RASSF1A Promoter Hypermethylation Research Articles

Low-dose cadmium exposure facilitates cell proliferation by promoter hypermethylation of RASSF1A and DAPK1 genes.

Cadmium (Cd) at low concentrations has a potential to promote cell proliferation. However, the molecular mechanisms of Cd-induced proliferation are not well understood. Here, we reported that Cd (0-500 nM) significantly promoted the proliferation of HepG2 cells as demonstrated by elevated cell viability, more EdU-positive cells and increased gene expression of KI-67 and COX-2. Meanwhile, the gene expression of DNA methyltransferases was found to be elevated while that of tumor suppressor genes DAPK1 and RASSF1A were decreased under Cd exposure. Correspondingly, the methylation level of promoters in DAPK1 and RASSF1A were increased. Specifically, the CpG sites at -461 (Chr3:50, 374, 481) of RASSF1A promoter, and that at -260 (Chr9:90, 113, 207), -239 (Chr9:90, 113, 228), and -68 (Chr9:90, 113, 399) of DAPK1 promoter, were significantly hypermethylated. Moreover, 5-azacytidine (an inhibitor of DNA methyltransferase) partly impaired Cd-induced promoter hypermethylation of RASSF1A and DAPK1 genes, increased their expressions and slowed down Cd-induced cell proliferation, suggesting that DNA methylation play an essential part in Cd-boosted proliferation. The study showed that Cd caused promoter hypermethylation of RASSF1A and DAPK1, decreasing their expression and leading to higher level of cell proliferation. Furthermore, Cd at low concentrations could influence DNA methylation, which may serve as the proliferative mechanism of Cd.

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.

Assessment of Aberrant Promoter Hypermethylation of RASSF1a and P16 in Endometrial Carcinoma

Endometrial carcinoma is one of the most common gynecological malignancies known to affect around 142000 women worldwide. Aberrant promoter hypermethylation of tumor suppressor genes is a common epigenetic alteration reported in cancers. In the present study we report the aberrant promoter hypermethylation of RASSF1a and p16 in 78 endometrial cancer patients. Methods: Endometrial carcinoma samples were collected after pathological examination and DNA was extracted. The DNA was subjected to sodium bisulfite modification and then used to perform methylation specific PCR. The PCR was performed using a two step procedure of nested and methylation specific PCR. The PCR product was visualized using agarose gel electrophoresis. The results obtained were correlated with various clinicopathological parameters. Chi square test was used for statistical analysis and a p-value of <0.05 was considered statistically significant. Result: 60 of the 78 (76.92%) samples showed methylation for RASSF1a gene. 28 of the 78 (35.8%) samples showed methylation for p16 gene. A higher methylation frequency was observed for RASSF1a in the endometrioid and poorly differentiated histological subtypes and stage I and II of the disease. Conclusion: Aberrant promoter hypermethylation of RASSF1a and p16 is known to play an important role in endometrial cancer initiation and progression. The methylation patterns can be used as an important molecular marker for early diagnosis and prognosis of endometrial cancer. Keywords: Epigenetic alterations, DNA Hypermethylation, Endometrial cancer, RASSF1a, p16, Methylation-specific PCR.

Aberrant Promoter Hypermethylation of p16 and RASSF1a Genes in Colorectal Cancer - Significance in Young Patients.

The clinical profile of colorectal cancers (CRC) in India is different from that described in western countries. Microsatellite instability and APC mutation explain the molecular biology of up to 50% of colorectal cancers. Global genome hypermethylation may be the cause in at least 20% of cases. Few studies from India have examined the epigenetic profile of colorectal cancers. This study was designed to study aberrant promoter hypermethylation of two select tumour suppressor genes (p16, RASSF1a) in patients with colorectal cancer and their association with clinicopathologic features. A total of 41 samples including controls were collected from colorectal cancer patients. DNA was isolated from tumour tissue, and methylation-specific PCR was performed for the 2 genes. p16 and RASSF1a promoter hypermethylation was found in 26% and 48% of CRC cases, respectively. RASSF1a promoter hypermethylation was more often seen in young CRC patients aged 40years or less, and this was found to be statistically significant (p value = 0.037). RASSF1a hypermethylation is peculiar to rectal cancers and left-sided colonic tumours in young patients. Large-scale population-based studies with extensive genetic and epigenetic characterization are required for a better understanding and further validation of our findings. For individuals diagnosed with sporadic CRC, these studies on specimen might help predict prognosis and response to therapy.

