Constitutional promoter methylation and risk of familial melanoma

Constitutional epigenetic changes detected in blood or non-disease involving tissues have been associated with disease susceptibility. We measured promoter methylation of CDKN2A (p16 and p14ARF) and 13 melanoma-related genes using bisulfite pyrosequencing of blood DNA from 114 cases and 122 controls in 64 melanoma-prone families (26 segregating CDKN2A germline mutations). We also obtained gene expression data for these genes using microarrays from the same blood samples. We observed that CDKN2A epimutation is rare in melanoma families, and therefore is unlikely to cause major susceptibility in families without CDKN2A mutations. Although methylation levels for most gene promoters were very low (<5%), we observed a significantly reduced promoter methylation (odds ratio = 0.63, 95% confidence interval = 0.50, 0.80, P < 0.001) and increased expression (fold change = 1.27, P = 0.048) for TNFRSF10C in melanoma cases. Future research in large prospective studies using both normal and melanoma tissues is required to assess the significance of TNFRSF10C methylation and expression changes in melanoma susceptibility.     

Identification of novel epigenetically modified genes in human melanoma via promoter methylation gene profiling

The inactivation of tumor-related genes through the aberrant methylation of promoter CpG islands is thought to contribute to tumor initiation and progression. We therefore investigated promoter methylation events involved in cutaneous melanoma by screening 30 genes of interest for evidence of promoter hypermethylation, examining 20 melanoma cell lines and 40 freshly procured melanoma samples. Utilizing quantitative methylation-specific PCR, we identified five genes (SOCS1, SOCS2, RAR-beta 2, TNFSF10C, and TNFSF10D) with hypermethylation frequencies ranging from 50% to 80% in melanoma cell lines as well as freshly procured tissue samples. Eighteen genes (LOX, RASSF1A, WFDC1, TM, APC, TFPI2, TNFSF10A, CDKN2A, MGMT, TIMP3, ASC, TPM1, IRF8, CIITA-PIV, CDH1, SYK, HOXB13, and DAPK1) were methylated at lower frequencies (2-30%). Two genes (CDKN1B and PTEN), previously reported as methylated in melanoma, and five other genes (RECK, IRF7, PAWR, TNFSF10B, and Rb) were not methylated in the samples screened here. Daughter melanoma cell lines showed identical methylation patterns when compared with original samples from which they were derived, as did synchronous metastatic lesions from the same patient. We identified four genes (TNFSF10C, TNFSF10D, LOX, and TPM1) that have never before been identified as hypermethylated in melanoma, with an overall methylation frequency of 60, 80, 50, and 10%, respectively, hypothesizing that these genes may play an important role in melanoma progression.     

CDKN2A mutations and MC1R variants in Italian patients with single or multiple primary melanoma

We evaluated the contribution of germline CDKN2A mutations and MC1R variants to the development of melanoma in a hospital-based study of single (SPM, n = 398) and multiple primary melanoma (MPM, n = 95). The overall frequency of CDKN2A mutations was 15.2%, and four-fold higher in MPM than in SPM cases (OR = 4.27; 95% CI 2.43-7.53). The likelihood of identifying a CDKN2A mutation increased with family history of melanoma and younger age at diagnosis in MPM cases. Compared to SPM patients, the risk of harboring a CDKN2A mutation rose as the number of primary melanomas increased and was not influenced by family history. The G101W and E27X founder mutations were the most common. Several other mutations (W15X, Q50X, R58X, A68L, A127P and H142R) were detected for the first time in Italian patients. One novel mutation, T77A, was identified. Several non-coding variants with unknown functional significance were also found (5'UTR -25C > T, -21C > T, -67G > C, IVS1 +37G > C); the novel 5'UTR -21C > T variant was not detected in controls. The CDKN2A A148T polymorphism was more frequent in MPM patients than in the control population (15.7% versus 6.6%). Compared to the SPM patients, MPM cases had a 2-fold increased probability of being MC1R variant carriers and a higher probability of carrying two or more variants. No specific association was observed between the type of variant and the number of melanomas, suggesting that the number rather than the type of MC1R variant increases the risk of MPM. We observed no interaction between CDKN2A status and the presence of MC1R variants. The high frequency of CDKN2A mutations in our MPM cases, independent of their family history, is of relevance to genetic counseling and testing in our population.     

Clinical and prognostic effects of CDKN2A,CDKN2B and CDH13 promoter methylation in ovarian cancer: a study using meta-analysis and TCGA data

Abstract

Background: Promoter methylation of tumour suppressor genes (TSGs) CDKN2A, CDKN2B and CDH13 has been reported in ovarian cancer. However, the clinicopathological characteristics and prognostic role of CDKN2A, CDKN2B and CDH13 promoter methylation in ovarian carcinoma remained unclear.

