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.     

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.     

Differential DNA methylation with age displays both common and dynamic features across human tissues that are influenced by CpG landscape

Background

DNA methylation is an epigenetic modification that changes with age in human tissues, although the mechanisms and specificity of this process are still poorly understood. We compared CpG methylation changes with age across 283 human blood, brain, kidney, and skeletal muscle samples using methylation arrays to identify tissue-specific age effects.

Results

We found age-associated CpGs (ageCGs) that are both tissue-specific and common across tissues. Tissue-specific ageCGs are frequently located outside CpG islands with decreased methylation, and common ageCGs show the opposite trend. AgeCGs are significantly associated with poorly expressed genes, but those with decreasing methylation are linked with higher tissue-specific expression levels compared with increasing methylation. Therefore, tissue-specific gene expression may protect against common age-dependent methylation. Distinguished from other tissues, skeletal muscle ageCGs are more associated with expression, enriched near genes related to myofiber contraction, and closer to muscle-specific CTCF binding sites. Kidney-specific ageCGs are more increasingly methylated compared to other tissues as measured by affiliation with kidney-specific expressed genes. Underlying chromatin features also mark common and tissue-specific age effects reflective of poised and active chromatin states, respectively. In contrast with decreasingly methylated ageCGs, increasingly methylated ageCGs are also generally further from CTCF binding sites and enriched within lamina associated domains.

Conclusions

Our data identified common and tissue-specific DNA methylation changes with age that are reflective of CpG landscape and suggests both common and unique alterations within human tissues. Our findings also indicate that a simple epigenetic drift model is insufficient to explain all age-related changes in DNA methylation.     

Methylation of the <Emphasis Type="Italic">RASSF1A</Emphasis> promoter region and the allelic imbalance frequencies in chromosome 3 critical regions correlate with progression of clear cell renal carcinoma

The short arm of chromosome 3 (3p) contains several critical regions that have increased frequencies of allelic deletions and harbor a set of tumor suppressor genes. In particular, the range of functions performed by RASSF1A (LUCA region, 3p21.31) includes those potentially associated with carcinogenesis. Among 3p genes, RASSF1A has the highest methylation frequency in epithelial tumors of various locations. For the first time, two different methods (methylation-specific PCR and methylation-sensitive restriction analysis) independently showed that the methylation level of the CpG island in the RASSF1A promoter region significantly correlated with grade and clinical stage of clear cell renal cell carcinoma (RCC). An analysis of 23 3p polymorphic markers in a representative set of 80 RCC cases characterized clinically and histologically revealed that RCC progression significantly correlated with the frequency of allelic imbalances in some critical regions of 3p (LUCA and AP20), but not in 3p as a whole. These data suggest that RCC progression is associated with the methylation of the RASSF1A promoter and, possibly, with structural and functional alterations in other 3p genes. In addition, significant correlation between RASSF1A methylation and allelic losses at the nearby polymorphic marker locus suggests the “two hit” model for the inactivation of this tumor suppressor gene in RCC.     

Complete Biallelic Insulation at the H19/Igf2 Imprinting Control Region Position Results in Fetal Growth Retardation and Perinatal Lethality

Background

The H19/Igf2 imprinting control region (ICR) functions as an insulator exclusively in the unmethylated maternal allele, where enhancer-blocking by CTCF protein prevents the interaction between the Igf2 promoter and the distant enhancers. DNA methylation inhibits CTCF binding in the paternal ICR allele. Two copies of the chicken β-globin insulator (ChβGI)₂ are capable of substituting for the enhancer blocking function of the ICR. Insulation, however, now also occurs upon paternal inheritance, because unlike the H19 ICR, the (ChβGI)₂ does not become methylated in fetal male germ cells. The (ChβGI)₂ is a composite insulator, exhibiting enhancer blocking by CTCF and chromatin barrier functions by USF1 and VEZF1. We asked the question whether these barrier proteins protected the (ChβGI)₂ sequences from methylation in the male germ line.

