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.     

Development of a multiplex methylation specific PCR suitable for (early) detection of non-small cell lung cancer

Lung cancer is a worldwide health problem and a leading cause of cancer-related deaths. Silencing of potential tumor suppressor genes (TSGs) by aberrant promoter methylation is an early event in the initiation and development of cancer. Thus, methylated cancer type-specific TSGs in DNA can serve as useful biomarkers for early cancer detection. We have now developed a “Multiplex Methylation Specific PCR” (MMSP) assay for analysis of the methylation status of multiple potential TSGs by a single PCR reaction. This method will be useful for early diagnosis and treatment outcome studies of non-small cell lung cancer (NSCLC). Genome-wide CpG methylation and expression microarrays were performed on lung cancer tissues and matched distant non-cancerous tissues from three NSCLC patients from China. Thirty-eight potential TSGs were selected and analyzed by methylation PCR on bisulfite treated DNA. On the basis of sensitivity and specificity, six marker genes, HOXA9, TBX5, PITX2, CALCA, RASSF1A, and DLEC1, were selected to establish the MMSP assay. This assay was then used to analyze lung cancer tissues and matched distant non-cancerous tissues from 70 patients with NSCLC, as well as 24 patients with benign pulmonary lesion as controls. The sensitivity of the assay was 99% (69/70). HOXA9 and TBX5 were the 2 most sensitive marker genes: 87% (61/70) and 84% (59/70), respectively. RASSF1A and DLEC1 showed the highest specificity at 99% (69/70). Using the criterion of identifying at least any two methylated marker genes, 61/70 cancer samples were positive, corresponding to a sensitivity of 87% and a specificity of 94%. Early stage I or II NSCLC could even be detected with a 100% specificity and 86% sensitivity. In conclusion, MMSP has the potential to be developed into a population-based screening tool and can be useful for early diagnosis of NSCLC. It might also be suitable for monitoring treatment outcome and recurrence.     

HYAL1 and HYAL2 inhibit tumour growth in vivo but not in vitro

Background

We identified two 3p21.3 regions (LUCA and AP20) as most frequently affected in lung, breast and other carcinomas and reported their fine physical and gene maps. It is becoming increasingly clear that each of these two regions contains several TSGs. Until now TSGs which were isolated from AP20 and LUCA regions (e.g.G21/NPRL2, RASSF1A, RASSF1C, SEMA3B, SEMA3F, RBSP3) were shown to inhibit tumour cell growth both in vitro and in vivo.

Methodology/Principal Findings

The effect of expression HYAL1 and HYAL2 was studied by colony formation inhibition, growth curve and cell proliferation tests in vitro and tumour growth assay in vivo. Very modest growth inhibition was detected in vitro in U2020 lung and KRC/Y renal carcinoma cell lines. In the in vivo experiment stably transfected KRC/Y cells expressing HYAL1 or HYAL2 were inoculated into SCID mice (10 and 12 mice respectively). Tumours grew in eight mice inoculated with HYAL1. Ectopic HYAL1 was deleted in all of them. HYAL2 was inoculated into 12 mice and only four tumours were obtained. In 3 of them the gene was deleted. In one tumour it was present but not expressed. As expected for tumour suppressor genes HYAL1 and HYAL2 were down-expressed in 15 fresh lung squamous cell carcinomas (100%) and clear cell RCC tumours (60–67%).

Conclusions/Significance

The results suggest that the expression of either gene has led to inhibition of tumour growth in vivo without noticeable effect on growth in vitro. HYAL1 and HYAL2 thus differ in this aspect from other tumour suppressors like P53 or RASSF1A that inhibit growth both in vitro and in vivo. Targeting the microenvironment of cancer cells is one of the most promising venues of cancer therapeutics. As major hyaluronidases in human cells, HYAL1 and HYAL2 may control intercellular interactions and microenvironment of tumour cells providing excellent targets for cancer treatment.     