Identification of RASSF1A promoter hypermethylation as a biomarker for hepatocellular carcinoma

BackgroundRAS association domain family protein 1A (RASSF1A) promoter hypermethylation is suggested to be linked to hepatocellular carcinoma (HCC), but the results remained controversial.MethodsWe evaluated how RASSF1A promoter hypermethylation affects HCC risk and its clinicopathological characteristics through meta-analysis. Data on DNA methylation in HCC and relevant clinical data were also collected based on The Cancer Genome Atlas (TCGA) database to investigate the prognostic role of RASSF1A promoter hypermethylation in HCC.ResultsForty-four articles involving 4777 individuals were enrolled in the pooled analyses. The RASSF1A promoter methylation rate was notably higher in the HCC cases than the non-tumor cases and healthy individuals, and was significantly related to hepatitis B virus (HBV) infection-positivity and large tumor size. Kaplan–Meier survival analysis revealed that HCC cases with RASSF1A promoter hypermethylation had worse outcomes. Receiver operating characteristic curves confirmed that RASSF1A promoter methylation may be a marker of HCC-related prognoses.ConclusionsRASSF1A promoter hypermethylation is a promising biomarker for the diagnosis of HCC from tissue and peripheral blood, and is an emerging therapeutic target against HCC.

ROLE OF HYPERMETHYLATION (RASSF1A) PROMOTER GENE AMONG PATIENTS WITH HEPATOCELLULAR CARCINOMA

Background: Hepatocellular carcinoma (HCC) is one of the most aggressive malignancies worldwide and one of the main causes of cancer-related mortality globally. Its incidence is increasing at alarming rates. The presence of cirrhosis is the major risk factor and this is largely due to chronic HCV and HBV infection. Serum alpha-fetoprotein (AFP) has insufficient sensitivity and specificity for detection of hepatocellular carcinoma (HCC). Aberrant hypermethylation of tumor suppressor genes e.g(RASSF1A ) is one of the most frequent and early mechanisms involved in HCC development, so that it could help to select high-risk populations and thus to modulate the indications of screening procedures. Objectives: To evaluate the frequency of tumor suppressor gene RASSF1A hypermethylation in whole blood from HCC patients Methods: The study included eighty subjects : 30 patients with HCC and elevated AFP. ; 30 with liver cirrhosis In addition, 20 healthy subjects were included as a control group. Clinical and radiological features (abdominal ultrasonography and/or abdominal triphasic computed tomography) were recorded. Liver function tests, complete blood cell count, and serum AFP were measured.. Detection of promotor methylation status of RASSF1A using methylation specific PCR . Results: The obtained results showed a significant RASSF1A promoter hypermethylation in HCC subjects that was 83.3% in comparison to healthy control subjects as well as in comparison to subjects with non HCC chronic liver disease. Conclusion:.Detection of methylated RASSF1A promoter is useful marker for HCC screening in high risk vulnerable patients and early HCC diagnosis.

Aberrant Promoter Hypermethylation of RASSF1a and BRCA1 in Circulating Cell-Free Tumor DNA Serves as a Biomarker of Ovarian Carcinoma.

