Gene promoter methylation in colorectal cancer and healthy adjacent mucosa specimens: Correlation with physiological and pathological characteristics,and with biomarkers of one-carbon metabolism

We evaluated the promoter methylation levels of the APC, MGMT, hMLH1, RASSF1A and CDKN2A genes in 107 colorectal cancer (CRC) samples and 80 healthy adjacent tissues. We searched for correlation with both physical and pathological features, polymorphisms of folate metabolism pathway genes (MTHFR, MTRR, MTR, RFC1, TYMS, and DNMT3B), and data on circulating folate, vitamin B12 and homocysteine, which were available in a subgroup of the CRC patients. An increased number of methylated samples were found in CRC respect to adjacent healthy tissues, with the exception of APC, which was also frequently methylated in healthy colonic mucosa. Statistically significant associations were found between RASSF1A promoter methylation and tumor stage, and between hMLH1 promoter methylation and tumor location. Increasing age positively correlated with both hMLH1 and MGMT methylation levels in CRC tissues, and with APC methylation levels in the adjacent healthy mucosa. Concerning gender, females showed higher hMLH1 promoter methylation levels with respect to males. In CRC samples, the MTR 2756AG genotype correlated with higher methylation levels of RASSF1A, and the TYMS 1494 6bp ins/del polymorphism correlated with the methylation levels of both APC and hMLH1. In adjacent healthy tissues, MTR 2756AG and TYMS 1494 6bp del/del genotypes correlated with APC and MGMT promoter methylation, respectively. Low folate levels were associated with hMLH1 hypermethylation. Present results support the hypothesis that DNA methylation in CRC depends from both physiological and environmental factors, with one-carbon metabolism largely involved in this process.     

Hypermethylation of Genes Detected in Urine from Ghanaian Adults with Bladder Pathology Associated with Schistosoma haematobium Infection

Purpose

Schistosoma haematobium is associated with chronic bladder damage and may subsequently induce bladder cancer in humans, thus posing a serious threat where the parasite is endemic. Here we evaluated aberrant promoter DNA methylation as a potential biomarker to detect severe bladder damage that is associated with schistosomiasis by analyzing urine specimens.

Materials and Methods

A quantitative methylation-specific PCR (QMSP) assay was used to examine the methylation status of seven genes (RASSF1A, RARβ2, RUNX3, TIMP3, MGMT, P16, ARF) in 57 urine samples obtained from volunteers that include infected and uninfected by S. haematobium from an endemic region. The Fishers Exact Test and Logistic Regression analysis were used to evaluate the methylation status with bladder damage (as assessed by ultrasound examination) in subjects with S. haematobium infection.

Results

RASSF1A and TIMP3 were significant to predict severe bladder damage both in univariate (p = 0.015 and 0.023 respectively) and in multivariate (p = 0.022 and 0.032 respectively) logistic regression analysis. Area under the receiver operator characteristic curves (AUC-ROC) for RASSF1A and TIMP3 to predict severe bladder damage were 67.84% and 63.73% respectively. The combined model, which used both RASSF1A and TIMP3 promoter methylation, resulted in significant increase in AUC-ROC compared to that of TIMP3 (77.55% vs. 63.73%.29; p = 0.023).

Conclusions

In this pilot study, we showed that aberrant promoter methylation of RASSF1A and TIMP3 are present in urine sediments of patients with severe bladder damage associated with S. haematobium infection and that may be used to develop non-invasive biomarker of S. haematobium exposure and early molecular risk assessmentof neoplastic transformation.     