Methodology/Principal Findings

We genetically dissected the ChβGI in the mouse by deleting the binding sites USF1 and VEZF1. The methylation of the mutant versus normal (ChβGI)₂ significantly increased from 11% to 32% in perinatal male germ cells, suggesting that the barrier proteins did have a role in protecting the (ChβGI)₂ from methylation in the male germ line. Contrary to the H19 ICR, however, the mutant (mChβGI)₂ lacked the potential to attain full de novo methylation in the germ line and to maintain methylation in the paternal allele in the soma, where it consequently functioned as a biallelic insulator. Unexpectedly, a stricter enhancer blocking was achieved by CTCF alone than by a combination of the CTCF, USF1 and VEZF1 sites, illustrated by undetectable Igf2 expression upon paternal transmission.

Conclusions/Significance

In this in vivo model, hypomethylation at the ICR position together with fetal growth retardation mimicked the human Silver-Russell syndrome. Importantly, late fetal/perinatal death occurred arguing that strict biallelic insulation at the H19/Igf2 ICR position is not tolerated in development.     

Frequent Epigenetic Silencing of the Folate-Metabolising Gene Cystathionine-Beta-Synthase in Gastrointestinal Cancer

Background

Both gastric and colorectal cancers (CRC) are the most frequently occurring malignancies worldwide with the overall survival of these patients remains unsatisfied. Identification of tumor suppressor genes (TSG) silenced by promoter CpG methylation uncovers mechanisms of tumorigenesis and identifies new epigenetic biomarkers for early cancer detection and prognosis assessment. Cystathionine-beta-synthase (CBS) functions in the folate metabolism pathway, which is intricately linked to methylation of genomic DNA. Dysregulation of DNA methylation contributes substantially to cancer development.

Methodology/Principal Findings

To identify potential TSGs silenced by aberrant promoter methylation in CRC, we analyzed tumor and adjacent tissues from CRC cases using the Illumina Human Methylation45 BeadChip. We identified hypermethylation of the CBS gene in CRC samples, compared to adjacent tissues. Methylation and decreased mRNA expression of CBS were detected in most CRC cell lines by methylation-specific PCR and semiquantitative RT-PCR, as well as in gastric cancer. Treatment with 5-aza-2''-deoxycytidine and/or trichostatin A reversed methylation and restored CBS mRNA expression indicating a direct effect. Aberrant methylation was further detected in 31% of primary CRCs (29 of 96) and 55% of gastric tumors (11 of 20). In contrast, methylation was seldom found in normal tissues adjacent to the tumor. CBS methylation was associated with KRAS mutations in primary CRCs (P = 0.04, by χ²-test). However, no association was found between CBS methylation or KRAS mutations with cancer relapse/metastasis in Stage II CRC patients.

Conclusion

A novel finding from this study is that the folate metabolism enzyme CBS mRNA levels are frequently downregulated through CpG methylation of the CBS gene in gastric cancer and CRC, suggesting that CBS functions as a tumor suppressor gene. These findings warrant further study of CBS as an epigenetic biomarker for molecular diagnosis of gastrointestinal cancers.     

Frequent and distinct aberrations of DNA methylation patterns in fibrolamellar carcinoma of the liver

Background

Gene silencing due to aberrant DNA methylation is a frequent event in hepatocellular carcinoma (HCC) and also in hepatocellular adenoma (HCA). However, very little is known about epigenetic defects in fibrolamellar carcinoma (FLC), a rare variant of hepatocellular carcinoma that displays distinct clinical and morphological features.

