RASSF1A Gene Promoter Methylation Research Articles

Aberrant promoter methylation, expression and function of RASSF1A gene in a series of Italian parathyroid tumors

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Epigenetic Regulation of the Tumour Suppressor RASSF1A in Bone Cancer Cells: DNA Methylation Study

Objective: Osteosarcoma is a bone cancer that affects children and adolescents. The RASSF1A is a tumor suppressor capable of mediating the regulation of cell cycle arrest, migration, including apoptosis. It is the most continually silenced gene that contributes to human cancer. Furthermore, RASSF1A functions as a scaffold protein that can regulate microtubules network and bind apoptotic kinases MST1 and MST2 via the Sav-RASSF-Hippo domain. Epigenetic inactivation of genes by DNA methylation is a key factor regulating gene expression and genomic stability. Our aim was to study the RASSF1A gene promoter methylation in three osteosarcomas (U2OS, Saos-2, and MG-63), two Ewing Sarcoma (A-673 and SK-ES-1), and one-fibrosarcoma (HT-1080) cell lines.
 Materials and Methods: Three osteosarcomas (U2OS, Saos-2, and MG-63), two Ewing Sarcoma (A-673 and SK-ES-1), and one-fibrosarcoma (HT-1080) cell lines were used to study RASSF1A gene promoter methylation, using bisulphite conversion of DNA, followed by methylation-specific polymerase chain reaction (PCR)
 Results: The RASSFIA’s gene promoter methylation was established as a frequent event. Hypermethylation of RASSF1A promoter, was detected in five out of six studied cell lines.
 Conclusions: These results demonstrated that altering the Sav-RASSF1-Hippo may be accomplished through hypermethylation of RASSF1A and may play an essential role in Ewing’s sarcoma and Osteosarcoma. The methylation pattern of Sav-RASSF1-Hippo tumor suppressor pathway in human bone cancer along with RASSF1A expression with its effector proteins merits further investigation. This may reveal how the RASSFIA has a physiological signal transduction, including how the process of its deregulation can contribute to transformation of the cell, eventually leading to the incorporation of novel therapeutic options with improved prognosis for bone cancer.

Impact of RASSF1A gene methylation on clinico-pathological features of tumor and non-tumor tissue of breast cancer

BackgroundBreast cancer is the most common malignancy in women caused by genetic and epigenetic changes. Promoter DNA methylation in tumor suppressor gene plays a major role in breast cancer. The study determined the association of promoter DNA methylation of RASSF1A gene with clinicopathological features in tumor and non-tumor tissue. Materials and methodsA cross sectional study was conducted in the Department of Pathology, Government Institute of Medical Sciences, Greater Noida and Molecular Pathology Laboratory, Department of Pathology, Jawaharlal Nehru Medical College, Datta Meghe Institute of Medical Sciences. Two sections, one from tumor and the other from non-tumor tissue, were obtained and processed for DNA extraction and bisulphite conversion. Methylation specific PCR was done and results of RASSF1A promoter methylation were statistically correlated with clinicopathological features. ResultsOf the 27 breast cancer tissue, 22 showed invasive ductal carcinoma, one showed invasive lobular carcinoma, another showed ductal carcinoma in situ and three cases showed malignant phyllodes tumor of breast. DNA promoter methylation was found in all the cases. 93% of tumor tissue samples and 67% of the non-tumor tissue samples were found to be aberrantly methylated. Tumor size and histological grade were found to be significantly (p-val <0.05) associated with the RASSF1A gene promoter methylation. ConclusionA significant association of higher tumor size and tumor histological grade with promoter methylation of RASSF1A gene exists suggestive of its being an important determinant of prognostic staging. This critical event in tumorigenesis may be of clinical utility in assessing breast cancer progression. Micro abstractThe study focuses on the RASSF1A gene promoter methylation and its impact on the clinicopathological features in Indian breast cancer patients highlighting the differences from other genetically different population. We found that RASFF1A gene methylation has significant impact on tumor size and tumor grade. The work carries high significance because it addresses the DNA methylation of tumor suppressor gene in relevance of breast cancer. It may also be the first such report on Indian patients with breast cancer.