Association between P16INK4a Promoter Methylation and Non-Small Cell Lung Cancer: A Meta-Analysis

Background

Aberrant methylation of CpG islands acquired in tumor cells in promoter regions plays an important role in carcinogenesis. Accumulated evidence demonstrates P^16INK4a gene promoter hypermethylation is involved in non-small cell lung carcinoma (NSCLC), indicating it may be a potential biomarker for this disease. The aim of this study is to evaluate the frequency of P^16INK4a gene promoter methylation between cancer tissue and autologous controls by summarizing published studies.

Methods

By searching Medline, EMBSE and CNKI databases, the open published studies about P^16INK4a gene promoter methylation and NSCLC were identified using a systematic search strategy. The pooled odds of P^16INK4A promoter methylation in lung cancer tissue versus autologous controls were calculated by meta-analysis method.

Results

Thirty-four studies, including 2 652 NSCLC patients with 5 175 samples were included in this meta-analysis. Generally, the frequency of P^16INK4A promoter methylation ranged from 17% to 80% (median 44%) in the lung cancer tissue and 0 to 80% (median 15%) in the autologous controls, which indicated the methylation frequency in cancer tissue was much higher than that in autologous samples. We also find a strong and significant correlation between tumor tissue and autologous controls of P^16INK4A promoter methylation frequency across studies (Correlation coefficient 0.71, 95% CI:0.51–0.83, P<0.0001). And the pooled odds ratio of P^16INK4A promoter methylation in cancer tissue was 3.45 (95% CI: 2.63–4.54) compared to controls under random-effect model.

Conclusion

Frequency of P^16INK4a promoter methylation in cancer tissue was much higher than that in autologous controls, indicating promoter methylation plays an important role in carcinogenesis of the NSCLC. Strong and significant correlation between tumor tissue and autologous samples of P^16INK4A promoter methylation demonstrated a promising biomarker for NSCLC.     

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.     

Downregulation of RBSP3/CTDSPL, NPRL2/G21, RASSF1A, ITGA9, HYAL1, and HYAL2 in non-small cell lung cancer

Chromosomal and genome abnormalities of 3p are frequent in many epithelial tumors, including lung cancer. Several critical regions with a high frequency of hemi-and homozygous deletions in tumors are known for 3p, and more than 20 cancer-related genes occur in 3p21.3. Quantitative real-time PCR was used to measure the mRNA level for tumor-suppressor and candidate genes of 3p21.3 (RBSP3/CTDSPL, NPRL2/G21, RASSF1A, ITGA9, HYAL1, and HYAL2) in major types of non-small cell lung cancer (NSCLC): squamous cell lung cancer (SCC) and lung adenocarcinoma (AC). A significant (2-to 100-fold) and frequent (44–100%) decrease in mRNA levels was observed in NSCLC. The mRNA level decrease and its frequency depended on the histological type of NSCLC for all genes. The downregulation of RASSF1A and ITGA9 was significantly associated with AC progression; the same tendency was observed for RBSP3/CTDSPL, NPRL2/G21, HYAL1, and HYAL2. In SCC, the downregulation of all genes was not associated with the clinical stage, tumor cells differentiation, and metastasis in lymph nodes. The RBSP3/CTDSPL, NPRL2/G21, ITGA9, HYAL1, and HYAL2 mRNA levels significantly (5-to 13-fold on average) decreased at a high frequency (83–100%) as early as SCC stage I. Simultaneous downregulation of all six genes was observed in some tumor samples and was independent of the gene position in 3p21.3 and the functions of the protein products. The Spearman correlation coefficient r _s was 0.63–0.91, p < 0.001. The highest r _s values were obtained for gene pairs ITGA9-HYAL2 and HYAL1-HYAL2, whose products mediate cell-cell adhesion and cell-matrix interactions; coregulation of the genes was assumed on this basis. Both genetic and epigenetic mechanisms proved to be important for downregulation of RBSP3/CTDSPL and ITGA9. This finding supported the hypothesis that the cluster of cancerrelated genes in the extended 3p21.3 locus is simultaneously inactivated during the development and progression of lung cancer and other epithelial tumors. A significant and frequent decrease in the mRNA level of the six genes in SCC could be important for developing specific biomarker sets for early SCC diagnosis and new approaches to gene therapy of NSCLC.     

