Aberrant Gene Promoter Methylation Research Articles

Aberrant gene promoter methylation of p16, FHIT, CRBP1, WWOX, and DLC-1 in Epstein-Barr virus-associated gastric carcinomas.

Alterations in global DNA methylation and specific regulatory gene methylation are frequently found in cancer, but the significance of these epigenetic changes in EBV-associated gastric carcinoma (EBVaGC) remains unclear. We evaluated global DNA methylation status in 49 EBVaGC and 45 EBV-negative gastric carcinoma (EBVnGC) tissue samples and cell lines by 5-methylcytosine immunohistochemical staining and methylation quantification. We determined promoter methylation status and protein expression for the p16, FHIT, CRBP1, WWOX, and DLC-1 genes in tissues and studied the correlation between CpG island methylator phenotype (CIMP) class and clinicopathological characteristics. Changes in gene methylation and mRNA expression in EBVaGC cell line SNU-719 and in EBVnGC cell lines SGC-7901, BGC-823, and AGS were assessed after treatment with 5-aza-2'-deoxycytidine (5-aza-dC), trichostatin A (TSA), or a combination of both, by methylation-specific PCR and quantitative real-time RT-PCR. Global genomic DNA hypomethylation was more pronounced in EBVnGC than in EBVaGC. Promoter methylation of all five genes was more frequent in EBVaGC than in EBVnGC (p<0.05). p16 and FHIT methylation was reversely correlated with protein expression in EBVaGC. Most (41/49) EBVaGC exhibited CIMP-high (CIMP-H), and the prognosis of CIMP-H patients was significantly worse than that of CIMP-low (p=0.027) and CIMP-none (p=0.003) patients. Treatment with 5-aza-dC and/or TSA induced upregulation of RNA expression of all five genes in SNU-719; meanwhile, individual gene expression increased in EBVnGC cell lines. In summary, EBV-induced hypermethylation of p16, FHIT, CRBP1, WWOX, and DLC-1 may contribute to EBVaGC development. Demethylation therapy may represent a novel therapeutic strategy for EBVaGC.

Epigenetic changes in gastrointestinal cancers

Epigenetic alterations, including DNA methylation, histone modification, loss of genome imprinting, chromatin remodeling and non-coding RNAs, are associated with human carcinogenesis. Among them, DNA methylation is a fundamental epigenetic process to modulate gene expression. In cancer cells, altered DNA methylation includes hypermethylation of site-specific CpG island promoter and global DNA hypo-methylation. Detection of aberrant gene promoter methylation has been applied to the clinic to stratify risk in cancer development, detect early cancer and predict clinical outcomes. Environmental factors associated with carcinogenesis are also significantly related to aberrant DNA methylation. Importantly, epigenetic changes, including altered DNA methylation, are reversible and thus, used as targets for cancer therapy or chemoprevention. An increasing number of recent studies reported DNA methylation level to be a useful biomarker for diagnosis, risk assessment and prognosis prediction for gastrointestinal (GI) cancers. This review summarized the accumulated evidence for clinical application to use aberrant DNA methylation levels in GI cancers, including colorectal, gastric and esophageal cancer.

Prognostic significance of CDH13 hypermethylation and mRNA in NSCLC.

Aberrant methylation of CpG dinucleotides is a commonly observed epigenetic modification in human cancer. Thus, detection of aberrant gene promoter methylation as a tool for diagnosis of tumors or as a prognostic marker has been widely described for many types of cancers, including nonsmall cell lung cancer (NSCLC). Emerging evidence indicates that CDH13 is a candidate tumor suppressor in several types of human tumors, including NSCLC. However, the correlation between CDH13 hypermethylation and clinicopathological characteristics of NSCLC remains unclear. In the current study, we conducted a systematic review and meta-analysis to quantitatively evaluate the effects of CDH13 hypermethylation on the incidence of NSCLC and clinicopathological characteristics. Final analysis of 803 NSCLC patients from eleven eligible studies was performed. CDH13 hypermethylation was observed to be significantly higher in NSCLC than in normal lung tissue, with the pooled odds ratio (OR) from seven studies including 448 NSCLC and 345 normal lung tissue (OR, 7.85; 95% confidence interval, 5.12–12.03; P<0.00001). CDH13 hypermethylation was also associated with pathological types. The pooled OR was obtained from four studies, including 111 squamous cell carcinoma and 106 adenocarcinoma (OR, 0.35; 95% confidence interval, 0.19–0.66; P=0.001), which indicated that CDH13 hypermethylation plays a more important role in the pathogenesis of adenocarcinoma. NSCLC with CDH13 hypermethylation was found more frequently in poorly differentiated NSCLC patients. NSCLC patients with CDH13 hypermethylation had a lower survival rate than those without CDH13 hypermethylation. In addition, CDH13 mRNA high expression was found to correlate with better overall survival for all NSCLC patients followed for 20 years (hazard ratio, 0.81; P=0.0056). Interestingly, CDH13 mRNA overexpression was found to correlate with better overall survival only in adenocarcinoma patients (hazard ratio, 0.42; P=9.6e–09), not in squamous cell carcinoma patients (hazard ratio, 0.93; P=0.59). The results of this meta-analysis suggest that CDH13 hypermethylation is associated with an increased risk and worse survival in NSCLC. CDH13 hypermethylation and mRNA expression play an important role in carcinogenesis, progression, and development, as well as clinical outcomes.

