Length of paternal lifespan is manifested in the DNA methylome of their nonagenarian progeny

The heritability of lifespan is 20-30%, but only a few genes associated with longevity have been identified. To explain this discrepancy, the inheritance of epigenetic features, such as DNA methylation, have been proposed to contribute to the heritability of lifespan.We investigated whether parental lifespan is associated with DNA methylation profile in nonagenarians. A regression model, adjusted for differences in blood cell proportions, identified 659 CpG sites where the level of methylation was associated with paternal lifespan. However, no association was observed between maternal lifespan and DNA methylation. The 659 CpG sites associated with paternal lifespan were enriched outside of CpG islands and were located in genes associated with development and morphogenesis, as well as cell signaling. The largest difference in the level of methylation between the progeny of the shortest-lived and longest-lived fathers was identified for CpG sites mapping to CXXC5. In addition, the level of methylation in three Notch-genes (NOTCH1, NOTCH3 and NOTCH4) was also associated with paternal lifespan.There are implications for the inheritance of acquired traits via epigenetic mechanisms in mammals. Here we describe DNA methylation features that are associated with paternal lifespan, and we speculate that the identified CpG sites may represent intergenerational epigenetic inheritance.     

DNA methylation patterns in lung carcinomas

The genome of epithelial tumors is characterized by numerous chromosomal aberrations, DNA base sequence changes, and epigenetic abnormalities. The epigenome of cancer cells has been most commonly studied at the level of DNA CpG methylation. In squamous cell carcinomas of the lung, CpG methylation patterns undergo substantial changes relative to normal lung epithelium. Using a genome-scale mapping technique for CpG methylation (MIRA-chip), we characterized CpG island methylation and methylation patterns of entire chromosome arms at a level of resolution of ～100 bp. In individual stage I lung carcinomas, several hundred and probably up to a thousand CpG islands become methylated. Interestingly, a large fraction (almost 80%) of the tumor-specifically methylated sequences are targets of the Polycomb complex in embryonic stem cells. Homeobox genes are particularly overrepresented and all four HOX gene loci on chromosomes 2, 7, 12, and 17 are hotspots for tumor-associated methylation because of the presence of multiple methylated CpG islands within these loci. DNA hypomethylation at CpGs in squamous cell tumors preferentially affects repetitive sequence classes including SINEs, LINEs, subtelomeric repeats, and segmental duplications. Since these epigenetic changes are found in early stage tumors, their contribution to tumor etiology as well as their potential usefulness as diagnostic or prognostic biomarkers of the disease should be considered.     

A modified MethyLight assay predicts the clinical outcomes of anti‐epidermal growth factor receptor treatment in metastatic colorectal cancer

DNA methylation status correlates with clinical outcomes of anti‐epidermal growth factor receptor (EGFR) treatment. There is a strong need to develop a simple assay for measuring DNA methylation status for the clinical application of drug selection based on it. In this study, we collected data from 186 patients with metastatic colorectal cancer (mCRC) who had previously received anti‐EGFR treatment. We modified MethyLite to develop a novel assay to classify patients as having highly methylated colorectal cancer (HMCC) or low‐methylated colorectal cancer (LMCC) based on the methylation status of 16 CpG sites of tumor‐derived genomic DNA in the development cohort (n = 30). Clinical outcomes were then compared between the HMCC and LMCC groups in the validation cohort (n = 156). The results showed that HMCC had a significantly worse response rate (4.2% vs 33.3%; P = .004), progression‐free survival (median: 2.5 vs 6.6 mo, P < .001, hazard ratio [HR] = 0.22), and overall survival (median: 5.6 vs 15.5 mo, P < .001, HR = 0.23) than did LMCC in patients with RAS wild‐type mCRC who were refractory or intolerable to oxaliplatin‐ and irinotecan‐based chemotherapy (n = 101). The DNA methylation status was an independent predictive factor and a more accurate biomarker than was the primary site of anti‐EGFR treatment. In conclusion, our novel DNA methylation measurement assay based on MethyLight was simple and useful, suggesting its implementation as a complementary diagnostic tool in a clinical setting.     