RASSF1A Promoter Hypermethylation Is a Strong Biomarker of Poor Survival in Patients with Salivary Adenoid Cystic Carcinoma in a Chinese Population

In addition to the clinicopathological parameters, molecular biomarkers are becoming increasingly important in the prognostic evaluation of cancer patients. This study aimed to determine the molecular alterations in the RAS association domain family protein1A gene (RASSF1A) in salivary adenoid cystic carcinoma (ACC) and to evaluate the potential of such alterations as prognostic markers. One hundred and sixty-seven ACC tumor tissues and 50 samples of matched normal salivary gland tissues from the same patients were analyzed for RASSF1A promoter methylation status by bisulfite sequencing PCR (BSP) and/or methylation-specific PCR (MSP). Fifty ACC tumor tissues and matched normal salivary gland tissues were analyzed for loss of heterozygosity (LOH) by examining two microsatellite markers (D3S1478, D3S1621) at 3p21. RASSF1A gene mutations were detected by direct sequencing of all six exons in 50 tumor and normal tissue specimens. Over-all, RASSF1A promoter hypermethylation was detected in 35.3% (59/167) of ACC tissues and was associated with histologically solid tumor pattern (P = 0.002) and advanced TNM stage (P = 0.014). RASSF1A LOH was observed in 18.0% (9/50) of cases, and no somatic mutation of RASSF1A was detected in any cases. RASSF1A promoter methylation was associated with the poor over-all survival (Log-rank test, P <0.001) and disease-free survival (Log-rank test, P <0.001) and identified as an independent predicator of over-all patient survival (P = 0.009) and disease-free survival (P <0.001). It was concluded that RASSF1A methylation is involved in the development, differentiation and progression of ACC and is a strong independent biomarker of poor survival in ACC patients in a Chinese population.     

Methylation of the Putative Tumor Suppressor Gene RASSF1A in Primary Cervical Tumors

Methylation-sensitive restriction endonuclease analysis (MSRA) followed by polymerase chain reaction (PCR) have been used to estimate the methylation level of 13 CpG dinucleotides in the promoter region of the putative suppressor gene RASSF1A (3p21.31) in squamous cell carcinomas of the uterine cervix (SCCs) carrying human papillomavirus (HPV) types 16, 18, and related types. Methylation of 3 to 13 CpG pairs has been found in 64% (25 out of 39) tumor DNA samples, 22% (2 out of 9) DNA samples from morphologically normal tissues adjacent to the tumor (P = 0.0306), and two out of three DNA samples from peripheral blood leukocytes of carcinoma patients. These CpG pairs are not methylated in the DNA of leukocytes of healthy donors (0 out of 10). The methylation level of the RASSF1A promoter region studied in tumors of the patients with regional lymph node metastases is significantly higher than in tumors of the patient that have no metastases (P = 8.5 × 10^–12). The methylation frequency of gene RASSF1A is two times higher than the frequency of hemi- and homozygous deletions in the chromosome 3 region where the gene is located. The data obtained indicate that methylation is one of the main mechanisms of the RASSF1A gene inactivation in HPV-positive human cervical tumors. The methylation of this gene may be an early event in the genesis of cervical tumors, the methylation level increasing with tumor progression.     

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.     

<Emphasis Type="Italic">RASSF1A</Emphasis> expression level in primary epithelial tumors of various locations

Tumor suppressor activity of RASSF1A in vitro and in vivo was established, in particular, in studies of knockout mice cells. Data on methylation of the promoter region and a lower expression of RASSF1A were mostly obtained with cancer cell lines. Here, the RASSF1A mRNA was quantified the first time in primary epithelial malignant tumors of five various locations from 130 patients by semi-quantitative RT-PCR. Representative samples of kidney, lung, and breast carcinomas were examined. Preliminary data were obtained for RASSF1A expression in ovarian and colorectal carcinomas. System studies showed unexpected expression profiles, namely, the mRNA level increased (two- to sevenfold) more frequently than decreased in renal, breast, ovarian, and colorectal carcinomas. A higher RASSF1A mRNA level was significantly more frequent in renal cell carcinomas (24/38, 63% vs 8/38, 21%, P = 0.0004 by Fisher’s exact test) and ovarian carcinomas (8/13, 62% vs 2/13, 15%, P = 0.0114). Equal frequencies of lower and higher RASSF1A expression levels were only observed in non-small cell lung cancer (16/38, 42%). Noteworthy, an increase in expression was more common at early clinical stages of squamous cell lung cancer and adenocarcinoma, while a decrease in RASSF1A expression was more frequent at advanced clinical stages. In clear cell renal cell carcinoma, an increase in RASSF1A expression occurred more often at both early and advanced stages and was significant at advanced stages (P = 0.0094). The findings suggested tumor specificity for changes in RASSF1A expression. The observed regularities may also indicate that RASSF1A has dual functions in tumors, acting as a tumor suppressor and as a protooncogene.     