Detection of aberrant promoter methylation of RNF180, DAPK1 and SFRP2 in plasma DNA of patients with gastric cancer.

Gastric cancer (GC) is one of the most frequently diagnosed malignancies in East Asia, particularly in China, and remains the second leading cause of cancer-associated mortality worldwide. However, no effective plasma biomarkers have been identified for the diagnosis of patients with GC. The aim of this study was to investigate the DNA methylation status of the ring finger protein 180 (RNF180), secreted frizzled-related protein 2 (SFRP2) and death-associated protein kinase 1 (DAPK1) genes in the plasma samples of 57 GC patients and 42 control individuals with no malignant disease, and to evaluate the clinical utility of these makers. A significantly higher level of methylation was observed in the plasma DNA of GC patients when compared with that of controls for the three genes investigated (RNF180, 57.89% vs. 23.81%; DAPK1, 49.12% vs. 28.57%; and SFRP2, 71.93% vs. 42.86%). No association was identified between the DAPK1 or SFRP2 methylation level in the plasma DNA and the clinicopathological parameters of patients. Notably, RNF180 methylation was found to positively correlate with tumor size (P=0.018), histological type (P=0.025), TNM stage (P=0.002), lymph node metastasis (P=0.008) and distant metastasis (P=0.018). Overall, 50 cancer patients (87.72%) exhibited methylation of at least one of the three markers, while 26 normal subjects presented methylation in plasma DNA [specificity, 38.1%; odds ratio (OR), 4.4]. The combined use of RNF180 and SFRP2 as methylation markers appeared to be the most preferable predictor with regard to predictive power and cost-performance (OR, 5.57; P=0.0002). The results of the present study indicate that aberrant promoter methylation of genes in the plasma may be detected in a substantial proportion of GC patients and thus, these genes must be evaluated in the screening and surveillance of GC.

A Preliminary Study of the Relationship between Promoter Methylation of the ABCG1, GALNT2 and HMGCR Genes and Coronary Heart Disease

AimsTo investigate the association of ABCG1, GALNT2 and HMGCR genes promoter DNA methylation with coronary heart disease (CHD) and explore the interaction between their methylation status and the CHD patients' clinical characteristics in Han Chinese population.Methods and resultsMethylation-specific polymerase chain reaction (MSP) technology was used to examine the role of the aberrant gene promoter methylation in CHD in Han Chinese population. A total of 85 CHD patients and 54 participants without CHD confirmed by angiography were recruited. 82.8% of the participants with ABCG1 gene promoter hypermethylation have CHD, while only 17.4% of the participants without hypermethylation have it. The average age of the participants with GALNT2 gene promoter hypermethylation is 62.10±8.21, while that of the participants without hypermethylation is 57.28±9.87; in the former group, 75.4% of the participants have CHD, compared to only 50% in the latter group. As for the HMGCR gene, the average age of the participants with promoter hypermethylation is 63.24±8.10 and that of the participants without hypermethylation is 57.79±9.55; its promoter hypermethylation is likely to be related to smoking. Our results indicated a significant statistical association of promoter methylation of the ABCG1 gene with increased risk of CHD (OR = 19.966; 95% CI, 7.319–54.468; P *<0.001; P *: adjusted for age, gender, smoking, lipid level, hypertension, and diabetes). Similar results were obtained for that of the GALNT2 gene (OR = 2.978; 95% CI, 1.335–6.646; P * = 0.008), but not of HMGCR gene (OR = 1.388; 95% CI, 0.572–3.371; P * = 0.469).ConclusionsThe present work provides evidence to support the association of promoter DNA methylation status with the risk profile of CHD. Our data indicates that promoter DNA hypermethylation of the ABCG1 and GALNT2 genes, but not the HMGCR gene, is associated with an increased risk of CHD. CHD, smoking and aging are likely to be the important factors influencing DNA hypermethylation.