Methodology/Principal Findings

We analyzed the methylation status of the APC, CDH1, cyclinD2, GSTπ1, hsa-mir-9-1, hsa-mir-9-2, and RASSF1A gene in a series of 15 FLC and paired normal liver tissue specimens by quantitative high-resolution pyrosequencing. Results were compared with common HCC arising in non-cirrhotic liver (n = 10). Frequent aberrant hypermethylation was found for the cyclinD2 (19%) and the RASSF1A (38%) gene as well as for the microRNA genes mir-9-1 (13%) and mir-9-2 (33%). In contrast to common HCC the APC and CDH1 (E-cadherin) genes were found devoid of any DNA methylation in FLC, whereas the GSTπ1 gene showed comparable DNA methylation in tumor and surrounding tissue at a moderate level. Changes in global DNA methylation level were measured by analyzing methylation status of the highly repetitive LINE-1 sequences. No evidence of global hypomethylation could be found in FLCs, whereas HCCs without cirrhosis showed a significant reduction in global methylation level as described previously.

Conclusions

FLCs display frequent and distinct gene-specific hypermethylation in the absence of significant global hypomethylation indicating that these two epigenetic aberrations are induced by different pathways and that full-blown malignancy can develop in the absence of global loss of DNA methylation. Only quantitative DNA methylation detection methodology was able to identify these differences.     

Elucidating the Landscape of Aberrant DNA Methylation in Hepatocellular Carcinoma

Background

Hepatocellular carcinoma (HCC) is one of the most common cancers and frequently presents with an advanced disease at diagnosis. There is only limited knowledge of genome-scale methylation changes in HCC.

Methods and Findings

We performed genome-wide methylation profiling in a total of 47 samples including 27 HCC and 20 adjacent normal liver tissues using the Illumina HumanMethylation450 BeadChip. We focused on differential methylation patterns in the promoter CpG islands as well as in various less studied genomic regions such as those surrounding the CpG islands, i.e. shores and shelves. Of the 485,577 loci studied, significant differential methylation (DM) was observed between HCC and adjacent normal tissues at 62,692 loci or 13% (p<1.03e-07). Of them, 61,058 loci (97%) were hypomethylated and most of these loci were located in the intergenic regions (43%) or gene bodies (33%). Our analysis also identified 10,775 differentially methylated (DM) loci (17% out of 62,692 loci) located in or surrounding the gene promoters, 4% of which reside in known Differentially Methylated Regions (DMRs) including reprogramming specific DMRs and cancer specific DMRs, while the rest (10,315) involving 4,106 genes could be potential new HCC DMR loci. Interestingly, the promoter-related DM loci occurred twice as frequently in the shores than in the actual CpG islands. We further characterized 982 DM loci in the promoter CpG islands to evaluate their potential biological function and found that the methylation changes could have effect on the signaling networks of Cellular development, Gene expression and Cell death (p = 1.0e-38), with BMP4, CDKN2A, GSTP1, and NFATC1 on the top of the gene list.

Conclusion

Substantial changes of DNA methylation at a genome-wide level were observed in HCC. Understanding epigenetic changes in HCC will help to elucidate the pathogenesis and may eventually lead to identification of molecular markers for liver cancer diagnosis, treatment and prognosis.     

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.     

Boundaries between chromosomal domains of X inactivation and escape bind CTCF and lack CpG methylation during early development

Escape from X inactivation results in expression of genes embedded within inactive chromatin, suggesting the existence of boundary elements between domains. We report that the 5' end of Jarid1c, a mouse escape gene adjacent to an inactivated gene, binds CTCF, displays high levels of histone H3 acetylation, and functions as a CTCF-dependent chromatin insulator. CpG island methylation at Jarid1c was very low during development and virtually absent at the CTCF sites, signifying that CTCF may influence DNA methylation and chromatin modifications. CTCF binding sites were also present at the 5' end of two other escape genes, mouse Eif2s3x and human EIF2S3, each adjacent to an inactivated gene, but not at genes embedded within large escape domains. Thus, CTCF was specifically bound to transition regions, suggesting a role in maintaining both X inactivation and escape domains. Furthermore, the evolution of X chromosome domains appears to be associated with repositioning of chromatin boundary elements.     