The Relationship Between Promoter Methylation of RASSF1A Gene and Prognosis of Patients Affected with Breast Cancer in Khuzestan Province

Background DNA methylation is one of the most frequent molecular changes that are associated with human cancers. Tumor suppressor genes are the important targets of hypermethylation in breast cancer and thus may result in cancer by deregulation of cell growth and division.Objectives The purpose of this research was to compare the methylation pattern of the RASSF1A gene in cancerous and normal tissues.Methods Twenty breast cancer patients with known clinicopathologic characteristics and 20 healthy women as control were studied for analysis of the methylation status of the RASSF1A gene promoter by methylation-specific polymerase chain reaction (MS-PCR) technique.Results The findings showed that the frequency of RASSF1A promoter methylation was in 15% of tumor tissues and 5% of normal tissues. These results indicate that there is no statistically significant relationship between the RASSF1A promoter methylation status of tumor tissues and normal tissues (P > 0.05).Conclusions Until now, the evidence for powerful methylation biomarkers is still incomplete and the recognized biomarkers require further validation.

An Effective Concentration of 5-Aza-CdR to Induce Cell Death and Apoptosis in Human Pancreatic Cancer Cell Line through Reactivating RASSF1A and Up-Regulation of Bax Genes.

Promoter hyper-methylation of tumor suppressor genes is a common event that occurs in cancer. As methylation is a reversible modification, agents capable of reversing an abnormal methylation status should help to combat cancer. 5-Aza-CdR is a DNA methyl-transferase inhibitor. The present study aimed to evaluate the effect of 5-Aza-CdR on the proliferation of human pancreatic cancer cell line (PANC-1) and the expression of RASSF1A and Bax genes. PANC-1 cells were cultured and treated with 5 and 10 µM/L of 5-Aza-CdR for 24, 48, 72, and 96 hours and the percentages of cell viability and apoptosis were measured by MTT and flow cytometry. RASSF1A gene promoter methylation was assessed by methyl-specific primer-PCR (MSP-PCR) and the expression of RASSF1A and Bax genes was measured using quantitative real-time PCR (qPCR). All quantitative data are presented as mean±SD (standard deviation). The one-way analysis of variance (ANOVA) with the LSD post hoc test was performed for statistical analysis using the SPSS software package, version 16.0. 3-[4,5-dimethythiaziazol-2yl]-2,5-diphenyl tetrazoliumbr omide (MTT) assay revealed that 5-Aza-CdR significantly inhibit the growth and proliferation of PANC-1. The flow cytometry results showed over 40% and 70% of early and late apoptotic cells after treatment with 5 and 10 µm/L of 5-Aza-CdR, respectively. MSP-PCR data indicated that the treatment of cells with 10 µm/L 5-Aza-CdR resulted in partial demethylation of RASSF1A gene promoter. qPCR results showed significant re-expression of RASSF1A and up-regulation of Bax genes after 96 hours treatment of cells with 10 µm/L 5-Aza-CdR versus control cells (P<0.01). The result demonstrated that 5 and 10 µM of 5-Aza-CdR induce cell death and apoptosis by epigenetic reactivation of RASSF1A and up-regulation of Bax genes.

Promoter Methylation of RASSF1A Gene in Egyptian Patients with Ovarian Cancer.

Ovarian malignancy is diagnosed in nearly a fourth of a million women internationally every year. Methylation of RASSF1A tumor suppressor gene prompts its inactivation in diseases. In this study, the RASSF1A promoter methylation was detected by methylated-specific PCR and investigated serum RASSF1A protein level through enzyme-linked immunosorbant assay in 160 Egyptian patients with ovarian cancer and 160 healthy controls. The present work proved that there was a higher frequency of RASSF1A methylation and a decrease in its serum level in patients with ovarian cancer compared to controls as well as in the high-grade tumor patients compared to low grade ones and also in advanced ovarian tumor stage compared to early stages. Our study exhibited that RASSF1A promoter hypermethylation and its protein levels may be a reliable and sensitive tool for diagnosing and monitoring of ovarian malignancy patients.

The relationship between RASSF1A gene promoter methylation and the susceptibility and prognosis of melanoma: A meta-analysis and bioinformatics.