Characterizing DNA methylation patterns in pancreatic cancer genome

We performed a global methylation profiling assay on 1505 CpG sites across 807 genes to characterize DNA methylation patterns in pancreatic cancer genome. We found 289 CpG sites that were differentially methylated in normal pancreas, pancreatic tumors and cancer cell lines. We identified 23 and 35 candidate genes that are regulated by hypermethylation and hypomethylation in pancreatic cancer, respectively. We also identified candidate methylation markers that alter the expression of genes critical to gemcitabine susceptibility in pancreatic cancer. These results indicate that aberrant DNA methylation is a frequent epigenetic event in pancreatic cancer; and by using global methylation profiling assay, it is possible to identify these markers for diagnostic and therapeutic purposes in this disease.     

Genome‐wide DNA methylation patterns in pancreatic ductal adenocarcinoma reveal epigenetic deregulation of SLIT‐ROBO,ITGA2 and MET signaling

The importance of epigenetic modifications such as DNA methylation in tumorigenesis is increasingly being appreciated. To define the genome‐wide pattern of DNA methylation in pancreatic ductal adenocarcinomas (PDAC), we captured the methylation profiles of 167 untreated resected PDACs and compared them to a panel of 29 adjacent nontransformed pancreata using high‐density arrays. A total of 11,634 CpG sites associated with 3,522 genes were significantly differentially methylated (DM) in PDAC and were capable of segregating PDAC from non‐malignant pancreas, regardless of tumor cellularity. As expected, PDAC hypermethylation was most prevalent in the 5′ region of genes (including the proximal promoter, 5′UTR and CpG islands). Approximately 33% DM genes showed significant inverse correlation with mRNA expression levels. Pathway analysis revealed an enrichment of aberrantly methylated genes involved in key molecular mechanisms important to PDAC: TGF‐β, WNT, integrin signaling, cell adhesion, stellate cell activation and axon guidance. Given the recent discovery that SLIT‐ROBO mutations play a clinically important role in PDAC, the role of epigenetic perturbation of axon guidance was pursued in more detail. Bisulfite amplicon deep sequencing and qRT‐PCR expression analyses confirmed recurrent perturbation of axon guidance pathway genes SLIT2, SLIT3, ROBO1, ROBO3, ITGA2 and MET and suggests epigenetic suppression of SLIT‐ROBO signaling and up‐regulation of MET and ITGA2 expression. Hypomethylation of MET and ITGA2 correlated with high gene expression, which was associated with poor survival. These data suggest that aberrant methylation plays an important role in pancreatic carcinogenesis affecting core signaling pathways with potential implications for the disease pathophysiology and therapy.     

Aberrant CpG island methylation in cancer cell lines arises in the primary cancers from which they were derived

A higher prevalence of epigenetic inactivation of tumor suppressor genes has been reported in cancer cell line populations compared to primary cancer populations. Cancer-related genes are commonly methylated in cancer cell lines but it is not known the extent to which tumor suppressor genes may be artificially methylated in vitro. We therefore examined 10 pancreatic cancer cell lines and corresponding primary tumors for aberrant DNA methylation of promoter CpG islands of eight genes and seven CpG islands. Using methylation-specific PCR (MSP), methylation was not detected at any of the 15 CpG islands in 15 normal pancreata or in an immortalized normal pancreatic duct epithelial (HPDE) cell line. Of 150 loci examined, 49 loci were methylated in both primary carcinomas and their corresponding cell lines, 95 loci were not methylated in either cell lines or their corresponding primary carcinomas. There were four loci methylated only in cell lines while another two loci were methylated only in primary carcinomas. Overall, the methylation status of primary carcinomas and their cell lines were concordant in 96% of cases (144 of 150) (J statistic; J=0.92, P<0.0001). We conclude that most of the DNA methylation of tumor suppressor genes observed in cancer cell lines is present in the primary carcinomas from which they were derived.     