Objective:Ovarian cancer is one of the leading causes of cancer deaths in women. Ovarian cancer is diagnosed at the late stages and generally relapses within 12-14 months of cytoreductive surgery. This is attributed to lack of precise molecular detection methodologies to detect and track the disease. Epigenetic alteration such as aberrant promoter hypermethylation is an important early event that occurs during cancer development and progression. This study focuses on development of a minimally invasive methylation marker that could be used for detection and prognosis of ovarian cancer patients. Methods:Aberrant promoter hypermethylation of RASSF1a and BRCA1 was assessed in circulating DNA of 72 EOC patients using methylation-specific PCR. The findings were correlated with various clinicopathological parameters. Statistical analysis was done using the Fisher exact test and chi-square test. Results:The aberrant methylation patterns of RASSF1a and BRCA1 was identified to be present in the cancerous samples. A total of 31.9 % and 56.9% methylation was observed for RASSF1a and BRCA1 respectively. A striking 50% methylation of BRCA1 was identified in the benign sample cohort, which marks the significance of assessing the hypermethylation pattern to detect cancer at its early stages. Methylation of the two tumor suppressor genes was evident across various stages and grades of ovarian tumors suggesting that this could also help as a prognostic marker. Conclusion:The results of the current study hold significance since the hypermethylation patterns can be identified in the cell-free circulating tumor DNA from a small volume of blood plasma and is a simple and minimally-invasive method. Assessment of hypermethylation patterns of a panel of TSG along with the existing screening markers could aid in better diagnosis and management of the disease. It could also aid in designing specifically tailored treatment strategies to fight the disease.

Correlation Between RASSF1A Gene Promoter Hypermethylation in Serum or Sputum and Non-Small Cell Lung Cancer (NSCLC): A Meta-Analysis.

BackgroundThe aim of this study was to evaluate the efficacy of RASSF1A promoter hypermethylation of serum or sputum in diagnosis of non-small cell lung cancer (NSCLC) by pooling open published data.Material/MethodsOpen-published studies relevant to RASSF1A promoter hypermethylation and NSCLC diagnosis were screened through Medline, EMBASE, the Cochrane Library, Web of Science, Google Scholar, and CBM. Number of cases of true positive (tp), false positive (fp), false negative (fn), and true negative (tn) by RASSF1A gene promoter hypermethylation was extracted from each of the include original studies. The combined diagnostic sensitivity, specificity, and symmetric receiver operating characteristic curve (SROC) were calculated, as was the effect size.ResultsTwelve studies with 826 NSCLC and 598 controls were included in the present work. The combined sensitivity and specificity were 0.45 (95%CI: 0.41–0.48) (random effects) and 0.99(95%CI: 0.98–1.00) (fixed effects) respectively. The pooled positive likelihood ratio (+LR) and negative likelihood ratio (−LR) were 20.27 (9.64–42.61) and 0.53 (0.42–0.66), respectively, through the random effects model. The combined DOR was 46.63 (95%CI: 17.30–125.65) through the fixed effects model. The AUC of the SROC was 0.9989, calculated through Moses’s model for RASSF1A promoter hypermethylation as a biomarker in diagnosis of NSCLC.ConclusionsThe low diagnostic sensitivity for RASSF1A gene promoter hypermethylation indicated that it is not suitable for NSCLC screening. However, the high specificity made it effective for NSCLC confirmation diagnosis, which could be used instead of pathological diagnosis.

Association of Peripheral Blood RASSF1A and CDKN2A Methylation Status with Smoking Behaviour in Nasopharyngeal Carcinoma