Quantitative analysis of mRNA expression levels and DNA methylation profiles of three neighboring genes: FUS1, NPRL2/G21 and RASSF1A in non-small cell lung cancer patients

Background

Tumor suppressor gene (TSG) inactivation plays a crucial role in carcinogenesis. FUS1, NPRL2/G21 and RASSF1A are TSGs from LUCA region at 3p21.3, a critical chromosomal region in lung cancer development. The aim of the study was to analyze and compare the expression levels of these 3 TSGs in NSCLC, as well as in macroscopically unchanged lung tissue surrounding the primary lesion, and to look for the possible epigenetic mechanism of TSG inactivation via gene promoter methylation.

Methods

Expression levels of 3 TSGs and 2 DNA methyltransferases, DNMT1 and DNMT3B, were assessed using real-time PCR method (qPCR) in 59 primary non-small cell lung tumors and the matched macroscopically unchanged lung tissue samples. Promoter methylation status of TSGs was analyzed using methylation-specific PCRs (MSP method) and Methylation Index (MI) value was calculated for each gene.

Results

The expression of all three TSGs were significantly different between NSCLC subtypes: RASSF1A and FUS1 expression levels were significantly lower in squamous cell carcinoma (SCC), and NPRL2/G21 in adenocarcinoma (AC). RASSF1A showed significantly lower expression in tumors vs macroscopically unchanged lung tissues. Methylation frequency was 38–76 %, depending on the gene. The highest MI value was found for RASSF1A (52 %) and the lowest for NPRL2/G21 (5 %). The simultaneous decreased expression and methylation of at least one RASSF1A allele was observed in 71 % tumor samples. Inverse correlation between gene expression and promoter methylation was found for FUS1 (rs = −0.41) in SCC subtype. Expression levels of DNMTs were significantly increased in 75–92 % NSCLCs and were significantly higher in tumors than in normal lung tissue. However, no correlation between mRNA expression levels of DNMTs and DNA methylation status of the studied TSGs was found.

Conclusions

The results indicate the potential role of the studied TSGs in the differentiation of NSCLC histopathological subtypes. The significant differences in RASSF1A expression levels between NSCLC and macroscopically unchanged lung tissue highlight its possible diagnostic role in lung cancer in situ recognition. High percentage of lung tumor samples with simultaneous RASSF1A decreased expression and gene promoter methylation indicates its epigenetic silencing. However, DNMT overexpression doesn’t seem to be a critical determinate of its promoter hypermethylation.     

Global hypomethylation and promoter methylation in small intestinal neuroendocrine tumors: An in vivo and in vitro study

Aberrant DNA methylation is a feature of human cancer affecting gene expression and tumor phenotype. Here, we quantified promoter methylation of candidate genes and global methylation in 44 small intestinal-neuroendocrine tumors (SI-NETs) from 33 patients by pyrosequencing. Findings were compared with gene expression, patient outcome and known tumor copy number alterations. Promoter methylation was observed for WIF1, RASSF1A, CTNNB1, CXCL14, NKX2–3, P16, LAMA1, and CDH1. By contrast APC, CDH3, HIC1, P14, SMAD2, and SMAD4 only had low levels of methylation. WIF1 methylation was significantly increased (P = 0.001) and WIF1 expression was reduced in SI-NETs vs. normal references (P = 0.003). WIF1, NKX2–3, and CXCL14 expression was reduced in metastases vs. primary tumors (P < 0.02). Low expression of RASSF1A and P16 were associated with poor overall survival (P = 0.045 and P = 0.011, respectively). Global methylation determined by pyrosequencing of LINE1 repeats was reduced in tumors vs. normal references, and was associated with loss in chromosome 18. The tumors fell into three clusters with enrichment of WIF1 methylation and LINE1 hypomethylation in Cluster I and RASSF1A and CTNNB1 methylation and loss in 16q in Cluster II. In Cluster III, these alterations were low-abundant and NKX2-3 methylation was low. Similar analyses in the SI-NET cell lines HC45 and CNDT2 showed methylation for CDH1 and WIF1 and/or P16, CXCL14, NKX2-3, LAMA1, and CTNNB1. Treatment with the demethylating agent 5-azacytidine reduced DNA methylation and increased expression of these genes in vitro. In conclusion, promoter methylation of tumor suppressor genes is associated with suppressed gene expression and DNA copy number alterations in SI-NETs, and may be restored in vitro.     