Epigenetic silencing of the kinase tumor suppressor WNK2 is tumor-type and tumor-grade specific

Both genetic and epigenetic mechanisms contribute to meningioma development by altering gene expression and protein function. To determine the relative contribution of each mechanism to meningioma development, we used an integrative approach measuring copy number and DNA methylation changes genomewide. We found that genetic alterations affected 1.9%, 7.4%, and 13.3% of the 691 loci studied, whereas epigenetic mechanisms affected 5.4%, 9.9%, and 10.3% of these loci in grade I, II, and III meningiomas, respectively. Genetic and epigenetic mechanisms rarely involved the same locus in any given tumor. The predilection for epigenetic rather than genetic silencing was exemplified at the 5′ CpG island of WNK2, a serine-threonine kinase gene on chromosome 9q22.31. WNK2 is known to negatively regulate epidermal growth factor receptor signaling via inhibition of MEK1 (mitogen-activated protein kinase kinase 1), and point mutations have been reported in WNK1, WNK2, WNK3, and WNK4. In meningiomas, WNK2 was aberrantly methylated in 83% and 71% of grade II and III meningiomas, respectively, but rarely in a total of 209 tumors from 13 other tumor types. Aberrant methylation of the CpG island was associated with decreased expression in primary tumors. WNK2 could be reactivated with a methylation inhibitor in IOMM-Lee, a meningioma cell line with a densely methylated WNK2 CpG island and lack of WNK2 expression. Expression of exogenous WNK2 inhibited colony formation, implicating it as a potential cell growth suppressor. These findings indicate that epigenetic mechanisms are common across meningiomas of all grades and that for specific genes such as WNK2, epigenetic alteration may be the dominant, grade-specific mechanism of gene inactivation.     

Targeting EGFR with photodynamic therapy in combination with Erbitux enhances in vivo bladder tumor response

Background

Renal cell carcinoma (RCC) is histopathologically heterogeneous with clear cell and papillary the most common subtypes. The most frequent molecular abnormality in clear cell RCC is VHL inactivation but promoter methylation of tumour suppressor genes is common in both subtypes of RCC. To investigate whether RCC CpG methylation status was influenced by histopathology and VHL status we performed high-throughput epigenetic profiling using the Illumina Goldengate Methylation Array in 62 RCC (29 RCC from von Hippel-Lindau (VHL) disease patients, 20 sporadic clear cell RCC with wild type VHL and 13 sporadic papillary RCC).

Results

43 genes were methylated in >20% of primary RCC (range 20–45%) and most (37/43) of these had not been reported previously to be methylated in RCC. The distribution of the number of methylated CpGs in individual tumours differed from the expected Poisson distribution (p < 0.00001; log-likelihood G test) suggesting that a subset of RCC displayed a CpG Island Methylator Phenotype. Comparison of RCC subtypes revealed that, on average, tumour specific CpG methylation was most prevalent in papillary RCC and least in VHL RCC. Many of the genes preferentially methylated in pRCC were linked to TGFβ or ERK/Akt signalling.

Conclusion

These findings demonstrate differing patterns of tumour-specific CpG methylation in VHL and non VHL clear cell RCC and papillary RCC, and identify multiple novel potential CpG methylation biomarkers for RCC.     