BACKGROUND: Hypermethylation of RASSF1A and CDKN2A is one of epigenetic factor underlies nasopharyngeal carcinoma (NPC) development. Smoking behavior as an NPC’s risk factor causes aberrant DNA methylation. RASSF1A and CDKN2A promoter hypermethylation from peripheral blood cells correlates with smoking behavior. The use of body fluids including peripheral blood as a specimen for DNA methylation analyzes are widely developed, as less invasive method compared to the use of tissue biopsy. This study aims to observe the association between RASSF1A and CDKN2A methylation in peripheral blood and smoking behavioramong NPC patients.METHODS: Newly diagnosed NPC subjects were recruited from ear-nose-throat (ENT) outpatient clinic of Dr. Sardjito Hospital, Yogyakarta. DNA from buffycoat of 19 smokers and 20 non-smokers NPC’s patients were isolated. Bisulphite modification was applied to 500 ng of the isolated DNA. The methylation status was detected by MSP (methylation-specific polymerase chain reaction (PCR)). The association between smoking status and promoter hypermethylation was analysis using Chi-Square test.RESULTS: MSP analysis of RASSF1A showed that 68.42% smoker and 75% non-smoker NPC’s patients were methylated. MSP analysis of CDKN2A showed that 21.05% smoker and 25% non-smoker NPC’s patients were methylated. There was no association between smoking behavior with RASSF1A and CDKN2A methylation (p>0.05).CONCLUSION: Statistical analysis showed that smoking behavior is not associated with methylation of RASSF1A and CDKN2A among NPC’s patients.KEYWORDS: DNA methylation, CDKN2A, RASSF1A, Nasopharyngeal carcinoma, Smoking

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.

Combination of LINE-1 hypomethylation and RASSF1A promoter hypermethylation in serum DNA is a non-invasion prognostic biomarker for early recurrence of hepatocellular carcinoma after curative resection.

Hepatocarcinogenesis, a multistep process, involves not only genetic mutations but also epigenetic alterations. Widespread of global DNA hypomethylation is accompanied with specific regional hypermethylation especially at tumor suppressor genes' promoters. The aim of this study is to determine the efficacy of combined DNA methylation analysis of a global DNA methylation marker - LINE-1 and a tumor suppressor gene highly associated with the malignancy of HCC- RASSF1A in serum as a novel prognostic marker for diagnosis of early recurrence after curative resection.LINE-1 was hypomethylated in 66.7% (70/105) and RASSF1A promoter was hypermethylated in 73.3% (77/105) of HCC serum DNA samples by methylation specific PCR, but in none of the healthy controls: LINE-1 hypometylation (0/50) and RASSF1A hypermethylation (0/50). A significant association was found between LINE-1 hypomethylation and clinical pathologic features including HBsAg positivity (p=0.009), tumor size (p=0.001) and AFP levels (p<0.001). Besides, significant correlation was detected between RASSF1A promoter hypermethylation and lymph nodes metastasis (p=0.045).The results of Kaplan-Meier estimates of survival suggested that LINE-1 hypomethylation was highly associated with poor survival of patients (disease-free survival p=0.002, overall survival p=0.0123). More importantly, co-evaluation of LINE-1 hypomethylation and RASSF1A promoter hypermethylation was found to be significantly correlated to early recurrence and poor prognosis (disease-free survival p=0.0001, overall survival p=0.05) in patients after curative resection.In conclusion, our study showed that the combined examination of LINE-1 hypomethylation and RASSF1A promoter hypermethylation was effective in predicting early recurrence of HCC after curative resection. Patients with dual positivity of LINE-1 hypomethylation and RASSF1A promoter hypermethylation should be supplied with more intensive care and close follow-up after they undergo tumor resection.

Diminished expression of MGMT & RASSF1A genes in gastric cancer in ethnic population of Kashmir.

Cancer initiation and progression are accompanied by profound changes in DNA. DNA methylation that was the first epigenetic alterations identified in cancer. DNA hypermethylation at promoter sites is closely associated with down regulation of protein and as major participant in the development and progression of series of human tumors. Therefore we hypothesized that promoter hypermethylation of RASSF1A & MGMT gene could influence susceptibility to gastric cancer (GC) as well, and we conducted this study to test the hypothesis in Kashmiri population. A hospital based case-control study; including 200 GC cases and 200 matched controls from patients who went surgical resection. Promoter hypermethylation was determined by Methylation Specific Polymerase chain reaction. The expression of MGMT & RASSF1A protein was examined by Western blotting technique. Frequency of promoter region hypermethylation of MGMT gene were 46.5% in cases and 5.5% in controls (P<0.05) while as in case of RASSF1A frequency was 44% in cases and 4.5% in controls (P<0.05). Further, frequency of hypermethylation of both genes was found predominant in males, aged and advanced pathological stage subjects. Loss of MGMT expression was found in 46.5% cases (P<0.05) while as loss of RASSF1A expression was found in 40.5% cases (P<0.05). In both genes a positive correlation was observed between promoter CpG island hypermethylation and down regulation of respective proteins. These findings indicate that promoter hypermethylation at CpG island may be responsible for reduction of expression at protein level which may be an initial event in carcinogenesis and the progression of GC.