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.     

Promoter methylation analysis of WNT/β-catenin pathway regulators and its association with expression of DNMT1 enzyme in colorectal cancer

Background

Aberrant DNA methylation as the most important reason making epigenetic silencing of genes is a main mechanism of gene inactivation in patients with colorectal cancer. In this study, we decided to identify promoter methylation status of ten genes encoding WNT negative regulators, and measure the expression of DNMT1 enzyme in colorectal cancer samples.

Results

Aberrant methylation of APC gene was statistically significant associated with age over 50 (p = 0.017), DDK3 with male (p < 0.0001), SFRP4, WIF1, and WNT5a with increasing tumor stage (p = 0.004, p = 0.029, and p = 0.004), SFRP4 and WIF1 with tumor differentiation (p = 0.009 and p = 0.031) and SFRP2 and SFRP5 with histological type (p = 0.001 and p = 0.025). The increasing number of methylated genes correlated with the expression levels of the DNMT1 mRNA.

Conclusions

The rate of gene promoter methylation of WNT pathway regulators is high in colorectal cancer cells. Hyper-methylation is associated with increased expression of the DNMT1 enzyme.     

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.     

Mrassf1a-Pap,a Novel Methylation-Based Assay for the Detection of Cell-Free Fetal DNA in Maternal Plasma

Objectives

RASSF1A has been described to be differentially methylated between fetal and maternal DNA and can therefore be used as a universal sex-independent marker to confirm the presence of fetal sequences in maternal plasma. However, this requires highly sensitive methods. We have previously shown that Pyrophosphorolysis-activated Polymerization (PAP) is a highly sensitive technique that can be used in noninvasive prenatal diagnosis. In this study, we have used PAP in combination with bisulfite conversion to develop a new universal methylation-based assay for the detection of fetal methylated RASSF1A sequences in maternal plasma.

Methods

Bisulfite sequencing was performed on maternal genomic (g)DNA and fetal gDNA from chorionic villi to determine differentially methylated regions in the RASSF1A gene using bisulfite specific PCR primers. Methylation specific primers for PAP were designed for the detection of fetal methylated RASSF1A sequences after bisulfite conversion and validated.

Results

Serial dilutions of fetal gDNA in a background of maternal gDNA show a relative percentage of ∼3% can be detected using this assay. Furthermore, fetal methylated RASSF1A sequences were detected both retrospectively as well as prospectively in all maternal plasma samples tested (n = 71). No methylated RASSF1A specific bands were observed in corresponding maternal gDNA. Specificity was further determined by testing anonymized plasma from non-pregnant females (n = 24) and males (n = 21). Also, no methylated RASSF1A sequences were detected here, showing this assay is very specific for methylated fetal DNA. Combining all samples and controls, we obtain an overall sensitivity and specificity of 100% (95% CI 98.4%–100%).

Conclusions

Our data demonstrate that using a combination of bisulfite conversion and PAP fetal methylated RASSF1A sequences can be detected with extreme sensitivity in a universal and sex-independent manner. Therefore, this assay could be of great value as an addition to current techniques used in noninvasive prenatal diagnostics.     

Development of a multiplex methylation specific PCR suitable for (early) detection of non-small cell lung cancer