Epigenetic alteration of p16 and retinoic acid receptor beta genes in the development of epithelial ovarian carcinoma.

Silencing of tumor suppressor and tumor-related genes by promoter hypermethylation is one of the major events in ovarian carcinogenesis. In this study, we analyzed aberrant promoter methylation of p16 and RAR-β genes in 134 epithelial ovarian carcinomas (EOCs), 23 low malignant potential (LMP) tumors, 26 benign cystadenomas, and 15 normal ovarian tissues. Methylation was investigated by methylation-specific PCR (MSP), and the results were confirmed by bisulfite DNA sequencing. Relative gene expression of p16 and RAR-β was done using quantitative reverse transcriptase PCR (qRT-PCR) on 51 EOC cases, 9 LMP tumors, and 7 benign cystadenomas with 5 normal ovarian tissues. Aberrant methylation for p16 and RAR-β was present in 43 % (58/134) and 31 % (41/134) in carcinoma cases, 22 % (05/23) and 52 % (12/23) in LMP tumors, and 42 % (11/26) and 69 % (18/26) in benign cystadenomas. No methylation was observed in any of the normal ovarian tissues. The mRNA expression level of p16 and RAR-β was significantly downregulated in EOC and LMP tumors than the corresponding normal tissues whereas the expression level was normal in benign cystadenomas for p16 and slightly reduced for RAR-β. A significant correlation of p16 promoter methylation was observed with reduced gene expression in EOC. For RAR-β, no significant correlation was observed between promoter methylation and gene expression. Our results suggest that epigenetic alterations of p16 and RAR-β have an important role in ovarian carcinogenesis and that mechanism along with methylation plays a significant role in downregulation of RAR-β gene in ovarian cancer.

Aberrant promoter methylation of p15 (INK⁴b) and p16 (INK⁴a) genes may contribute to the pathogenesis of multiple myeloma: a meta-analysis.

We carried out the current meta-analysis aiming to comprehensively assess the potential role of p15 (INK4b) and p16 (INK4a) aberrant promoter methylation in the pathogenesis of multiple myeloma (MM). The MEDLINE (1966 ~ 2013), Cochrane Library (Issue 12, 2013), EMBASE (1980 ~ 2013), CINAHL (1982 ~ 2013), Web of Science (1945 ~ 2013), and Chinese Biomedical (CBM) (1982 ~ 2013) databases were searched without language restrictions. Meta-analyses were conducted using Stata software (Version 12.0, Stata Corporation, College Station, TX, USA). Odds ratios (ORs) and their 95 % confidence intervals (95 %CIs) were calculated. Thirteen clinical case-control studies, which enrolled a total of 465 MM patients and 180 healthy subjects, were included in the meta-analysis. The results of our meta-analysis demonstrated that the frequencies of p15 (INK4b) and p16 (INK4a) promoter methylation in cancer samples were significantly higher than in normal samples (p15 (INK4b) : OR = 6.26, 95 %CI = 3.87 ~ 10.12, P < 0.001; p16 (INK4a) : OR = 2.26, 95 %CI = 1.22 ~ 4.20, P < 0.001). Ethnicity-stratified analysis showed that the aberrant methylation of p15 (INK4b) was significantly related with the risk of MM among both Caucasians and Asians (all P < 0.05). Furthermore, our results also illustrated a strong positive correlation between p16 (INK4a) promoter methylation and the pathogenesis of MM among Asians (OR = 5.17, 95 %CI = 3.45 ~ 7.74, P < 0.001), but not among Caucasians (P > 0.05). The current meta-analysis confirms and reinforces existing findings that p15 (INK4b) and p16 (INK4a) promoter methylation may be closely implicated in the pathogenesis of MM.

Development of a DUSP9 methylation screening assay.

A methylation screening assay for DUSP9 (dual-specificity phosphatase 9) has been developed and applied on 79 FFPE samples from patients with colorectal cancer (CRC) and 22 corresponding tumor free colon samples in this study. Quantitative pyrosequencing was used for the determination of the methylation in the promoter CpG island, including 83 CpG motifs. In this way, the methylation pattern of the 11 tumor samples with the weakest and the strongest methylation could be identified and were compared to their corresponding tumor free colon samples. Forty six percent of the weakly methylated samples showed no significant difference to their tumor free counterparts, whereas in 27% of the cases an increased or reduced methylation was detectable. For the strongly methylated tumor samples only 18% showed no significant difference to their tumor free counterparts, whereas 82% were significantly stronger methylated. In CRC, the aberrant promoter methylation of tumor suppressor genes is one aspect that defines the CpG island methylator phenoptype (CIMP) and is frequently observed in a subpopulation of cases. Patients harboring a CIMP phenotype often show additional clinicopathological characteristics, the so called CIMP features. Interestingly, no CIMP features were found for the weakly methylated samples analyzed in this study but could be seen in 82% of the strongly methylated cases, indicating a possible use for DUSP9 as CIMP marker.