BackgroundThe function of the tumor suppressor gene RASSF1A in cancer cells has been detailed in many studies. However, due to the methylation of its promoter, the expression of RASSF1A is missing in most cancers. In the literature, we found that the conclusion regarding the relationship between RASSF1A gene promoter methylation and the susceptibility and prognosis of melanoma was not unified. This study adopts the use of a meta-analysis and bioinformatics to explore the relationship between RASSF1A gene promoter methylation and the susceptibility and prognosis of melanoma.MethodsData on melanoma susceptibility were downloaded from the PubMed, Cochrane Library, Web of Science and Google Scholar databases, which were analyzed via a meta-analysis. The effect sizes were estimated by measuring an odds ratio (OR) with a 95% confidence interval (CI). We also used a chi-squared-based Q test to examine the between-study heterogeneity, and used funnel plots to evaluate publication bias. The data on melanoma prognosis, which were analyzed by bioinformatics methods, were downloaded from The Cancer Genome Atlas (TCGA) project. The effect sizes were estimated by measuring the hazard ratios (HRs) with a 95% confidence interval (CI).ResultsOur meta-analysis included 10 articles. We found that RASSF1A gene promoter methylation was closely related to melanoma susceptibility (OR = 12.67, 95% CI: 6.16 ∼ 26.05, z = 6.90, P<0.0001 according to a fixed effects model and OR = 9.25, 95% CI: 4.37 ∼ 19.54, z = 5.82, P<0.0001 according to a random effects model). The results of the meta-analysis did not reveal any heterogeneity (tau2 = 0.00; H = 1 [1; 1.55]; I2 = 0% [0%; 58.6%], P = 0.5158) or publication bias (t = 0.87, P = 0.4073 by Egger’s test; Z = 0.45, P = 0.6547 by Begg’s test); therefore, we believe that the results of our meta-analysis were more reliable. To explore the relationship between RASSF1A gene methylation, the prognosis of melanoma and the clinical features of this cancer type, we used the melanoma DNA methylation data and clinical data from TCGA project. We found that RASSF1A gene promoter methylation and melanoma prognosis did not demonstrate any relationship (HR was 0.94 (95% CI = [0.69; 1.27], P = 0.694) with disease-free survival and 0.74 (95% CI = [0.53; 1.05], P = 0.106) for overall survival), and no significant difference was observed between RASSF1A gene promoter methylation and the clinical-pathological features of melanoma.ConclusionsIn conclusion, the meta-analysis of the data in these articles provides strong evidence that the methylation status of the RASSF1A gene promoter was strongly related to melanoma susceptibility. Our bioinformatics analysis revealed no significant difference between RASSF1A gene promoter methylation and the prognosis and clinical-pathological features of melanoma.

Application of artificial neural network model combined with four biomarkers in auxiliary diagnosis of lung cancer.

The purpose of the study was to explore the application of artificial neural network model in the auxiliary diagnosis of lung cancer and compare the effects of back-propagation (BP) neural network with Fisher discrimination model for lung cancer screening by the combined detections of four biomarkers of p16, RASSF1A and FHIT gene promoter methylation levels and the relative telomere length. Real-time quantitative methylation-specific PCR was used to detect the levels of three-gene promoter methylation, and real-time PCR method was applied to determine the relative telomere length. BP neural network and Fisher discrimination analysis were used to establish the discrimination diagnosis model. The levels of three-gene promoter methylation in patients with lung cancer were significantly higher than those of the normal controls. The values of Z(P) in two groups were 2.641 (0.008), 2.075 (0.038) and 3.044 (0.002), respectively. The relative telomere lengths of patients with lung cancer (0.93±0.32) were significantly lower than those of the normal controls (1.16±0.57), t=4.072, P<0.001. The areas under the ROC curve (AUC) and 95% CI of prediction set from Fisher discrimination analysis and BP neural network were 0.670 (0.569-0.761) and 0.760 (0.664-0.840). The AUC of BP neural network was higher than that of Fisher discrimination analysis, and Z(P) was 0.76. Four biomarkers are associated with lung cancer. BP neural network model for the prediction of lung cancer is better than Fisher discrimination analysis, and it can provide an excellent and intelligent diagnosis tool for lung cancer.

Abstract 442: RASSF1A gene promoter methylation in primary tumors, adjacent morphologically normal tissues and plasma samples of patients with high grade serous ovarian cancer