Characteristic methylation profile in CpG island methylator phenotype‐negative distal colorectal cancers

Aberrant DNA methylation is involved in colon carcinogenesis. Although the CpG island methylator phenotype (CIMP) is defined as a subset of colorectal cancers (CRCs) with remarkably high levels of DNA methylation, it is not known whether epigenetic processes are also involved in CIMP‐negative tumors. We analyzed the DNA methylation profiles of 94 CRCs and their corresponding normal‐appearing colonic mucosa with 11 different markers, including the five classical CIMP markers. The CIMP markers were frequently methylated in proximal CRCs (p < 0.01); however, RASSF1A methylation levels were significantly higher in distal CRCs, the majority of which are CIMP‐negative (p < 0.05). Similarly, methylation levels of RASSF1A and SFRP1 in the normal‐appearing mucosae of distal CRC cases were significantly higher than those in the proximal CRC cases (p < 0.05). They were also positively correlated with age (RASSF1A, p < 0.01; SFRP1, p < 0.01). Microarray‐based genome‐wide DNA methylation analysis of 18 CRCs revealed that 168 genes and 720 genes were preferentially methylated in CIMP‐negative distal CRCs and CIMP‐positive CRCs, respectively. Interestingly, more than half of the hypermethylated genes in CIMP‐negative distal CRCs were also methylated in the normal‐appearing mucosae, indicating that hypermethylation in CIMP‐negative distal CRCs is more closely associated with age‐related methylation. By contrast, more than 60% of the hypermethylated genes in CIMP‐positive proximal CRCs were cancer specific (p < 0.01). These data altogether suggest that CpG island promoters appear to be methylated in different ways depending on location, a finding which may imply the presence of different mechanisms for the acquisition of epigenetic changes during colon tumorigenesis.     

DNA demethylase is expressed in ovarian cancers and the expression correlates with demethylation of CpG sites in the promoter region of c-erbB-2 and survivin genes.

The objectives of this study were to examine DNA demethylase (dMTase) expression in ovarian cancers and evaluate methylation of CpG sites in the promoter of the c-erbB-2 gene and survivin gene exon 1. Forty-three epithelial ovarian cancers and 43 non-cancerous ovarian tissues were studied for dMTase expression by RT-PCR. Genomic DNA was extracted and digested with HindIII and then HpaII. CpG site-sensitive primers were constructed to amplify the promoter of the c-erbB-2 gene and survivin gene exon 1. Immunohistochemical evaluation of ErbB-2 protein and RT-PCR for survivin were also performed. dMTase was positive in 88.4% of ovarian cancers but only in 9.3% of non-cancerous ovaries (P<0.001, Fisher's exact test). The expression was similarly observed in both early stage (stage I+II: 17/19) and advanced stage (stage III+IV: 21/24) groups of ovarian malignancy. It was found that 78.9% of dMTase-positive cancers had both c-erbB-2 promoter and survivin gene exon 1 unmethylated, whereas 40% of dMTase-negative cancers had both sites methylated. In non-cancerous ovaries, these sites were mostly methylated (90.6%) and the difference from cancer cases was highly significant (P<0.001). Immunohistochemical evaluation of ErbB-2 showed significant correlation of unmethylated c-erbB-2 promoter and ErbB-2 expression. The RT-PCR for survivin expression showed that 86% of cancers were positive and six cases were negative. Exon 1 was methylated in 83% of the survivin-negative cases. This is the first report of dMTase expression in ovarian cancers. The correlation of dMTase expression with unmethylation of c-erbB-2 promoter and survivin gene exon 1 suggests that these sites may be targets for demethylation by the enzyme. The up-regulation of oncogenes may be the consequence of epigenetic control of gene expression by the dMTase.     