Lung cancer is a worldwide health problem and a leading cause of cancer-related deaths. Silencing of potential tumor suppressor genes (TSGs) by aberrant promoter methylation is an early event in the initiation and development of cancer. Thus, methylated cancer type-specific TSGs in DNA can serve as useful biomarkers for early cancer detection. We have now developed a “Multiplex Methylation Specific PCR” (MMSP) assay for analysis of the methylation status of multiple potential TSGs by a single PCR reaction. This method will be useful for early diagnosis and treatment outcome studies of non-small cell lung cancer (NSCLC). Genome-wide CpG methylation and expression microarrays were performed on lung cancer tissues and matched distant non-cancerous tissues from three NSCLC patients from China. Thirty-eight potential TSGs were selected and analyzed by methylation PCR on bisulfite treated DNA. On the basis of sensitivity and specificity, six marker genes, HOXA9, TBX5, PITX2, CALCA, RASSF1A, and DLEC1, were selected to establish the MMSP assay. This assay was then used to analyze lung cancer tissues and matched distant non-cancerous tissues from 70 patients with NSCLC, as well as 24 patients with benign pulmonary lesion as controls. The sensitivity of the assay was 99% (69/70). HOXA9 and TBX5 were the 2 most sensitive marker genes: 87% (61/70) and 84% (59/70), respectively. RASSF1A and DLEC1 showed the highest specificity at 99% (69/70). Using the criterion of identifying at least any two methylated marker genes, 61/70 cancer samples were positive, corresponding to a sensitivity of 87% and a specificity of 94%. Early stage I or II NSCLC could even be detected with a 100% specificity and 86% sensitivity. In conclusion, MMSP has the potential to be developed into a population-based screening tool and can be useful for early diagnosis of NSCLC. It might also be suitable for monitoring treatment outcome and recurrence.     

Investigating the potential role of genetic and epigenetic variation of DNA methyltransferase genes in hyperplastic polyposis syndrome

Background

Hyperplastic Polyposis Syndrome (HPS) is a condition associated with multiple serrated polyps, and an increased risk of colorectal cancer (CRC). At least half of CRCs arising in HPS show a CpG island methylator phenotype (CIMP), potentially linked to aberrant DNA methyltransferase (DNMT) activity. CIMP is associated with methylation of tumor suppressor genes including regulators of DNA mismatch repair (such as MLH1, MGMT), and negative regulators of Wnt signaling (such as WIF1). In this study, we investigated the potential for interaction of genetic and epigenetic variation in DNMT genes, in the aetiology of HPS.

Methods

We utilized high resolution melting (HRM) analysis to screen 45 cases with HPS for novel sequence variants in DNMT1, DNMT3A, DNMT3B, and DNMT3L. 21 polyps from 13 patients were screened for BRAF and KRAS mutations, with assessment of promoter methylation in the DNMT1, DNMT3A, DNMT3B, DNMT3L MLH1, MGMT, and WIF1 gene promoters.

Results

No pathologic germline mutations were observed in any DNA-methyltransferase gene. However, the T allele of rs62106244 (intron 10 of DNMT1 gene) was over-represented in cases with HPS (p<0.01) compared with population controls. The DNMT1, DNMT3A and DNMT3B promoters were unmethylated in all instances. Interestingly, the DNMT3L promoter showed low levels of methylation in polyps and normal colonic mucosa relative to matched disease free cells with methylation level negatively correlated to expression level in normal colonic tissue. DNMT3L promoter hypomethylation was more often found in polyps harbouring KRAS mutations (p = 0.0053). BRAF mutations were common (11 out of 21 polyps), whilst KRAS mutations were identified in 4 of 21 polyps.

Conclusions

Genetic or epigenetic alterations in DNMT genes do not appear to be associated with HPS, but further investigation of genetic variation at rs62106244 is justified given the high frequency of the minor allele in this case series.     

Genome-Wide Analysis of DNA Methylation Differences in Muscle and Fat from Monozygotic Twins Discordant for Type 2 Diabetes

Background

Monozygotic twins discordant for type 2 diabetes constitute an ideal model to study environmental contributions to type 2 diabetic traits. We aimed to examine whether global DNA methylation differences exist in major glucose metabolic tissues from these twins.