Abstract Introduction: RASSF1A promoter methylation is frequent in high grade serous ovarian cancer (HGSC), the most common histological subtype. We examined RASSF1A promoter methylation in primary tumors, adjacent normal tissues and corresponding plasma samples of patients with HGSC, using real-time methylation specific PCR (real-time MSP) and methylation-sensitive high-resolution melting analysis (MS-HRMA). Materials and methods: A training group of 78 primary HGSC FFPEs was first analyzed using both real-time MSP and MS-HRMA. Our validation group consisted of 61 primary HGSC FFPEs, 58 adjacent tissues (analyzed using both real-time MSP and MS-HRMA), and 59 corresponding plasma samples (analyzed using real-time MSP). The specificity of both assays was evaluated by analyzing a group of 16 fallopian tube samples from healthy individuals. OVCAR29 and IGROV1 cell lines were used as positive controls. Results: In the training group, RASSF1A promoter methylation was detected in 33/78 (42.3%) by real-time MSP and in 33/78 (42.3%) by MS-HRMA (Agreement = 94.9%, P = 0.001, k = 0.895). The validation group results are shown in Table 1. According to the semi-quantitative MS-HRMA, RASSF1A promoter methylation was detected at significantly lower percentages in the adjacent morphologically normal tissues, compared to the paired primary tumors. In corresponding plasma samples, methylation was detected in 15/59 (25.4%) (Agreement with paired tumors = 60.7%, P = 0.259, k = 0.143). Both real-time MSP and MS-HRMA revealed no RASSF1A promoter methylation in the small group of normal fallopian tubes (n = 16). Conclusion: RASSF1A promoter is highly methylated in primary tumors and at lower percentages in the adjacent normal tissues. In all cases, MS-HRMA gave comparable results with real-time MSP. Evaluation of the clinical significance of RASSF1A promoter methylation in corresponding plasma requires further investigation. Table 1RASSF1A promoter methylationMethodPrimary tumor samples (validation group) N = 61Adjacent tissues N = 58Agreement (paired samples, N = 57)Real time MSP25/61 (41%)17/58 (29.3%)48/57(84.2%), P = 0.001, k = 0.652MS-HRMA28/61 (45.9%)21/58 (36.2%)51/57(89.5%), P = 0.001, k = 0.782Agreement (methods)58/61(95.1%), P = 0.001, Cohen's kappa = 0.90050/58(86.2%),P = 0.001, Cohen's kappa = 0.689 Citation Format: Lydia Giannopoulou, Issam Chebouti, Kitty Pavlakis, Sabine Kasimir-Bauer, Evi S. Lianidou. RASSF1A gene promoter methylation in primary tumors, adjacent morphologically normal tissues and plasma samples of patients with high grade serous ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 442.

Retraction Note to: Aberrant promoter methylation of RASSF1A gene may be correlated with colorectal carcinogenesis: a meta-analysis.

We conducted a meta-analysis of cohort studies to evaluate the potential role of RASSF1A promoter methylation in colorectal carcinogenesis. A range of electronic databases were searched: PubMed (1966–2013), the Cochrane Library Database (Issue 12, 2013), EMBASE (1980–2013), CINAHL (1982–2013), Web of Science (1945–2013) and the Chinese Biomedical Database (CBM) (1982–2013) without language restrictions. Meta-analysis was conducted using the STATA 12.0 software. Crude odds ratio (OR) with 95 % confidence interval (95 % CI) was calculated. Eleven clinical cohort studies with a total of 1,505 colorectal cancer (CRC) patients that met all inclusion criteria were included in our meta-analysis. The results of our meta-analysis revealed that the frequency of RASSF1A promoter methylation was strongly correlated with clinical stage (OR = 1.69, 95 % CI 1.16–2.44, P = 0.006), histological grade (OR = 1.92, 95 % CI 1.22–3.04, P = 0.005) and distant metastasis (OR = 2.59, 95 % CI 1.46–4.60, P = 0.037) of CRC patients. However, we observed no positive correlations of RASSF1A promoter methylation with gender (OR = 1.04, 95 % CI 0.74–1.46, P = 0.842), age (OR = 1.70, 95 % CI 0.98–2.93, P = 0.057) and lymph node metastasis (OR = 1.65, 95 % CI 0.87–3.14, P = 0.127) of CRC patients. Further subgroup analysis by ethnicity demonstrated that RASSF1A promoter methylation was correlated with clinicopathological characteristics of CRC patients among Asians (clinical stage: OR = 2.55, 95 % CI 1.55–4.20, P < 0.001; histological grade: OR = 2.70, 95 % CI 1.44–5.06, P = 0.002; lymph node metastasis: OR = 4.09, 95 % CI 1.49–11.26, P = 0.006; distant metastasis: OR = 5.38, 95 % CI 1.73–16.70, P = 0.004), but not among Caucasians and Africans (all P > 0.05). Our meta-analysis has shown positive correlations between aberrant promoter methylation of RASSF1A gene and clinicopathological characteristics of CRC patients, especially among Asians.