Detection of MGMT, RASSF1A, p15INK4B, and p14ARF promoter methylation in circulating tumor-derived DNA of central nervous system cancer patients

Despite the growing understanding of the mechanisms of carcinogenesis, cancers of the central nervous system are usually associated with unfavorable prognosis. The use of an appropriate molecular marker may improve the treatment outcome by allowing early diagnosis and treatment susceptibility monitoring. Since methylation of tumor-derived DNA can be detected in the serum of cancer patients, this makes DNA methylation-based biomarkers one of the most promising diagnostic strategies. In this study, the methylation profiles of MGMT, RASSF1A, p15INK4B, and p14ARF genes were evaluated in serum free-circulating DNA and the corresponding tumor tissue in a group of 33 primary or metastatic central nervous system cancer patients. Gene promoter methylation was assessed using methylation-specific polymerase chain reaction (PCR). All the tested genes were found to be methylated to a different extent in both serum and tumor samples. In comparison to metastatic brain tumor patients, the patients with glial tumors were characterized by a higher frequency of gene hypermethylation. The hypermethylation of RASSF1A differentiated primary from metastatic brain cancers. Moreover, the gene methylation profiles observed in serum, in most cases, matched the methylation profiles detected in paired tumor samples.     

Global and gene-specific promoter methylation analysis in primary hyperparathyroidism

Epigenetic mechanisms involved in primary hyperparathyroidism are poorly understood as studies are limited. In order to understand the role of aberrant DNA promoter methylation in the pathogenesis of parathyroid tumors, we have quantified the CpG island promoter methylation density of several candidate genes including APC (promoter 1A and 1B), β-catenin (CTNNB1), CASR, CDC73/HRPT2, MEN1, P16 (CDKN2A), PAX1, RASSF1A, SFRP1 and VDR in 72 parathyroid tumors and 3 normal parathyroid references using bisulfite pyrosequencing. Global methylation levels were assessed for LINE-1. We also compared methylation levels with gene expression levels measured by qRT-PCR for genes showing frequent hypermethylation. The adenomas displayed frequent hypermethylation of APC 1A (37/66; 56%), RASSF1A (34/66; 52%) and β-catenin (19/66; 29%). One of the three atypical adenomas was hypermethylated for APC 1A. The three carcinomas were hypermethylated for RASSF1A and SFRP1, and the latter was only observed in this subtype. The global methylation density was similar in tumors (mean 70%) and parathyroid reference samples (mean 70%). In general, hypermethylated genes had reduced expression in the parathyroid adenomas using qRT-PCR. Among the adenomas, methylation of APC 1A correlated with adenoma weight (r = 0.306, p < 0.05). Furthermore, the methylation status of RASSF1A correlated with each of APC 1A (r = 0.289, p < 0.05) and β-catenin (r = 0.315, p < 0.01). Our findings suggest a role for aberrant DNA promoter methylation of APC 1A, β-catenin and RASSF1A in a subset of parathyroid tumors.     