Methodology/Principal Findings

Skeletal muscle (n = 11 pairs) and subcutaneous adipose tissue (n = 5 pairs) biopsies were collected from 53–80 year-old monozygotic twin pairs discordant for type 2 diabetes. DNA methylation was measured by microarrays at 26,850 cytosine-guanine dinucleotide (CpG) sites in the promoters of 14,279 genes. Bisulfite sequencing was applied to validate array data and to quantify methylation of intergenic repetitive DNA sequences. The overall intra-pair variation in DNA methylation was large in repetitive (LINE1, D4Z4 and NBL2) regions compared to gene promoters (standard deviation of intra-pair differences: 10% points vs. 4% points, P<0.001). Increased variation of LINE1 sequence methylation was associated with more phenotypic dissimilarity measured as body mass index (r = 0.77, P = 0.007) and 2-hour plasma glucose (r = 0.66, P = 0.03) whereas the variation in promoter methylation did not associate with phenotypic differences. Validated methylation changes were identified in the promoters of known type 2 diabetes-related genes, including PPARGC1A in muscle (13.9±6.2% vs. 9.0±4.5%, P = 0.03) and HNF4A in adipose tissue (75.2±3.8% vs. 70.5±3.7%, P<0.001) which had increased methylation in type 2 diabetic individuals. A hypothesis-free genome-wide exploration of differential methylation without correction for multiple testing identified 789 and 1,458 CpG sites in skeletal muscle and adipose tissue, respectively. These methylation changes only reached some percentage points, and few sites passed correction for multiple testing.

Conclusions/Significance

Our study suggests that likely acquired DNA methylation changes in skeletal muscle or adipose tissue gene promoters are quantitatively small between type 2 diabetic and non-diabetic twins. The importance of methylation changes in candidate genes such as PPARGC1A and HNF4A should be examined further by replication in larger samples.     

Colorectal cancer DNA methylation patterns from patients in Manaus,Brazil

Background

DNA methylation is commonly linked with the silencing of the gene expression for many tumor suppressor genes. As such, determining DNA methylation patterns should aid, in times to come, in the diagnosis and personal treatment for various types of cancers. Here, we analyzed the methylation pattern from five colorectal cancer patients from the Amazon state in Brazil for four tumor suppressor genes, viz.: DAPK, CDH1, CDKN2A, and TIMP2 by employing a polymerase chain reaction (PCR) specific to methylation. Efforts in the study of colorectal cancer are fundamental as it is the third most of highest incidence in the world.

Results

Tumor biopsies were methylated in 1/5 (20 %), 2/5 (40 %), 4/5 (80 %), and 4/5 (80 %) for CDH1, CDKN2A, DAPK, and TIMP2 genes, respectively. The margin biopsies were methylated in 3/7 (43 %), 2/7 (28 %), 7/7 (100 %), and 6/7 (86 %) for CDH1, CDKN2A, DAPK, and TIMP2, respectively.

Conclusions

Our findings showed DAPK and TIMP2 to be methylated in most samples from both tumor tissues and adjacent non-neoplastic margins; thus presenting distinct methylation patterns. This emphasizes the importance of better understanding of the relation of these patterns with cancer in the context of different populations.     

CpG island hypermethylation of multiple tumor suppressor genes associated with loss of their protein expression during rat lung carcinogenesis induced by 3-methylcholanthrene and diethylnitrosamine

The epigenetic mechanisms underlying the tumorigenesis caused by polycyclic aromatic hydrocarbons and nitrosamine compounds such as 3-methylcholanthrene (MCA) and diethylnitrosamine (DEN) are currently unknown. We reported previously that dynamic changes in DNA methylation occurred during MCA/DEN-induced rat lung carcinogenesis. Here, we used the same animal model to further study the evolution of methylation alterations in tumor suppressor genes (TSGs) DAPK1, FHIT, RASSF1A, and SOCS-3. We found that none of these genes were methylated in either normal or hyperplasia tissue. However, as the severity of the cancer progressed through squamous metaplasia and dysplasia to carcinoma in situ (CIS) and infiltrating carcinoma, so methylation became more prevalent. Particularly dramatic increases in the level of methylation, the average number of methylated genes, and the incidence of concurrent methylation in three genes were observed in CIS and infiltrating carcinoma. Similar but less profound changes were seen in squamous metaplasia and dysplasia. Furthermore, methylation status was closely correlated to loss of protein expression for these genes, with protein levels markedly declining along the continuum of carcinogenesis. These results suggest that progressive CpG island hypermethylation leading to inactivation of TSGs might be a vital molecular mechanism in the pathogenesis of MCA/DEN-induced multistep rat lung carcinogenesis.