The study of hypermethylation in blood leukocytes of irradiated parents and their children

AbstractBackground: Accumulation of evidence about the epigenetic regulation of genome function suggests the necessity to explore new aspects of the genotoxic action of radiation on the human body. Methodology: A methylation-sensitive PCR assay was used to analyze promoter methylation of p16/CDKN2A, p14/ARF, RASSF1A and GSTP1 genes in blood leukocytes from 103 unirradiated volunteers and 104 irradiated subjects (83 Chernobyl Nuclear Power Plant liquidators and 21 nuclear specialists). Additionally, 21 families whose fathers were nuclear specialists were examined. Results: A significantly elevated frequency of individuals with abnormal methylation of p16/CDKN2A and GSTP1 genes was revealed in the exposed group compared to the control group (p = 0.0097 and p = 0.005, respectively). The occurrence of promoter methylation of RASSF1A gene significantly correlated with aging both in the control group (r = 0, 213; p = 0.006) and in the exposed individuals (r = 0, 212; p = 0,031). No methylated genes were found in the offspring of control families. Conclusion: Our study showed for the fist time that prolonged radiation exposure at low and medium doses is associated with hypermethylation of genes involved in the basic protective functions of cells; an effect that is persistent in blood leukocytes for significant periods after irradiation.

RETRACTED ARTICLE: Aberrant promoter methylation of RASSF1A gene may be correlated with colorectal carcinogenesis: a meta-analysis

We conducted a meta-analysis of cohort studies to evaluate the potential role of RASSF1A promoter methylation in colorectal carcinogenesis. A range of electronic databases were searched: PubMed (1966-2013), the Cochrane Library Database (Issue 12, 2013), EMBASE (1980-2013), CINAHL (1982-2013), Web of Science (1945-2013) and the Chinese Biomedical Database (CBM) (1982-2013) without language restrictions. Meta-analysis was conducted using the STATA 12.0 software. Crude odds ratio (OR) with 95% confidence interval (95% CI) was calculated. Eleven clinical cohort studies with a total of 1,505 colorectal cancer (CRC) patients that met all inclusion criteria were included in our meta-analysis. The results of our meta-analysis revealed that the frequency of RASSF1A promoter methylation was strongly correlated with clinical stage (OR = 1.69, 95% CI 1.16-2.44, P = 0.006), histological grade (OR = 1.92, 95% CI 1.22-3.04, P = 0.005) and distant metastasis (OR = 2.59, 95% CI 1.46-4.60, P = 0.037) of CRC patients. However, we observed no positive correlations of RASSF1A promoter methylation with gender (OR = 1.04, 95% CI 0.74-1.46, P = 0.842), age (OR = 1.70, 95% CI 0.98-2.93, P = 0.057) and lymph node metastasis (OR = 1.65, 95% CI 0.87-3.14, P = 0.127) of CRC patients. Further subgroup analysis by ethnicity demonstrated that RASSF1A promoter methylation was correlated with clinicopathological characteristics of CRC patients among Asians (clinical stage: OR = 2.55, 95% CI 1.55-4.20, P < 0.001; histological grade: OR = 2.70, 95% CI 1.44-5.06, P = 0.002; lymph node metastasis: OR = 4.09, 95% CI 1.49-11.26, P = 0.006; distant metastasis: OR = 5.38, 95% CI 1.73-16.70, P = 0.004), but not among Caucasians and Africans (all P > 0.05). Our meta-analysis has shown positive correlations between aberrant promoter methylation of RASSF1A gene and clinicopathological characteristics of CRC patients, especially among Asians.

Promoter hypermethylation of the tumor-suppressor genes RASSF1A, GSTP1 and CDH1 in endometrial cancer