DACT2 is frequently methylated in human gastric cancer and methylation of DACT2 activated Wnt signaling

Dapper, Dishevelled-associated antagonist of β-catenin (DACT), is a key regulator of Wnt signaling pathway. The purpose of this study is to explore the epigenetic changes and the function ofDACT2 in human gastric cancer (GC). Eight human gastric cancer cell lines, 167 cases of primary gastric cancer and 8 cases of normal gastric mucosa were involved in this study. In addition, methylation Specific PCR (MSP), semi-quantitative RT-PCR, colony formation assay, flow cytometry assay, siRNA, immunofluorescence techniques and xenograft mice models were employed. The results indicate that DACT2 is frequently methylated in human primary gastric cancer (55.7%), and that methylation of DACT2 is associated with lost or reduction in its expression (X² test, P<0.01). We found that DACT2 expression was regulated by promoter region hypermethylation. Methylation of DACT2 is associated with tumor differentiation, invasion and intravascular cancerous emboli (X² test, P<0.05, P<0.05 and P<0.05). In gastric cancer patients treated with 5-FU and cisplatin, the five-year survival rates are higher in DACT2 methylated cases. DACT2 inhibits cell proliferation, migration and invasion in gastric cancer cells and suppresses gastric cancer xenografts in mice. Restoration of DACT2 expression inhibits both canonical and noncanonical WNT signaling in SGC7901 cells. Restoration of DACT2 expression sensitized gastric cancer cells to paclitaxel and 5-FU. In conclusion, DACT2 is frequently methylated in human gastric cancer and DACT2 expression is silenced by promoter region hypermethylation. DACT2 suppressed gastric cancer proliferation, invasion and metastasis by inhibiting Wnt signaling both in vitro and in vivo.     

Methylated CpG site count of dapper homolog 1 (DACT1) promoter prediction the poor survival of gastric cancer

Objective: To elucidate the clinical significance of the methylated status of CpG sites of dapper homolog 1 (DACT1) promoter in the survival prediction in gastric cancer (GC). Methods: The large scale GC patients (n=459) were analyzed for the quantitatively methylated status of CpG sites of DACT1 DNA promoter with the bisulphite sequencing PCR (BSP). With gene sequencing analysis, the methylated statuses of 12 cytosine-phosphate-guanine (CpG) sites in DACT1 promoter were detected to supply detailed information for the precisely prognostic prediction. Associations between molecular, clinicopathologic, and survival data were analyzed. Results: With the MSP detection, different methylated levels of DACT1 promoter were identified in the 25 GC tissues, while none of 25 normal gastric mucosal tissues were found to be methylated. DACT1 promoter methylation was found in 28.32% in 459 patients. GC patients with 4 or more methylated CpG sites of DACT1 promoter was significantly associated with the poorer survival (P=0.19). The methylated statuses of CpG -515, CpG -435, and CpG -430 sites were also identified to provide the elaborate survival discrimination for 459 GC patients, respectively (P=0.049, =0.006, and =0.037). In addition, we demonstrated that the methylated CpG site count had smallest AIC and BIC values than other three methylated status of CpG sites for prediction the survival of 459 GC patients. Conclusions: The methylated CpG site count of DACT1 promoter had the significant applicability for clinical evaluation the prognosis of GC.     

Screening for genomic fragments that are methylated specifically in colorectal carcinoma with a methylated MLH1 promoter

A subset of colorectal carcinomas (CRCs) is associated with microsatellite instability (MSI) of the genome. Although extensive methylation of CpG islands within the promoter regions of DNA mismatch repair genes such as MLH1 is thought to play a central role in tumorigenesis for MSI-positive sporadic CRCs, it has been obscure whether such aberrant epigenetic regulation occurs more widely and affects other cancer-related genes in vivo. Here, by using methylated CpG island amplification coupled with representational difference analysis (MCA-RDA), we screened genomic fragments that are selectively methylated in CRCs positive for MLH1 methylation, resulting in the identification of hundreds of CpG islands containing genomic fragments. Methylation status of such CpG islands was verified for 28 genomic clones in 8 CRC specimens positive for MLH1 methylation and the corresponding paired normal colon tissue as well as in 8 CRC specimens negative for methylation. Many of the CpG islands were preferentially methylated in the MLH1 methylation-positive CRC specimens, although methylation of some of them was more widespread. These data provide insights into the complex regulation of the methylation status of CpG islands in CRCs positive for MSI and MLH1 methylation.