A protein expression. We also examined for the first time NORE1A promoter region methylation in thyroid cell lines and primary tumors and showed that two of three thyroid cell lines were methylated in the NORE1A promoter region, while all primary thyroid tumors analyzed (n=51) were unmethylated. BRAF mutation was present in 38% of papillary thyroid carcinomas (PTC), including 20% of PTC with a follicular variant pattern and 67% of the tall cell variant of PTC. Hyalinizing trabecular tumors (n=23), which had nuclear features similar to PTC, did not have BRAF mutations, indicating that the presence of BRAF mutations can help to separate these two tumor types. Phospho-MEK expression was increased in the NPA cell line, which had a BRAF mutation, supporting the importance of the BRAF pathway alterations in PTC pathogenesis. These results indicate that RASSF1A epigenetic changes are an early event in thyroid tumor pathogenesis and progression and that NORE1A methylation is uncommon in primary thyroid tumors. BRAF mutation occurs later in thyroid tumor progression and is restricted mainly to PTC and anaplastic thyroid carcinoma.     

Hypermethylation of RASSF1A in human and rhesus placentas

The pseudomalignant nature of the placenta prompted us to search for tumor suppressor gene hypermethylation, a phenomenon widely reported in cancer, in the human placenta. Nine tumor suppressor genes were studied. Hypermethylation of the Ras association domain family 1 A (RASSF1A) gene was found in human placentas from all three trimesters of pregnancy but was absent in other fetal tissues. Hypermethylation of Rassf1 was similarly observed in placentas from the rhesus monkey but not the mouse. An inverse relationship between RASSF1A promoter methylation and gene expression was demonstrated by bisulfite sequencing of microdissected placental cells and immunohistochemical staining of placental tissue sections using an anti-RASSF1A antibody. Treatment of choriocarcinoma cell lines, JAR and JEG3, by 5-aza-2'-deoxycytidine and trichostatin A led to reduction in RASSF1A methylation but increased expression. These observations extend the analogy between the primate placenta and malignant tumors to the epigenetic level.     

RASSF1A promoter methylation and 3p21.3 loss of heterozygosity are features of foregut, but not midgut and hindgut, malignant endocrine tumours

The Ras-association domain family 1A (RASSF1A) tumour suppressor gene is inactivated in a variety of solid tumours, usually by epigenetic silencing of the promoter and/or allelic loss of its locus at 3p21.3. RASSF1A induces cell cycle arrest through inhibition of cyclin D1 accumulation. In this work, 62 endocrine tumours from different sites in the gut were investigated for methylation of the RASSF1A promoter using the polymerase chain reaction, the presence of 3p21.3 deletions by loss of heterozygosity analysis, and cyclin D1 expression by immunohistochemistry. Methylation was found in 20/62 (32%) cases and was restricted to foregut tumours; deletion at 3p21.3 was found in 15/58 (26%) informative cases and restricted to malignant foregut tumours; cyclin D1 hyper-expression was found in 31/58 (53%) cases and correlated with RASSF1A methylation. Our data suggest that RASSF1A is involved in the development of endocrine tumours derived from the foregut only, and that the presence of both RASSF1A methylation and 3p21.3 deletion is associated with malignancy. These results may provide a rationale for foregut-targeted therapy for aggressive endocrine carcinomas entailing the use of demethylating agents.     

食管鳞状细胞癌中SFRP基因家族启动子区甲基化状态的检测

目的: 检测食管鳞状细胞癌(ESCC)中Wnt通路拮抗基因家族分泌型卷曲相关蛋白(SFRP)基因的甲基化状态,探讨其与食管鳞癌发生的关系。方法: 应用甲基化特异性PCR(MS-PCR)及RT-PCR的方法检测78例食管鳞癌及相应癌旁非肿瘤组织中 SFRP1 、 SFRP2 、 SFRP4 和 SFRP5 基因的甲基化状态及mRNA表达情况,并分析其与Wnt通路中心因子β-catenin蛋白表达的关系。结果: 在食管鳞癌组织中, SFRP1 、 SFRP2 、 SFRP4 和 SFRP5 基因的甲基化率分别为65.4%(51/78)、69.2%(54/78)、62.8%(49/78)和52.6%(41/78),均明显高于癌旁非肿瘤组织(P<0.01)。这4个基因的甲基化率与肿瘤患者的组织学分级及临床分期均无关,但它们共同发生甲基化的频率则与临床分期显著相关(P<0.05)。这4个基因mRNA的阳性表达率分别为42.3%(33/78)、46.2%(36/78)、50.0%(39/78)和39.7%(31/78),均明显低于癌旁非肿瘤组织(P<0.01)。在发生甲基化的食管癌组织中这4个基因的mRNA表达阳性率及β-catenin蛋白的异质表达率均明显低于未发生甲基化的癌组织,且差异显著(P<0.05)。结论: 食管鳞癌组织中 SFRP1 、 SFRP2 、 SFRP4 和 SFRP5 基因均呈高甲基化状态,并有可能通过Wnt/β-catenin信号转导通路参与了食管癌的发生。联合检测SFRP基因家族甲基化状态对于食管癌的预后判断有一定指导意义。     

Prognostic CpG Methylation Biomarkers Identified by Methylation Array in Esophageal Squamous Cell Carcinoma Patients

Background: Esophageal squamous cell carcinoma (ESCC) is an aggressive cancer with poor prognosis. We aimed to identify a panel of CpG methylation biomarkers for prognosis prediction of ESCC patients.Methods: Illumina''s GoldenGate methylation array, supervised principal components, Kaplan-Meier survival analyses and Cox regression model were conducted on dissected tumor tissues from a training cohort of 40 ESCC patients to identify potential CpG methylation biomarkers. Pyrosequencing quantitative methylation assay were performed to validate prognostic CpG methylation biomarkers in 61 ESCC patients. The correlation between DNA methylation and RNA expression of a validated marker, SOX17, was examined in a validation cohort of 61 ESCC patients.Results: We identified a panel of nine CpG methylation probes located at promoter or exon1 region of eight genes including DDIT3, FES, FLT3, NTRK3, SEPT5, SEPT9, SOX1, and SOX17, for prognosis prediction in ESCC patients. Risk score calculated using the eight-gene panel statistically predicted poor outcome for patients with high risk score. These eight-gene also showed a significantly higher methylation level in tumor tissues than their corresponding normal samples in all patients analyzed. In addition, we also detected an inverse correlation between CpG hypermethylation and the mRNA expression level of SOX17 gene in ESCC patients, indicating that DNA hypermethylation was responsible for decreased expression of SOX17.Conclusions: This study established a proof-of-concept CpG methylation biomarker panel for ESCC prognosis that can be further validated by multiple cohort studies. Functional characterization of the eight prognostic methylation genes in our biomarker panel could help to dissect the mechanism of ESCC tumorigenesis.