Methods: The pooled odds ratio (OR) or hazard ratios (HRs) with their 95% confidence intervals (95% CIs) were calculated in this meta-analysis. The Cancer Genome Atlas data were obtained to confirm the role of CDKN2A, CDKN2B and CDH13 methylation in ovarian cancer.

Results: CDKN2A, CDKN2B and CDH13 promoter methylation was higher in ovarian cancer than in normal ovarian tissues. CDH13 promoter methylation was correlated with tumour histology (serous vs. non-serous type: OR?=?0.33, p?=?0.031). CDKN2A promoter methylation was not linked to overall survival (OS), but it was correlated with a poor prognosis in progression-free survival (HR?=?1.55, p?=?0.004). TCGA data showed no correlation between CDKN2A, CDKN2B and CDH13 methylation and OS as well as disease-free survival (DFS).

Conclusions: CDKN2A, CDKN2B and CDH13 promoter methylation may correlate with the increased risk of ovarian cancer. CDKN2A promoter methylation may be an independent prognostic biomarker for predicting progression-free survival.     

Gene promoter methylation in colorectal cancer and healthy adjacent mucosa specimens

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.     

Genetics of familial melanoma: 20 years after CDKN2A

Twenty years ago, the first familial melanoma susceptibility gene, CDKN2A, was identified. Two years later, another high‐penetrance gene, CDK4, was found to be responsible for melanoma development in some families. Progress in identifying new familial melanoma genes was subsequently slow; however, with the advent of next‐generation sequencing, a small number of new high‐penetrance genes have recently been uncovered. This approach has identified the lineage‐specific oncogene MITF as a susceptibility gene both in melanoma families and in the general population, as well as the discovery of telomere maintenance as a key pathway underlying melanoma predisposition. Given these rapid recent advances, this approach seems likely to continue to pay dividends. Here, we review the currently known familial melanoma genes, providing evidence that most additionally confer risk to other cancers, indicating that they are likely general tumour suppressor genes or oncogenes, which has significant implications for surveillance and screening.     

Blood glutathione redox status and global methylation of peripheral blood mononuclear cell DNA in Bangladeshi adults

Oxidative stress and DNA methylation are metabolically linked through the relationship between one-carbon metabolism and the transsulfuration pathway, but possible modulating effects of oxidative stress on DNA methylation have not been extensively studied in humans. Enzymes involved in DNA methylation, including DNA methyltransferases and histone deacetylases, may show altered activity under oxidized cellular conditions. Additionally, in vitro studies suggest that glutathione (GSH) depletion leads to global DNA hypomethylation, possibly through the depletion of S-adenosylmethionine (SAM). We tested the hypothesis that a more oxidized blood GSH redox status is associated with decreased global peripheral blood mononuclear cell (PBMC) DNA methylation in a sample of Bangladeshi adults. Global PBMC DNA methylation and whole blood GSH, glutathione disulfide (GSSG), and SAM concentrations were measured in 320 adults. DNA methylation was measured by using the [³H]-methyl incorporation assay; values are inversely related to global DNA methylation. Whole blood GSH redox status (E_h) was calculated using the Nernst equation. We found that a more oxidized blood GSH E_h was associated with decreased global DNA methylation (B ± SE, 271 ± 103, p = 0.009). Blood SAM and blood GSH were associated with global DNA methylation, but these relationships did not achieve statistical significance. Our findings support the hypothesis that a more oxidized blood GSH redox status is associated with decreased global methylation of PBMC DNA. Furthermore, blood SAM does not appear to mediate this association. Future research should explore mechanisms through which cellular redox might influence global DNA methylation.     

Genome‐wide promoter methylation analysis identifies epigenetic silencing of MAPK13 in primary cutaneous melanoma

The involvement of epigenetic alterations in the pathogenesis of melanoma is increasingly recognized. Here, we performed genome‐wide DNA methylation analysis of primary cutaneous melanoma and benign melanocytic nevus interrogating 14 495 genes using BeadChip technology. This genome‐wide view of promoter methylation in primary cutaneous melanoma revealed an array of recurrent DNA methylation alterations with potential diagnostic applications. Among 106 frequently hypermethylated genes, there were many novel methylation targets and tumor suppressor genes. Highly recurrent methylation of the HOXA9, MAPK13, CDH11, PLEKHG6, PPP1R3C, and CLDN11 genes was established. Promoter methylation of MAPK13, encoding p38δ, was present in 67% of primary and 85% of metastatic melanomas. Restoration of MAPK13 expression in melanoma cells exhibiting epigenetic silencing of this gene reduced proliferation, indicative of tumor suppressive functions. This study demonstrates that DNA methylation alterations are widespread in melanoma and suggests that epigenetic silencing of MAPK13 contributes to melanoma progression.     