Endometrial cancer is a common gynecological malignancy with a good prognosis in early stages of the disease. The CpG island in the promoter region of tumor-suppressor genes are frequently methylated in various types of human cancers. In the present study, we examined the methylation status of the GSTP1, CDH1 and RASSF1A genes in endometrioid endometrial cancer (EEC), endometrial complex hyperplasia (EHP) and healthy endometrium with the aim to identify correlations between promoter hypermethylation, disease risk and clinicopathological parameters. A nested two-stage methylation-specific PCR (MSP) was performed to analyze the promoter CpG methylation status of GSTP1, CDH1 and RASSF1A genes in the population studied. A total of 92 subjects were initially included in the study of which 41 EEC, 19 EHP and 20 controls were processed for final analyses. A significant difference was found between the study groups and the presence of promoter CpG hypermethylation status in the GSTP1 (p<0.05) and RASSF1A (p<0.0001) genes. RASSF1A, GSTP1 and CDH1 gene promoter methylation was present in 85.4, 68.3 and 31.4% of EEC samples when compared to that in the controls with 30.0, 35.0 and 20.0%, respectively. CpG methylation of all three investigated tumor-suppressor genes was found in 12.2% of EEC patients, in 4.2% of EHP patients and in 3.7% of the controls, respectively. Positive findings for the promoter methylation of two investigated genes were found in 48.7% of EEC patients, 26.0% of EHP patients and in 18.5% of the controls. With regard to histopathological variables and CpG methylation, we found significant correlations between the RASSF1A and GSTP1 genes and higher tumor grade, deeper myometrial invasion and positive metastatic involvement of pelvic lymph nodes. No associations were noted between promoter hypermethylation of the CDH1 gene and biological features of the endometrial cancer cases. The results indicate that aberrant CpG methylation of the promoter region in the GSTP1 and RASSF1A tumor-suppressor genes is an important event in carcinogenesis of endometrial cancer and may have an impact on tumor aggressiveness. Finally, the present study suggests that epigenetic alterations may be of diagnostic value for the better clinical management of premalignant endometrial lesions.

Polymorphism of A133S and promoter hypermethylation in Ras association domain family 1A gene (RASSF1A) is associated with risk of esophageal and gastric cardia cancers in Chinese population from high incidence area in northern China

BackgroundThe role of tumor suppressor gene RASSF1A in the esophageal and gastric cardia carcinogenesis is still inconclusive. In this study, the polymorphism, promoter methylation and gene expression of RASSF1A were characterized in esophageal squamous cell carcinoma (ESCC) and gastric cardia adenocarcinoma (GCA).MethodsWe firstly analyzed the prevalence of RASSF1A A133S in a total of 228 cancer patients with ESCC (n=112) and GCA (n=116) and 235 normal controls by polymerase chain reaction (PCR) and restriction enzyme-digestion assay. Then, the promoter methylation status of the RASSF1A in ESCC (n=143), GCA (n=92) and corresponding adjacent normal tissues were further investigated using methylation-specific PCR (MSP) approach. Finally, the RASSF1A protein expression were determined in ESCC (n=27), GCA (n=24) and the matched adjacent normal tissues by immunohistochemical method.ResultsThe frequency of 133Ala/Se and Ser/Ser genotype was significantly higher in GCA patients than in normal controls (19.0% vs. 10.2%, P=0.02). Compared with Ala/Ala genotype, Ala/Se and Ser/Ser genotype significantly increased susceptibility to GCA (OR=2.06, 95% CI=1.09–3.97). However, this polymorphism had no association with ESCC (P=0.69). The promoter methylation of RASSF1A gene was significantly increased the risk to both ESCC (OR=5.90, 95% CI=2.78–12.52) and GCA (OR=7.50, 95% CI= 2.78–20.23). Promoter methylation of RASSF1A gene in ESCC was also associated with age and cancer cell differentiation (for age: OR=3.11, 95% CI=1.10–8.73; for differentiation: OR=0.29, 95% CI=0.12–0.69). RASSF1A positive expression was significantly decreased the risk of GCA (OR=0.16, 95% CI=0.03–0.83). In contrast, there was no statistical significance between RASSF1A positive expression and ESCC. The expression of RASSF1A protein trend to be positively related with older GCA patients (OR=16.20, 95% CI=1.57–167.74).ConclusionsThe present findings suggest that alterations of RASSF1A may play an important role in gastric cardia carcinogenesis in terms of polymorphism, promoter hypermethylation and protein expression. Whereas, RASSF1A hypermethylation may probably also be involved in esophageal squamous cell carcinogenesis.