A Phase I Protocol of Hydralazine and Valproic Acid in Advanced, Previously Treated Solid Cancers

Smokers experience aberrant gene promoter methylation in their bronchial cells, which may predispose to the development of neoplasia. Hydralazine is a DNA demethylating agent, and valproic acid is a histone deacetylase inhibitor, and both have modest but synergistic anticancer activity in vitro. We conducted a phase I trial combining valproic acid and hydralazine to determine the maximally tolerated dose (MTD) of hydralazine in combination with a therapeutic dose of valproic acid in patients with advanced, unresectable, and previously treated solid cancers. Twenty females and nine males were enrolled, with a median age of 57 years and a median ECOG performance status of 0. Grade 1 lymphopenia and fatigue were the most common adverse effects. Three subjects withdrew for treatment-related toxicities occurring after the DLT observation period, including testicular edema, rash, and an increase in serum lipase accompanied by hyponatremia in one subject each. A true MTD of hydralazine in combination with therapeutic doses of valproic acid was not reached in this trial, and the planned upper limit of hydralazine investigated in this combination was 400 mg/day without grade 3 or 4 toxicities. A median number of two treatment cycles were delivered. One partial response by Response Evaluation Criteria In Solid Tumors criteria was observed, and five subjects experienced stable disease for 3 to 6 months. The combination of hydralazine and valproic acid is simple, nontoxic, and might be appropriate for chemoprevention or combination with other cancer treatments. This trial supports further investigation of epigenetic modification as a new therapeutic strategy.

Abnormal promoter methylation of multiple tumor suppressor genes in human bronchial epithelial malignant cells.

Carcinoma of the lung is the leading cause of cancer-related mortality worldwide. In order to understand the pathogenesis of radiation-induced lung cancer, we adopted a model of transformed human bronchial epithelial cells (BEP2D) induced by α-particles. Methylation-specific polymerase chain reaction was performed to detect aberrant promoter methylation of multiple tumor suppressor genes, including p14ARF, p16INK4a, O6-methylguanine-DNA methyltransferase, glutathione S-transferase P1 and death-associated protein kinase genes in the BEP2D cell line and its malignant transformant, the BERP35T1 cell line. Our results demonstrated the distinctive methylation pattern for these tumor suppressor genes in radiation-induced malignant cells, as compared to their wild-type counterparts. Our study revealed epigenetic signatures for the characterization of radiation-mediated carcinogenesis and it may facilitate early diagnosis of patients at high risk for lung cancer.

Changes in expression levels of excision repair cross-complementing group 1 (ERCC1) and dihydropyrimidine dehydrogenase (DPD) after first-line oxaliplatin-based chemotherapy for metastatic colorectal cancer: Results of a multicenter study.

463 Background: Our previous single-center study showed that expression levels of ERCC1 (a nucleotide excision repair pathway gene) and DPD (a pyrimidine catabolic pathway gene) in liver metastases from colorectal cancers (CRCs) were significantly higher in patients given oxaliplatin as first-line chemotherapy than those not given oxaliplatin. We conducted this multicenter study to validate our findings and better define a basic rationale for second-line therapy for metastatic CRC resistant to first-line therapy. Methods: Irinotecan-naive patients with mCRC (n = 190) were enrolled at 17 sites in Japan. Resected liver metastases were available for 91 patients who had received FOLFOX (or FOLFOX plus bevacizumab; FOLFOX group) and 99 who had not received oxaliplatin-based chemotherapy (non-oxaliplatin group). We measured mRNA expression levels of ERCC1 and DPD by real-time RT-PCR. Protein expression levels of ERCC1 and DPD were evaluated immunohistochemically. The methylation level of the long interspersed nucleotide element-1 (LINE-1; a surrogate m arker of global DNA methylation) was determined by pyrosequencing. Results: The median age at liver resection was 64 years (range, 32–89). Baseline features (e.g., sex, histology) were similar in the groups. Expression levels of DPD at the mRNA and protein levels were significantly higher in the FOLFOX group than in the non-oxaliplatin group (p &lt; 0.05). The mRNA expression level of ERCC1 was slightly, but not significantly higher in the FOLFOX group than in the non-oxaliplatin group (p = 0.053). Interestingly, ERCC1 expression levels correlated with DPD expression levels (r = 0.38, p&lt; 0.0001). LINE-1 methylation levels were similar in the groups. Conclusions: This multicenter study confirmed increased expression levels of DPD after first-line oxaliplatin-based chemotherapy, which may affect chemosensitivity to subsequent therapy. ERCC1 expression and epigenetic control by mechanisms such as aberrant promoter methylation of specific genes warrant further investigation.