Cocaine-mediated downregulation of microglial miR-124 expression involves promoter DNA methylation

Neuroinflammation plays a critical role in the development of reward-related behavior in cocaine self-administration rodents. Cocaine, one of most commonly abused drugs, has been shown to activate microglia both in vitro and in vivo. Detailed molecular mechanisms underlying cocaine-mediated microglial activation remain poorly understood. microRNAs (miRs) belonging to a class of small noncoding RNA superfamily have been shown to modulate the activation status of microglia. miR-124, one of the microglia-enriched miRs, functions as an anti-inflammatory regulator that maintains microglia in a quiescent state. To date, the possible effects of cocaine on microglial miR-124 levels and the associated underlying mechanisms have not been explored. In the current study, we demonstrated that cocaine exposure decreased miR-124 levels in both BV-2 cells and rat primary microglia. These findings were further validated in vivo, wherein we demonstrated decreased abundance of miR-124 in purified microglia isolated from cocaine-administered mice brains compared with cells from saline administered animals. Molecular mechanisms underlying these effects involved cocaine-mediated increased mRNA and protein expression of DNMTs in microglia. Consistently, cocaine substantially increased promoter DNA methylation levels of miR-124 precursors (pri-miR-124-1 and ?2), but not that of pri-miR-124-3, both in vitro and in vivo. In summary, our findings demonstrated that cocaine exposure increased DNA methylation of miR-124 promoter resulting into its downregulation, which, in turn, led to microglial activation. Our results thus implicate that epigenetic modulation of miR-124 could be considered as a potential therapeutic approach to ameliorate microglial activation and, possibly, the development of cocaine addiction.     

Promoter methylation as biomarkers for diagnosis of melanoma: A systematic review and meta-analysis

Melanoma is one of the most common skin cancer that is characterized by rapid growth, early metastasis, high malignant, and mortality. Accumulating evidence demonstrated that promoter methylation of tumor-suppressor genes is implicated in the pathogenesis of melanoma. In the current study, we performed a meta-analysis to identify promising methylation biomarkers in the diagnosis of melanoma. We carried out a systematic literature search using Pubmed, Embase, and ISI web knowledge database and found that gene promoter methylation of 50 genes was reported to be associated with the risk of melanoma. Meta-analysis revealed that hypermethylation of claudin 11 (CLDN11; odds ratio [OR], 16.82; 95% confidence interval [CI], 1.97–143.29; p = 0.010), O-6-methylguanine-DNA methyltransferase (MGMT; OR, 5.59; 95% CI, 2.51–12.47; p < 0.0001), cyclin-dependent kinase inhibitor 2A (p16; OR, 6.57; 95% CI, 2.19–19.75; p = 0.0008), retinoic acid receptor β (RAR-β2; OR, 24.31; 95% CI, 4.58–129.01; p = 0.0002), and Ras association domain family member (RASSF1A; OR, 9.35; 95% CI, 4.73–18.45; p < 0.00001) was significantly higher in melanoma patients compared with controls. CLDN11 (OR, 14.52; 95% CI, 1.84–114.55; p = 0.01), MGMT (OR, 8.08; 95% CI, 1.84–35.46; p = 0.006), p16 (OR, 9.44; 95% CI, 2.68–33.29; p = 0.0005), and RASSF1A (OR, 7.72; 95% CI, 1.05–56.50; p = 0.04) hypermethylation was significantly increased in primary melanoma compared with controls. Methylation frequency of CLDN11 (OR, 25.56; 95% CI, 2.32–281.66; p = 0.008), MGMT (OR, 4.64; 95% CI, 1.98–10.90; p = 0.0004), p16 (OR, 4.31; 95% CI, 1.33–13.96; p = 0.01), and RASSF1A (OR, 10.10; 95% CI, 2.87–35.54; p = 0.0003) was significantly higher in metastasis melanoma compared with controls. These findings indicated that CLDN11, MGMT, p16, RAR-β2, and RASSF1A hypermethylation is a risk factor and a potential biomarker for melanoma. CLDN11, MGMT, p16, and RASSF1A promoter methylation may take part in the development of melanoma and become useful biomarkers in the early diagnosis of the disease.     