Aberrant promoter methylation of the RASSF1A and APC genes in epithelial ovarian carcinoma development

Tumor suppressor gene (TSG) silencing through promoter hypermethylation plays an important role in cancer development. The aim of this study was to assess the extent of methylation of the RASSF1A and APC TSG promoters in ovarian epithelial adenomas, low malignant potential tumours and carcinomas in order to reveal a role for epigenetic TSG silencing in the development of these ovarian malignancies. The promoter methylation status of the RASSF1A and APC genes was assessed in 19 benign cystadenomas, 14 low malignant potential (LMP) tumours, and 86 carcinomas using methylation specific PCR (MSP). The methylation frequencies of the RASSF1A and APC gene promoters in benign cystadenomas were found to be 37% and 16%, respectively. The LMP tumours exhibited RASSF1A and APC gene promoter methylation frequencies of 50% and 28%, respectively, whereas the carcinomas exhibited methylation frequencies of 58% and 29%, respectively. Methylation of either the RASSF1A or the APC gene promoter was encountered in 58% of the invasive carcinomas. The observed aberrant methylation frequencies of the RASSF1A and APC gene promoters indicate that an accumulation of epigenetic events at these specific TSG promoters may be associated with the malignant transformation of benign cystadenomas and LMP tumours to carcinomas.

Abstract 3148A: MYOD1, hTERT, and RASSF1A gene promoter methylation is useful for prediction of outcome/ prognostication in patients with invasive cervical cancer

Abstract Background: Cervical cancer is the leading cause of cancer related mortality in women worldwide including India. DNA methylation, an epigenetic event regulates gene expression and chromatin structure. We hypothesized that the promoter methylation of genes involved in tumor suppression, DNA repair, cell cycle and apoptosis may affect response to radiation therapy and therefore the outcome of women with invasive cervical cancer. Objective: To perform DNA methylation profiling of BRCA1, RASSF1A, MLH1, ESR1, MYOD1 and hTERT genes in tissue samples of invasive cervical cancer and correlate the methylation pattern to radiation response and clinical outcome. Methodology: After obtaining informed consent, patients with invasive cervical cancer who underwent chemo-radiation as per protocol and with a minimum of 2 years follow-up after completion of treatment were included in the analysis [N=91; 2008-11]. The patients were followed up 3 monthly for evidence of local or systemic disease and their outcome documented. 60 patients were alive and well with No evidence of disease [NED] at the end of 24 months whereas 31 patients had evidence of disease [ED]. Out of these 31 patients, 3 died of disease, 9 had evidence of systemic disease [SED] and 19 patients had local disease recurrence [LED]. Genomic DNA was extracted from the snap frozen cervical biopsy tissues using TRIzol reagent, modified using the EZDNA methylation kit and promoter methylation of the individual genes determined by Real Time Polymerase Chain Reaction using Taqman probe chemistry using previously published primer and probe sets and appropriate controls. The probes were labelled with 5′-FAM and 3′-BHQ respectively. The housekeeping gene α-ACTIN was taken as the reference gene. Statistical analysis: Pearson's Chi-square test with Yates correction and Step-wise Logistic Regression was performed using the SPSS package version 18.0. A p-value &amp;lt;0.05 was considered as statistically significant. Results: In invasive cervical cancer, BRCA1, RASSF1A, MLH1, MYOD1, ESR1 and hTERT gene promoters were methylated in 51%, 47%, 40%, 51%, 38% and 60% cases respectively. Comparison of the NED vs LED groups revealed that methylated MYOD1 and hTERT genes in combination predicted for better outcome (Chi-Square test, p=0.03). Further logistic regression analysis revealed that methylation of RASSF1A, MYOD1 and hTERT genes predicted for better outcome following radiation therapy indicating that these genes play a role in determining radiation responsiveness of a tumor. The cross-classification table showed that 80% of the cases could be correctly classsified by the logistic model. Conclusion: A panel of 3 genes which includes MYOD1, hTERT and RASSF1A may be useful to predict the outcome of invasive cervical carcinoma patients treated with standard chemo-radiation therapy. 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 3148A. doi:1538-7445.AM2012-3148A

Relationship between the expression of RASSF1A protein and promoter hypermethylation of RASSF1A gene in bladder tumor

To investigate the relationship between the expression of RASSF1A protein and promoter hypermethylation of RASSF1A gene, RASSF1A protein expression was measured by Western blotting in 10 specimens of normal bladder tissues and 23 specimens of bladder transitional cell carcinoma (BTCC). The promoter methylation in BTCC and normal bladder tissues was detected by methylation-specific PCR (MSP). The results showed that the expression level of RASSF1A protein was significantly lower in BTCC tissues than that in normal bladder tissues. However, it was not correlated with its clinical stages and pathological grades. The frequency of promoter methylation of RASSF1A gene was higher in BTCC tissues than that in normal bladder tissues. In 14 patients with the aberrant promoter methylation, 13 showed loss or low expression of RASSF1A protein. It is concluded that RASSF1A gene promoter methylation may contribute to the low level or loss of RASSF1A protein expression, the inactivation of RASSF1A gene and the genesis of BTCC. But, it may bear no correlation with its clinical stages and pathological grades.