Gene promoter methylation assayed in exhaled breath,with differences in smokers and lung cancer patients

Background

There is a need for new, noninvasive risk assessment tools for use in lung cancer population screening and prevention programs.

Methods

To investigate the technical feasibility of determining DNA methylation in exhaled breath condensate, we applied our previously-developed method for tag-adapted bisulfite genomic DNA sequencing (tBGS) for mapping of DNA methylation, and adapted it to exhaled breath condensate (EBC) from lung cancer cases and non-cancer controls. Promoter methylation patterns were analyzed in DAPK, RASSF1A and PAX5β promoters in EBC samples from 54 individuals, comprised of 37 controls [current- (n = 19), former- (n = 10), and never-smokers (n = 8)] and 17 lung cancer cases [current- (n = 5), former- (n = 11), and never-smokers (n = 1)].

Results

We found: (1) Wide inter-individual variability in methylation density and spatial distribution for DAPK, PAX5β and RASSF1A. (2) Methylation patterns from paired exhaled breath condensate and mouth rinse specimens were completely divergent. (3) For smoking status, the methylation density of RASSF1A was statistically different (p = 0.0285); pair-wise comparisons showed that the former smokers had higher methylation density versus never smokers and current smokers (p = 0.019 and p = 0.031). For DAPK and PAX5β, there was no such significant smoking-related difference. Underlying lung disease did not impact on methylation density for this geneset. (4) In case-control comparisons, CpG at -63 of DAPK promoter and +52 of PAX5β promoter were significantly associated with lung cancer status (p = 0.0042 and 0.0093, respectively). After adjusting for multiple testing, both loci were of borderline significance (p_adj = 0.054 and 0.031). (5) The DAPK gene had a regional methylation pattern with two blocks (1)~-215~-113 and (2) -84 ~+26); while similar in block 1, there was a significant case-control difference in methylation density in block 2 (p = 0.045); (6)Tumor stage and histology did not impact on the methylation density among the cases. (7) The results of qMSP applied to EBC correlated with the corresponding tBGS sequencing map loci.

Conclusion

Our results show that DNA methylation in exhaled breath condensate is detectable and is likely of lung origin. Suggestive correlations with smoking and lung cancer case-control status depend on individual gene and CpG site examined.     

Epigenetic Changes in Basal Cell Carcinoma Affect SHH and WNT Signaling Components

Background

The genetic background of Basal Cell Carcinoma (BCC) has been studied extensively, while its epigenetic makeup has received comparatively little attention. Epigenetic alterations such as promoter hypermethylation silence tumor suppressor genes (TSG) in several malignancies.

Objective

We sought to analyze the promoter methylation status of ten putative (tumor suppressor) genes that are associated with Sonic Hedgehog (SHH), WNT signaling and (hair follicle) tumors in a large series of 112 BCC and 124 healthy control samples by methylation-specific PCR.

Results

Gene promoters of SHH (P = 0.016), adenomatous polyposis coli (APC) (P = 0.003), secreted frizzled-related protein 5 (SFRP5) (P = 0.004) and Ras association domain family 1A (RASSF1A) (P = 0.023) showed significantly more methylation in BCC versus normal skin. mRNA levels of these four genes were reduced for APC and SFRP5 in BCC (n = 6) vs normal skin (n = 6). Down regulation of SHH, APC and RASSF1A could be confirmed on protein level as well (P<0.001 for all genes) by immunohistochemical staining. Increased canonical WNT activity was visualized by β-catenin staining, showing nuclear β-catenin in only 28/101 (27.7%) of BCC. Absence of nuclear β-catenin in some samples may be due to high levels of membranous E-cadherin (in 94.1% of the samples).

Conclusions

We provide evidence that promoter hypermethylation of key players within the SHH and WNT pathways is frequent in BCC, consistent with their known constitutive activation in BCC. Epigenetic gene silencing putatively contributes to BCC tumorigenesis, indicating new venues for treatment.