RASSF2 methylation is a strong prognostic marker in younger age patients with Ewing sarcoma

Ras-association domain family of genes consist of 10 members (RASSF1-RASSF10), all containing a Ras-association (RA) domain in either the C- or the N-terminus. Several members of this gene family are frequently methylated in common sporadic cancers; however, the role of the RASSF gene family in rare types of cancers, such as bone cancer, has remained largely uninvestigated. In this report, we investigated the methylation status of RASSF1A and RASSF2 in Ewing sarcoma (ES). Quantitative real-time methylation analysis (MethyLight) demonstrated that both genes were frequently methylated in Ewing sarcoma tumors (52.5% and 42.5%, respectively) as well as in ES cell lines and gene expression was upregulated in methylated cell lines after treatment with 5-aza-2′-deoxcytidine. Overexpression of either RASSF1A or RASSF2 reduced colony formation ability of ES cells. RASSF2 methylation correlated with poor overall survival (p = 0.028) and this association was more pronounced in patients under the age of 18 y (p = 0.002). These results suggest that both RASSF1A and RASSF2 are novel epigenetically inactivated tumor suppressor genes in Ewing sarcoma and RASSF2 methylation may have prognostic implications for ES patients.     

Association between RASSF1A Promoter Methylation and Ovarian Cancer: A Meta-Analysis

Background

The RAS association domain family protein 1a gene (RASSF1A) is one of the tumor suppressor genes (TSG). Inactivation of RASSF1A is critical to the pathogenesis of cancer. Aberrant TSG methylation was considered an important epigenetic silencing mechanism in the progression of ovarian cancer. A number of studies have discussed association between RASSF1A promoter methylation and ovarian cancer. However, they were mostly based on a small number of samples and showed inconsist results, Therefore, we conducted a meta-analysis to better identify the association.

Methods

Eligible studies were identified by searching the PubMed, EMBASE, Web of Science, and CNKI databases using a systematic searching strategy. We pooled the odds ratio (ORs) from individual studies using a fixed-effects model. We performed heterogeneity and publication bias analysis simultaneously.

Results

Thirteen studies, with 763 ovarian cancer patients and 438 controls were included in the meta-analysis. The frequencies of RASSF1A promoter methylation ranged from 30% to 58% (median is 48%) in the cancer group and 0 to 21% (median is 0) in the control group. The frequencies of RASSF1A promoter methylation in the cancer group were significantly higher than those in the control group. The pooled odds ratio was 11.17 (95% CI = 7.51–16.61) in the cancer group versus the corresponding control group under the fixed-effects model.

Conclusion

The results suggested that RASSF1A promoter methylation had a strong association with ovarian cancer.     

Methylation of the Promoter Region of the RASSF1A Candidate Tumor Suppressor Gene in Primary Epithelial Tumors

The methylation of the promoter CpG island of the RASSF1A tumor suppressor gene in primary tumors of 172 Muscovites with renal cell carcinoma (RCC), breast cancer (BC), or ovarian epithelial tumors (OET) was assayed by means of methylation-specific PCR (MSP) and PCR-based methylation-sensitive restriction enzyme analysis (MSRA). The MSP, MSRA, and previous bisulfite sequencing data correlated significantly with each other (P 10^–6 for Spearman's rank correlation coefficients). By MSP and MSRA, the respective methylation frequencies of the RASSF1A promoter were 86% (25/29) and 94% (50/53) in RCC, 64% (18/28) and 78% (32/41) in BC, and 59% (17/29) and 73% (33/45) in OET. Methylation-sensitive restriction enzymes (HpaII, HhaI, Bsh1236I, AciI) increased the analysis sensitivity and made it possible to establish the methylation status for 18 CpG dinucleotides of the RASSF1A promoter region. With the MSRA data, the density of methylation of the CpG island was estimated at 72% in RCC, 63% in BC, and 58% in OET (the product of the number of CpG dinucleotides and the number of specimens with RASSF1A methylation was taken as 100%). Methylation of the RASSF1A promoter region was observed in 11–35% of the DNA specimens from the histologically normal tissue adjacent to the tumor but not in the peripheral blood DNA of 15 healthy subjects. The RASSF1A methylation frequency showed no significant correlation with the stage, grade, and metastatic potential of the tumor. On the other hand, epigenetic modification of RASSF1A was considerably more frequent than hemizygous or homozygous deletions from the RASSF1A region. These results testify that methylation of the RASSF1A promoter region takes place early in carcinogenesis and is a major mechanism inactivating RASSF1A in epithelial tumors.     