42P Promoter hypermethylation of BRCA1, DAPK1 and RASSF1A is associated with increase mortality among breast cancer patients

42P Promoter hypermethylation of BRCA1, DAPK1 and RASSF1A is associated with increase mortality among breast cancer patients

42P Promoter hypermethylation of BRCA1, DAPK1 and RASSF1A is associated with increase mortality among breast cancer patients

42P Promoter hypermethylation of BRCA1, DAPK1 and RASSF1A is associated with increase mortality among breast cancer patients

Decitabine and 5-azacitidine both alleviate LPS induced ARDS through anti-inflammatory/antioxidant activity and protection of glycocalyx and inhibition of MAPK pathways in mice.

Decitabine (5-aza-2'-deoxycytidine, DAC) and 5-azacitidine (Aza), an inhibitor of DNA methyltransferases, possess a wide range of anti-metabolic and anti-cancer activities. This study examined the effects of DAC and Aza on inflammatory and oxidative injuries, as well as on glycocalyx and MAPK signaling pathways, in a LPS-stimulated ARDS mouse model. Results of ELISA revealed that DAC and Aza significantly inhibited the production of TNF-α and IL-1β and prevented LPS-induced elevation of myeloperoxidase and malondialdehyde levels in serum. The W/D ratio of lung and histopathologic examination with hematoxylin and eosin staining showed that DAC and Aza pretreatment substantially improved lung tissue injury. DAC and Aza reduced the level of glycocalyx degradation products (e.g., heparan sulfate and haluronic acid) and protected glycocalyx integrity. Western blot assay demonstrated that DAC and Aza both significantly suppressed LPS-induced activation of the MAPK signaling pathways by blocking the phosphorylation of JNK, ERK and P38 in lung tissues. Bisulfite sequencing PCR and real time-PCR showed that DAC reversed the RASSF1A promoter hypermethylation and furthermore elevated the expression of RASSF1A, which is a tumor suppressor that regulates MAPK signaling pathway. These results suggested that DAC inhibited the MAPK signaling pathway in LPS-induced ARDS mice might via demethylation in RASSF1A promoter region and by restoring its expression. This study highlighted the close relationship between DNA methylation and the development and progression of ARDS.

RASSF1A and DOK1 Promoter Methylation Levels in Hepatocellular Carcinoma, Cirrhotic and Non-Cirrhotic Liver, and Correlation with Liver Cancer in Brazilian Patients.

Hepatocellular carcinoma (HCC) is the second most common cause of cancer mortality worldwide. Most cases of HCC are associated with cirrhosis related to chronic hepatitis B virus or hepatitis C virus infections. Hypermethylation of promoter regions is the main epigenetic mechanism of gene silencing and has been involved in HCC development. The aim of this study was to determine whether aberrant methylation of RASSF1A and DOK1 gene promoters is associated with the progression of liver disease in Brazilian patients. Methylation levels were measured by pyrosequencing in 41 (20 HCC, 9 cirrhotic, and 12 non-cirrhotic) liver tissue samples. Mean rates of methylation in RASSF1A and DOK1 were 16.2% and 12.0% in non-cirrhotic, 26.1% and 19.6% in cirrhotic, and 59.1% and 56.0% in HCC tissues, respectively, showing a gradual increase according to the progression of the disease, with significantly higher levels in tumor tissues. In addition, hypermethylation of RASSF1A and DOK1 was found in the vast majority (88%) of the HCC cases. Interestingly, DOK1 methylation levels in HCC samples were significantly higher in the group of younger (<40 years) patients, and higher in moderately differentiated than in poorly differentiated tumors (p < 0.05). Our results reinforce the hypothesis that hypermethylation of RASSF1A and DOK1 contributes to hepatocarcinogenesis and is associated to clinicopathological characteristics. RASSF1A and DOK1 promoter hypermethylation may be a valuable biomarker for early diagnosis of HCC and a potential molecular target for epigenetic-based therapy.