Aberrant promoter methylation of cell adhesion-related genes associated with clinicopathologic features in non-small cell lung cancer in China

The aim of this study was to investigate the methylation status of three cell adhesion-related genes including CDH1, TSLC1 and TIMP3 in non-small cell lung cancer and explore its association with clinicopathologic features and various environmental risk factors. We detected the aberrant methylation presence of these genes by methylation-specific polymerase chain reaction and analyzed the potential correlations with multivariate logistic regression model as well as stepwise logistic regression. For CDH1, promoter methylation was less frequent in adenosquamous carcinomas than adenocarcinomas (OR=0.35, 95%CI=0.13-0.96); pickled food increased the methylation frequency (OR=2.23, 95%CI=1.09-4.54) while light smoking and fruit intake decreased that (OR=0.43, 95%CI=0.19-0.97; OR=0.37, 95%CI=0.15-0.95). For TSLC1, males and toxin exposure increased methylation frequency (OR=6.25, 95%CI=1.05-37.13; OR=2.42, 95%CI=1.01-5.77) while light smoking and radiation exposure decreased that (OR=0.14, 95%CI=0.03-0.60; OR=0.17, 95%CI=0.04-0.87). For TIMP3, males showed lower methylation frequency than females (OR=0.18, 95%CI=0.04-0.88) while central lung cancer, heavy smoking and radiation exposure presented higher aberrant DNA methylation status (OR=2.19, 95%CI=1.07-4.52; OR=6.99, 95%CI=1.32-37.14; OR=2.30, 95%CI=1.04-5.08). Aberrant promoter methylation of cell adhesion-related tumor suppressor genes in lung cancer displayed varieties of gene-specific correlations with clinicopathologic features and various environmental risk factors.

High-Resolution Melting-Based Quantitative Analysis of RASSF1 Methylation in Breast Cancer

Breast cancer is the leading cause of death from cancer among women worldwide. The aberrant promoter methylation of tumor suppressor genes is a typical epigenetic alteration for breast cancer and can be detected in early carcinogenesis. High-throughput and cost-effective methods are needed for the early and sensitive detection of epigenetic changes in clinical material. The main purpose of our study was to optimize a high-resolution melting (HRM) assay for the reliable and quantitative assessment of RASSF1 gene methylation, which is considered one of the earliest epigenetic alterations in breast cancer. A total of 76 breast carcinomas and 10 noncancerous breast tissues were studied by means of HRM and compared with the results obtained by means of quantitative methylation-specific polymerase chain reaction (QMSP) and methylation-specific polymerase chain reaction (MSP). Both quantitative methods, HRM and QMSP, showed a similar specificity and sensitivity for the detection of RASSF1 methylation in breast cancer (about 80% and 70%, respectively). In breast cancer, the mean methylation intensity of RASSF1 was 42.5% and 48.6% according to HRM and QMSP, respectively. Both methods detected low levels of methylation (less than 5%) in noncancerous breast tissues. In comparison with quantitative methods, MSP showed a lower sensitivity (70%), but a higher specificity (80%) for the detection of RASSF1 methylation in breast cancer. HRM is as a simple, cost-effective method for the reliable high-throughput quantification of DNA methylation in clinical material.

Abstract C12: Aberrant promoter methylation of HOPX-β, MUC1 genes and global methylation in esophageal squamous cell carcinoma