Role of RASSF1A promoter methylation in the pathogenesis of hepatocellular carcinoma: a meta-analysis of 21 cohort studies

We carried out the current meta-analysis aiming to comprehensively assess the potential role of RASSF1A aberrant promoter methylation in the pathogenesis of hepatocellular carcinoma (HCC). A range of electronic databases were searched: Web of Science (1945–2013), the Cochrane Library Database (Issue 12, 2013), PubMed (1966–2013), EMBASE (1980–2013), CINAHL (1982–2013) and the Chinese Biomedical Database (CBM) (1982–2013) without language restrictions. Meta-analysis was conducted using the STATA 12.0 software. Crude risk difference (RD) with their 95 % confidence interval (95 % CI) was calculated. In the present meta-analysis, 21 clinical cohort studies with a total of 1,205 HCC patients were included. The results of our meta-analysis illustrated that the frequency of RASSF1A promoter methylation in cancer tissues were significantly higher than those of normal, adjacent and benign tissues (cancer tissues vs. normal tissues: RD = 0.63, 95 % CI 0.53–0.73, P < 0.001; cancer tissues vs. adjacent tissues: RD = 0.43, 95 % CI 0.33–0.53, P < 0.001; cancer tissues vs. benign tissues: RD = 0.48, 95 % CI 038–0.58, P < 0.001; respectively). Further subgroup by ethnicity demonstrated that RASSF1A aberrant promoter methylation was correlated with the pathogenesis of HCC among both Asians and Caucasians (all P < 0.05). The current meta-analysis suggests that RASSF1A aberrant promoter methylation may be implicated in the pathogenesis of HCC. Thus, detection of RASSF1A promoter methylation may be a helpful and valuable biomarker for diagnosis and prognosis of HCC.     

Methylation status and protein expression of RASSF1A in breast cancer patients

Recently genetics and epigenetics alterations have been found to be characteristic of malignancy and hence can be used as targets for detection of neoplasia. RAS association domain family protein 1A (RASSF1A) gene hypermethylation has been a subject of interest in recent researches on cancer breast patients. The aim of the present study was to evaluate whether RASSF1A methylation status and RASSF1A protein expression are associated with the major clinico-pathological parameters. One hundred and twenty breast cancer Egyptian patients and 100-control subjects diagnosed with benign lesions of the breast were enrolled in this study. We evaluated RASSF1A methylation status in tissue and serum samples using Methyl specific PCR together with RASSF1A protein expression in tissues by immunohistochemistry. Results were studied in relation to known prognostic clinicopathological features in breast cancer. Frequency of RASSF1A methylation in tissues and serum were 70 and 63.3 % respectively and RASSF1A protein expression showed frequency of 46.7 %. There was an association between RASSF1A methylation in tissues, serum and loss of protein expression in tissues with invasive carcinoma, advanced stage breast cancer, L.N. metastasis, ER/PR and HER2 negativity. RASSF1A methylation in serum showed high degree of concordance with methylation in tissues (Kappa = 0.851, P < 0.001). RASSF1A hypermethylation in tissues and serum and its protein expression may be a valid, reliable and sensitive tool for detection and follow up of breast cancer patients.     

The Role of Dietary Extra Virgin Olive Oil and Corn Oil on the Alteration of Epigenetic Patterns in the Rat DMBA-Induced Breast Cancer Model