Aberrant Promoter Methylation of the Tumour Suppressor RASSF10 and Its Growth Inhibitory Function in Breast Cancer.

Breast cancer is the most common cancer in women, with 1.7 million new cases each year. As early diagnosis and prognosis are crucial factors in cancer treatment, we investigated potential DNA methylation biomarkers of the tumour suppressor family Ras-association domain family (RASSF). Promoter hypermethylation of tumour suppressors leads to their inactivation and thereby promotes cancer development and progression. In this study we analysed the tumour suppressors RASSF1A and RASSF10. Our study shows that RASSF10 is expressed in normal breast but inactivated by methylation in breast cancer. We observed a significant inactivating promoter methylation of RASSF10 in primary breast tumours. RASSF10 is inactivated in 63% of primary breast cancer samples but only 4% of normal control breast tissue is methylated (p < 0.005). RASSF1A also shows high promoter methylation levels in breast cancer of 56% vs. 8% of normal tissue (p < 0.005). Interestingly more than 80% of breast cancer samples harboured a hypermethylation of RASSF10 and/or RASSF1A promoter. Matching samples exhibited a strong tumour specific promoter methylation of RASSF10 in comparison to the normal control breast tissue. Demethylation treatment of breast cancer cell lines MCF7 and T47D reversed RASSF10 promoter hypermethylation and re-established RASSF10 expression. In addition, we could show the growth inhibitory potential of RASSF10 in breast cancer cell lines MCF7 and T47D upon exogenous expression of RASSF10 by colony formation. We could further show, that RASSF10 induced apoptotic changes in MCF7 and T47D cells, which was verified by a significant increase in the apoptotic sub G1 fraction by 50% using flow cytometry for MCF7 cells. In summary, our study shows the breast tumour specific inactivation of RASSF10 and RASSF1A due to DNA methylation of their CpG island promoters. Furthermore RASSF10 was characterised by the ability to block growth of breast cancer cell lines by apoptosis induction.

Decreased expression of RASSF1A tumor suppressor gene is associated with worse prognosis in clear cell renal cell carcinoma.

Clear-cell renal cell carcinoma (ccRCC) is the most common subtype of RCC (70–80%) and is associated with poor prognosis in 40% of cases mainly due to metastasis in the course of the disease. RASSF1, with its isoforms RASSF1A and RASSF1C, is a tumor suppressor gene which has not been fully analyzed in ccRCC yet. The epigenetic downregulation of RASSF1A is commonly associated with promoter hyper-methylation. The aim of the present study was to compare the ccRCC outcomes with the expression of RASSF1A and RASSF1C. Tissues were obtained from 86 ccRCC patients. RASSF1A and RASSF1C mRNA levels were assessed in tumor and matched normal kidney tissue, and in 12 samples of local metastases by quantitative PCR (qPCR). RASSF1A and RASSF1C proteins levels were semi-quantified in 58 samples by western blot analysis and their tissue localization was assessed by immunohistochemistry. Hypermethylation of RASSF1A promoter was measured by high-resolution-melting methylation-specific qPCR. RASSF1A mRNA levels were 4 and 5 times lower in 66% of tumor and 75% metastasized samples. RASSF1A hypermethylation was found in 40% of analyzed T cases. RASSF1A protein expression was 5 or 20 times decreased in 70% tumor and 75% metastatic samples, respectively. RASSF1A hypermethylation, mRNA and protein levels were associated with TNM progression and higher Fuhrman's grading. Decreased RASSF1A expression, hyper-methylation, TNM and Fuhrman's grading were associated with poorer overall survival (OS). Cox hazard ratio (HR) analysis revealed predictor role of RASSF1A mRNA levels on OS and progression-free survival (PFS) in relation to Fuhrman's grading (OS HR=2.25, PFS HR=2.93). RASSF1C levels were increased in ccRCC; no correlations with clinicopathological variables were found. We conclude that RASSF1C gene is not involved in ccRCC progression and we propose that the measurements of RASSF1A mRNA levels in paired tumor-normal kidney tissue could serve as a new prognostic factor in ccRCC.