Abstract Esophageal cancer ranks as the sixth most deadly cancer and esophageal squamous cell carcinoma (ESCC) is the sixth most common cause of cancer deaths in males in worldwide. Therefore, it is important to identify molecular markers for clinical diagnosis. Homeodomain-only protein X (HOPX)-β is a tumor suppressor gene and its promoter methylation is shown to be frequent in human cancer. Mucin 1 (MUC1) is a transmembrane mucin that is highly expressed in various cancers and correlates with malignant potential. In addition, the degree of whole genome methylation decreases as the lesion progressing from a benign proliferation of cells to an invasive cancer. The aim of this study was to investigate HOPX-β and MUC1 promoter methylation status and global methylation level in different stages of ESCC patients. Methylation-specific PCR (MSP) revealed that the methylation rate of HOPX-β was 26.5% (27/102) in tumor samples and only 9% (9/100) in paired non-tumor samples (p&amp;lt;0.05). In HOPX-β promoter hypermethylation samples, the frequency of lymph node invasion and tumor metastasis was increased about 3-fold and 2-fold, respectively. Furthermore, HOPX-β promoter methylation frequency was increased about 3-fold in advanced stage. On the other hand, the methylation frequency of MUC1 was 90.7% (97/107) in paired non-tumor samples while the methylation frequency was decreased to 53.3% (57/107) in tumor samples. 78.5% (84/107) of tumor samples showed significantly decline in the degree of methylation compared with paired non-tumor samples (p&amp;lt;0.0001). We found a decrease tendency in methylation frequency of MUC1 correlated with clinical stage (early/advanced), the frequency of lymph node invasion and metastasis. Importantly, the methylation rate of MUC1 was inversely related with the tumor size (p=0.0105). The level of global long interspersed nuclear element 1 (LINE-1) methylation in ESCC tissues (mean 65.8%) was significantly lower than paired non-tumor samples (mean 79.5%). However, there is no relation between the level of global LINE-1 methylation and tumor stage or survival rate. Our findings indicate that LINE-1, MUC1 methylation level and HOPX-β hypermethylation in ESCC are useful markers in clinical diagnosis. Citation Format: Yu Chun Kao, Sheng Hsiung Lin, Chun Chieh Yu, Ruei Nian Li, Ming Tsang Wu. Aberrant promoter methylation of HOPX-β, MUC1 genes and global methylation in esophageal squamous cell carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C12.

Aberrant Methylation in Promoters of GSTP1, p16, p14, and RASSF1A Genes in Smokers of North India

Promoter hypermethylation plays an important role in the inactivation of tumor suppressor/metabolic genes during tumorigenesis. The screening of high-risk population (smokers) for hypermethylation pattern in tumor suppressor/metabolic genes can be a good noninvasive biomarker tool, which should be included in prognosis so that therapeutic measures can be initiated at an early stage. The purpose of this study was to determine the prevalence of aberrant promoter methylation of GSTP1, p16, p14, and RASSF1A genes in smokers and nonsmokers of North India. Our study showed that compared with nonsmokers, smokers have an increased risk of hypermethylation in these genes. We found that 57.3% of the smokers samples showed methylation for GSTP1, 38% for p16, 18% for p14, and 32% for RASSF1A. Our population study allowed us to reveal the relationship between smoking and the subsequent appearance of an epigenetic change. Smoking speeds up the hypermethylation of these genes, which are thus unable to express, making the person more susceptible to the risk of lung and other solid carcinomas. Hypermethylation studies on DNA from two lung cancer cell lines (A549 and H460) were also done to compare the results, and the results are similar to samples of smokers.

Abstract 3123: Air pollution and DNA methylation in tumor suppressor genes

Abstract Ambient and occupational exposure to particular matter (PM) has been associated with increased risk of lung cancer. Although the carcinogenic potential of several toxic metals in PM was well-recognized, the molecular mechanisms underlying their association with lung cancer risk remain poorly understood. Aberrant tumor suppressor gene promoter methylation has emerged as one of the most important epigenetic mechanisms in the development of human cancers, including lung cancer. However, whether exposure to PM is associated with peripheral blood leukocyte (PBL) DNA methylation in tumor suppressor genes has not been examined. Foundry workers, even in modern foundry facilities, are still exposed to substantially higher levels of airborne PM compared to those found outdoors, and have been associated with increased risk of lung cancer. In the present study of 63 foundry workers with well-characterized exposure to metal-rich particles nearby Brescia, Italy, we evaluated whether exposure to PM and metal components was associated with PBL DNA methylation in 4 tumor suppressor genes (i.e., APC, p16, p53 and RASSF1A). Air pollution measures included concentrations of airborne PM with aerodynamic diameters&amp;lt;10 μm (PM10), &amp;lt;1 μm (PM1), and individual metal particle components in PM10. Blood samples were obtained on the 1st (baseline) and 4th day (post-exposure) of the same work week. DNA methylation was measured using pyrosequencing. A linear mixed model was used to examine the correlations of the exposure with promoter methylation levels. Mean promoter DNA methylation levels of APC or p16 were significantly higher in post-exposure samples compared to that in baseline samples (from 4.65 %5mC to 4.89 %5mC, p-value=0.005 for APC; from 2.20 %5mC to 2.34 %5mC, p-value=0.006 for p16). By contrast, the mean levels of p53 or RASSF1A promoter methylation was decreased in post-exposure samples compared to that in baseline samples (from 6.36 %5mC to 6.16 %5mC, p-value=0.015 for p53; from 8.17 %5mC to 7.08 %5mC, p-value&amp;lt;0.001 for RASSF1A). In post-exposure samples, APC methylation was positively associated with PM10 (α=0.27, 95% CI: 0.13-0.40) and PM1 (α=0.23, 95% CI: 0.09-0.38). The mean methylation level of p16 or RASSF1A promoter region showed positive (α=0.19, 95% CI: 0.02-0.37) or negative (α=−0.21, 95% CI: −0.37–0.05) association with the level of zinc exposure. We observed hypermethylation of p16 and APC, and hypomethylation of RASSF1A and p53 in a group of healthy foundry workers. Our results suggest that methylation alterations in tumor suppressor genes may be involved in PM-induced lung carcinogenesis. Further studies in larger populations with lung cancer outcomes are required before any firm conclusion can be reached on whether PM exposed individuals with such DNA methylation alterations are at higher risk for lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3123. doi:1538-7445.AM2012-3123