Disruption of epigenetic patterns is a major change occurring in all types of cancers. Such alterations are characterized by global DNA hypomethylation, gene-promoter hypermethylation and aberrant histone modifications, and may be modified by environment. Nutritional factors, and especially dietary lipids, have a role in the etiology of breast cancer. Thus, we aimed to analyze the influence of different high fat diets on DNA methylation and histone modifications in the rat dimethylbenz(a)anthracene (DMBA)-induced breast cancer model. Female Sprague-Dawley rats were fed a low-fat, a high corn-oil or a high extra-virgin olive oil (EVOO) diet from weaning or from induction with DMBA. In mammary glands and tumors we analyzed global and gene specific (RASSF1A, TIMP3) DNA methylation by LUMA and bisulfite pyrosequencing assays, respectively. We also determined gene expression and enzymatic activity of DNA methyltransferases (DNMT1, DNMT3a and DNMT3b) and evaluated changes in histone modifications (H3K4me2, H3K27me3, H4K20me3 and H4K16ac) by western-blot. Our results showed variations along time in the global DNA methylation of the mammary gland displaying decreases at puberty and with aging. The olive oil-enriched diet, on the one hand, increased the levels of global DNA methylation in mammary gland and tumor, and on the other, changed histone modifications patterns. The corn oil-enriched diet increased DNA methyltransferase activity in both tissues, resulting in an increase in the promoter methylation of the tumor suppressor genes RASSF1A and TIMP3. These results suggest a differential effect of the high fat diets on epigenetic patterns with a relevant role in the neoplastic transformation, which could be one of the mechanisms of their differential promoter effect, clearly stimulating for the high corn-oil diet and with a weaker influence for the high EVOO diet, on breast cancer progression.     

RASSF6 exhibits promoter hypermethylation in metastatic melanoma and inhibits invasion in melanoma cells

Brain metastasis is a major contributor to cancer mortality, yet, the genetic changes underlying the development of this capacity remain poorly understood. RASSF proteins are a family of tumor suppressors that often suffer epigenetic inactivation during tumorigenesis. However, their epigenetic status in brain metastases has not been well characterized. We have examined the promoter methylation of the classical RASSF members (RASSF1A-RASSF6) in a panel of metastatic brain tumor samples. RASSF1A and RASSF2 have been shown to undergo promoter methylation at high frequency in primary lung and breast tumors and in brain metastases. Other members exhibited little or no methylation in these tumors. In examining melanoma metastases, however, we found that RASSF6 exhibits the highest frequency of inactivation in melanoma and in melanoma brain metastases. Most melanomas are driven by an activating mutation in B-Raf. Introduction of RASSF6 into a B-Raf^V600E-containing metastatic melanoma cell line inhibited its ability to invade through collagen and suppressed MAPK pathway activation and AKT. RASSF6 also appears to increase the association of mutant B-Raf and MST1, providing a potential mechanism by which RASSF6 is able to suppress MAPK activation. Thus, we have identified a novel potential role for RASSF6 in melanoma development. Promoter methylation leading to reduced expression of RASSF6 may play an important role in melanoma development and may contribute to brain metastases.     

Correlation between BRAF mutation and promoter methylation of TIMP3, RARβ2 and RASSF1A in thyroid cancer

Our aim was to comprehensively analyze promoter hypermethylation of a panel of novel and known methylation markers for thyroid neoplasms and to establish their relationship with BRAF mutation and clinicopathologic parameters of thyroid cancer. A cohort of thyroid tumors, consisting of 44 cancers and 44 benign thyroid lesions, as well as 15 samples of adjacent normal thyroid tissue, was evaluated for BRAF mutation and promoter hypermethylation. Genes for quantitative methylation specific PCR (QMSP) were selected by a candidate gene approach. Twenty-two genes were tested: TSHR, RASSF1A, RARβ2, DAPK, hMLH1, ATM, S100, p16, CTNNB1, GSTP1, CALCA, TIMP3, TGFßR2, THBS1, MINT1, CTNNB1, MT1G, PAK3, NISCH, DCC, AIM1 and KIF1A. The PCR-based “mutector assay” was used to detect BRAF mutation. All p values reported are two sided. Considerable overlap was seen in the methylation markers among the different tissue groups. Significantly higher methylation frequency and level were observed for KIF1A and RARß2 in cancer samples compared with benign tumors. A negative correlation between BRAF mutation and RASSF1A methylation, and a positive correlation with RARß2 methylation were observed in accordance with previous results. In addition, positive correlation with TIMP3 and a marginal correlation with DCC methylation were observed. The present study constitutes a comprehensive promoter methylation profile of thyroid neoplasia and shows that results must be analyzed in a tissue-specific manner to identify clinically useful methylation markers. Integration of genetic and epigenetic changes in thyroid cancer will help identify relevant biologic pathways that drive its development.