Association between RASSF1A Promoter Hypermethylation and Oncogenic HPV Infection Status in Invasive Cervical Cancer: a Meta-analysis.

Cervical carcinoma is the main cause of cancer-related mortality in women and is correlated with more than 15 risk cofactors, including infection of cervical cells with high-risk types of HPV (hrHPV). Indeed, both aberrant methylation of the RASSF1A promoter and hrHPV infection are often observed in cervical carcinomas. The purpose of our meta-analysis was to evaluate the role of RASSF1A promoter methylation and hrHPV infection in cervical cancer. Our meta-analysis involved 895 cervical cancer patients and 454 control patients from 15 studies. Our results suggested that RASSF1A promoter hypermethylation increased the risk of cervical cancer (OR=9.77, 95%CI=[3.06, 31.26], P=0.0001, I2=78%). By grouping cases according to cancer subtypes, we found that HPV infection was higher in cervical squamous cell carcinomas (SCCs) than in cervical adenocarcinomas/ adenosquamous cancers (ACs/ASCs) (OR=4.00, 95%CI=[1.41, 11.30], P=0.009, I2=55%). Interestingly, HPV infection tended to occur in cervical cancers with relatively low levels of RASSF1A promoter methylation (OR=0.59, 95%CI=[0.36, 0.99], P=0.05, I2=0%). Our study provides evidence of a possible interaction between HPV infection and RASSF1A promoter methylation in the development of cervical cancers.

Prognostic role of APC and RASSF1A promoter methylation status in cell free circulating DNA of operable gastric cancer patients

Gastric carcinogenesis is a multistep process including not only genetic mutations but also epigenetic alterations. The best known and more frequent epigenetic alteration is DNA methylation affecting tumor suppressor genes that may be involved in various carcinogenetic pathways. The aim of the present study was to investigate the methylation status of APC promoter 1A and RASSF1A promoter in cell free DNA of operable gastric cancer patients.Using methylation specific PCR, we examined the methylation status of APC promoter 1A and RASSF1A promoter in 73 blood samples obtained from patients with gastric cancer.APC and RASSF1A promoters were found to be methylated in 61 (83.6%) and 50 (68.5%) of the 73 gastric cancer samples examined, but in none of the healthy control samples (p<0.001). A significant association between methylated RASSF1A promoter status and lymph node positivity was observed (p=0.005). Additionally, a significant correlation between a methylated APC promoter and elevated CEA (p=0.033) as well as CA-19.9 (p=0.032) levels, was noticed. The Kaplan–Meier estimates of survival, significantly favored patients with a non-methylated APC promoter status (p=0.008). No other significant correlations between APC and RASSF1A methylation status and different tumor variables examined was observed.Serum RASSF1A and APC promoter hypermethylation is a frequent epigenetic event in patients with early operable gastric cancer. The observed correlations between APC promoter methylation status and survival as well as between a hypermethylated RASSF1A promoter and nodal positivity may be indicative of a prognostic role for those genes in early operable gastric cancer. Additional studies, in a larger cohort of patients are required to further explore whether these findings could serve as potential molecular biomarkers of survival and/or response to specific treatments.