Aberrant Promoter Methylation of CDH13 and MGMT Genes is Associated With Clinicopathologic Characteristics of Primary Non–Small-Cell Lung Carcinoma

IntroductionSystemic methylation changes may be a diagnostic marker for tumor development or prognosis. Here, we investigate the relationship between gene methylation in lung tumors relative to normal lung tissue and whether DNA methylation changes can be detected in paired blood samples. Material and MethodsSixty-five patients were enrolled in a surgical case series of non–small-cell lung carcinoma at a single institution. By using bisulfite pyrosequencing, CpG methylation was quantified at 5 genes (RASSF1A, CDH13, MGMT, ESR1, and DAPK) in lung tumor, pathologically normal lung tissue, and circulating blood from enrolled cases. ResultsThe analyses of methylation in tumors compared with normal lung tissue identified higher methylation of CDH13, RASSF1A, and DAPK genes, whereas ESR1 and MGMT methylation did not differ significantly between these tissue types. We then examined whether the 3 aberrantly methylated genes could be detected in blood. The difference in methylation observed in tumors was not reflected in methylation status of matching blood samples, which indicated a low feasibility of detecting lung cancer by analyzing these genes in a blood-based test. Lastly, we probed whether tumor methylation was associated with clinical and demographic characteristics. Histology and sex were associated with methylation at the CDH13 gene, whereas, stage was associated with methylation at MGMT. ConclusionOur results showed higher methylation of RASSF1A, CDH13, and DAPK genes in lung tumors compared with normal lung. The lack of reflection of these methylation changes in blood samples from patients with non–small-cell lung carcinoma indicates their poor suitability for a screening test.

Detection of aberrant promoter methylation of GSTP1, RASSF1A, and RARβ2 in serum DNA of patients with breast cancer by a newly established one-step methylation-specific PCR assay

Aberrant promoter methylation of genes is a common molecular event in breast cancer. Thus, DNA methylation analysis is expected to be a new tool for cancer diagnosis. In this article, we have established a new, high-performance DNA methylation assay, the one-step methylation-specific polymerase chain reaction (OS-MSP) assay, which is optimized for analyzing gene methylation in serum DNA. The OS-MSP assay is designed to detect aberrant promoter methylation of GSTP1, RASSF1A, and RARβ2 genes in serum DNA. Moreover, two quality control markers were designed for monitoring the bisulfite conversion efficiency and measuring the DNA content in the serum. Serum samples were collected from patients with primary (n = 101, stages I-III) and metastatic breast cancers (n = 58) as well as from healthy controls (n = 87). If methylation of at least one of the three genes was observed, the OS-MSP assay was considered positive. The sensitivity of this assay was significantly higher than that of the assay involving conventional tumor markers (CEA and/or CA15-3) for stages I (24 vs. 8%) and II (26 vs. 8%) breast cancer and similar to that of the assay involving the conventional tumor markers for stage III (18 vs. 19%) and metastatic breast cancers (55 vs. 59%). The results of the OS-MSP assay and those of the assay involving CEA and/or CA15-3 seemed to compensate for each other because sensitivity of these assays increased to 78% when used in combination for metastatic breast cancer. In conclusion, we have developed a new OS-MSP assay with improved sensitivity and convenience; thus, this assay is more suitable for detecting aberrant promoter methylation in serum DNA. Moreover, the combination of the OS-MSP assay and the assay involving CEA and/or CA15-3 is promising for enhancing the sensitivity of diagnosis of metastatic breast cancer.