ΔDNMT3B Variants Regulate DNA Methylation in a Promoter-Specific Manner

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

Promoter methylation of RASSF1A and DAPK and mutations of K-ras, p53, and EGFR in lung tumors from smokers and never-smokers

BackgroundEpidemiological studies indicate that some characteristics of lung cancer among never-smokers significantly differ from those of smokers. Aberrant promoter methylation and mutations in some oncogenes and tumor suppressor genes are frequent in lung tumors from smokers but rare in those from never-smokers. In this study, we analyzed promoter methylation in the ras-association domain isoform A (RASSF1A) and the death-associated protein kinase (DAPK) genes in lung tumors from patients with primarily non-small cell lung cancer (NSCLC) from the Western Pennsylvania region. We compare the results with the smoking status of the patients and the mutation status of the K-ras, p53, and EGFR genes determined previously on these same lung tumors.MethodsPromoter methylation of the RASSF1A and DAPK genes was analyzed by using a modified two-stage methylation-specific PCR. Data on mutations of K-ras, p53, and EGFR were obtained from our previous studies.ResultsThe RASSF1A gene promoter methylation was found in tumors from 46.7% (57/122) of the patients and was not significantly different between smokers and never-smokers, but was associated significantly in multiple variable analysis with tumor histology (p = 0.031) and marginally with tumor stage (p = 0.063). The DAPK gene promoter methylation frequency in these tumors was 32.8% (40/122) and did not differ according to the patients' smoking status, tumor histology, or tumor stage. Multivariate analysis adjusted for age, gender, smoking status, tumor histology and stage showed that the frequency of promoter methylation of the RASSF1A or DAPK genes did not correlate with the frequency of mutations of the K-ras, p53, and EGFR gene.ConclusionOur results showed that RASSF1A and DAPK genes' promoter methylation occurred frequently in lung tumors, although the prevalence of this alteration in these genes was not associated with the smoking status of the patients or the occurrence of mutations in the K-ras, p53 and EGFR genes, suggesting each of these events may represent independent event in non-small lung tumorigenesis.

Quantitative evaluation of RASSF1Amethylation in the non-lesional, regenerative and neoplastic liver

BackgroundEpigenetic changes during ageing and their relationship with cancer are under the focus of intense research. RASSF1A and NORE1A are novel genes acting in concert in the proapoptotic pathway of the RAS signalling. While NORE1A has not been previously investigated in the human liver, recent reports have suggested that RASSF1A is frequently epigenetically methylated not only in HCC but also in the cirrhotic liver.MethodsTo address whether epigenetic changes take place in connection to age and/or to the underlying disease, we investigated RASSF1A and NORE1A gene promoter methylation by conventional methylation specific PCR and Real-Time MSP in a series of hepatitic and non-hepatitic livers harboring regenerative/hyperplastic (cirrhosis/focal nodular hyperplasia), dysplastic (large regenerative, low and high grade dysplastic nodules) and neoplastic (hepatocellular adenoma and carcinoma) growths.ResultsIn the hepatitic liver (chronic hepatitic/cirrhosis, hepatocellular nodules and HCC) we found widespread RASSF1A gene promoter methylation with a methylation index that increased from regenerative conditions (cirrhosis) to hepatocellular nodules (p < 0.01) to HCC (p < 0.001). In the non-hepatitic liver a consistent pattern of gene methylation was also found in both lesional (focal nodular hyperplasia and hepatocellular adenoma) and non-lesional tissue. Specifically, hepatocellular adenomas (HA) showed a methylation index significantly higher than that detected in focal nodular hyperplasia (FNH) (p < 0.01) and in non-lesional tissue (p < 0.001). In non-lesional liver also the methylation index gradually increased by ageing (p = 0.002), suggesting a progressive spreading of methylated cells over time. As opposed to RASSF1A gene promoter methylation, NORE1A gene was never found epigenetically alterated in both hepatitic and non-hepatitic liver.ConclusionWe have shown that in non-lesional, regenerative and neoplastic liver the RASSF1A gene is increasingly methylated, that this condition takes place as an age-related phenomenon and that the early setting and spreading over time of an epigenetically methylated hepatocyte subpopulation, might be related to liver tumorigenesis.