Highly frequent promoter methylation and PIK3CA amplification in non-small cell lung cancer (NSCLC)

BackgroundLung cancer is the leading cause of cancer-related death worldwide. Genetic and epigenetic alterations have been identified frequently in lung cancer, such as promoter methylation, gene mutations and genomic amplification. However, the interaction between genetic and epigenetic events and their significance in lung tumorigenesis remains poorly understood.MethodsWe determined the promoter methylation of 6 genes and PIK3CA amplification using quantitative methylation-specific PCR (Q-MSP) and real-time quantitative PCR, respectively, and explore the association of promoter methylation with PIK3CA amplification in a large cohort of clinically well-characterized non-small cell lung cancer (NSCLC).ResultsHighly frequent promoter methylation was observed in NSCLC. With 100% diagnostic specificity, excellent sensitivity, ranging from 45.8 to 84.1%, was found for each of the 6 genes. The promoter methylation was associated with histologic type. Methylation of CALCA, CDH1, DAPK1, and EVX2 was more common in squamous cell carcinomas (SCC) compared to adenocarcinomas (ADC). Conversely, there was a trend toward a higher frequency of RASSF1A methylation in ADC than SCC. In addition, PIK3CA amplification was frequently found in NSCLC, and was associated with certain clinicopathologic features, such as smoking history, histologic type and pleural indentation. Importantly, aberrant promoter methylation of certain genes was significantly associated with PIK3CA amplification.ConclusionsOur data showed highly frequent promoter methylation and PIK3CA amplification in Chinese NSCLC population, and first demonstrated the associations of gene methylation with PIK3CA amplification, suggesting that these epigenetic events may be a consequence of overactivation of PI3K/Akt pathway.

Abstract 816: Anti-inflammatory effects of black raspberries in ulcerative colitis are associated with demethylation of genes in the Wnt signaling and protective modulation of toll-like receptor pathway

Abstract Ulcerative Colitis (UC) causes pervasive, chronic inflammation of the colon and is a potential precursor to colon cancer. Reduced Interleukin 10 (IL10), an anti-inflammatory cytokine, is associated with an increased risk of developing UC in rodents and humans. IL10 knockout (KO) mice develop UC spontaneously with subsequent development of colon tumors. Toll-like receptor and Wnt pathways are deregulated in UC patients and IL10 KO mice. Toll-like receptor pathway plays critical roles in the inflammatory response partly by mediating the Wnt pathway. We previously showed that 5-10% dietary freeze-dried black raspberries (BRBs) reduced chronic inflammation and tumor development in IL10 KO mice; aberrant promoter methylation of negative Wnt regulators, e.g., dkk2, dkk3, and sfrp1, were observed in colon from these animals. The goals of the current study were to determine if the anti-inflammatory effects of BRBs on IL10 KO mice are associated with modulation of the Wnt pathway through DNA demethylation, with alteration of enzymes regulating DNA methylation, and with modulation of toll-like receptor pathway. IL10 KO mice were fed control or 5% BRB diet for 4 wks. Cells from bone marrow and spleen as well as colon tissue were collected. Promoter methylation of dkk2, dkk3, and sfrp1 was quantified by Pyrosequencing. Protein expression of enzymes regulating DNA methylation, e.g., histone deacetylase 1, 2 (HDAC1, 2), methyl binding domain 2 (MBD2), and DNA methyltransferase 3B (DNMT3B), and β-catenin were assessed using quantitative immunohistochemistry. mRNA expression of genes associated with the Wnt and toll-like receptor pathways were quantified using real-time PCR based arrays, each of which contained 84 genes. Increased promoter methylation of dkk2, dkk3, and sfrp1 was observed in colon from KO mice when compared with colon from wild-type mice. The methylation levels of these genes in bone marrow and spleen were lower than those in colon. BRBs significantly decreased promoter methylation of dkk2 in colon and dkk3 in spleen. This was associated with decreased protein expression of HDAC1, HDAC2, MBD2, and DNMT3B. The expression of 84 genes in both pathways was different in colon of IL10 KO mice when compared to colon of wild-type mice. BRBs modulated 80% (67/84) and 95% (80/84) of differentially expressed genes toward normal levels of expression in the Wnt and toll-like receptor pathways, respectively. Protein expression of nuclear β-catenin was decreased by BRBs. In summary, berries are capable of reversing aberrant promoter methylation of genes in the Wnt pathway presumably through inhibition of proteins regulating DNA methylation. These changes could result in protective modulation of the Wnt pathway and/or its interaction with the toll-like receptor pathway which, in turn, reduce inflammation in ulcerative colitis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 816. doi:10.1158/1538-7445.AM2011-816