A novel CDKN2A variant (p16L117P) in a patient with familial and multiple primary melanomas

Germline mutations in CDKN2A (p16) are commonly found in patients with family history of melanoma or personal history of multiple primary melanomas. The p16 tumor suppressor gene regulates cell cycle progression and senescence through binding of cyclin‐dependent kinases (CDK) and also regulates cellular oxidative stress independently of cell cycle control. We identified a germline missense (c.350T>C, p.Leu117Pro) CDKN2A mutation in a patient who had history of four primary melanomas, numerous nevi, and self‐reported family history of melanoma. This particular CDKN2A mutation has not been previously reported in prior large studies of melanoma kindreds or patients with multiple primary melanomas. Compared with wild‐type p16, the p16^L117P mutant largely retained binding capacity for CDK4 and CDK6 but exhibited impaired capacity for repressing cell cycle progression and inducing senescence, while retaining its ability to reduce mitochondrial reactive oxygen species. Structural modeling predicted that the Leu117Pro mutation disrupts a putative adenosine monophosphate (AMP) binding pocket involving residue 117 in the fourth ankyrin domain. Identification of this new likely pathogenic variant extends our understanding of CDKN2A in melanoma susceptibility and implicates AMP as a potential regulator of p16.     

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.     

Proteomic map of peripheral blood mononuclear cells

In the field of proteomics extensive efforts have been focused on the knowledge of proteins expressed by different cell types. In particular, enormous progress has been done in the characterization of blood cellular components. In this work, we have established a public 2-DE database for human peripheral blood mononuclear cells (PBMCs) proteins. Two hundred and forty-six spots corresponding to 174 different proteins have been identified on 2-DE gels from PBMCs isolated from six healthy individuals. All the identified proteins have been classified in thirteen categories on the basis of their differential functions or cellular localization and annotated at the http://physiology.unile.it/proteomics. The role of several proteins has been discussed in relation to their biological function. We intend to show the potentiality of PBMCs to investigate the proteomics changes possibly associated with a large number of pathologies such as autoimmune, neurodegenerative and cancer diseases.     

Genome‐wide screen of promoter methylation analysis of ES cells and ES derived epidermal‐like cells

Embryonic stem cells (ESCs) are a population of pluripotent cells which can differentiate into different cell types. However, there are few reports with regard to differentiate ESCs into epidermal cells in vitro. In this study, we aimed to investigate differentially methylated promoters involved in process of differentiation from ESCs into epidermal‐like cells (ELCs) induced by human amnion. We successfully induced ESCs into ELCs, which expressed the surface markers of CK19, CK15 and β1‐integrin. With MeDIP‐chip arrays, we identified 3435 gene promoters to be differentially methylated, involving 894 HCP (high CpG‐containing promoter), 974 ICP (intermediate CpG‐containing promoter) and 1567 LCP (low CpG‐containing promoter) among all the 17 500 DNA methylation regions of gene promoters in both ESCs and ELCs. Gene oncology and pathway analysis demonstrated that these genes were involved in all the three categories of GO enrichment analysis, including biological process, molecular function and cellular component. All these data suggested that embryonic stem cells can differentiate into epidermal‐like cells and promoter methylation is of great importance in this process. Copyright © 2015 John Wiley & Sons, Ltd.     

Screening and verification of microRNA promoter methylation sites in hepatocellular carcinoma

The promoter methylation mode of microribonucleic acid (miRNA) plays a crucial role in the process of hepatocellular carcinoma (HCC). Therefore, the primary purpose of this study was to screen and verify the miRNA methylation sites associated with the overall survival (OS) and clinical characteristics of HCC patients. Methylation-related data were from the Cancer Genome Atlas (TCGA). R software was utilized to screen the methylation sites. The least absolute shrinkage and selection operator algorithm was utilized to develop the miRNA promoter methylation models. Then, methylation-specific polymerase chain reaction was performed with 146 HCC tissues to verify the accuracy of the vascular infiltration-related model. Additionally, we verified the functions of vascular infiltration-related miRNA by utilizing cells transfected with miR-199a-3p mimic. The model for predicting OS of HCC patients contained eight methylation sites. The Kaplan-Meier analysis suggested that the model could divide HCC patients into high- and low-risk groups (P < .0001). COX regression analysis suggested that the model (P < .001; 95% CI, 1.264-2.709) and T category (P < .001; 95% CI, 1.472-3.119) were independent risk factors for affecting OS of HCC patients. The model for predicting vascular infiltration, pathological grade, and clinical stage contained 7, 10, and 9 methylation sites respectively, with their area under the receiver operating characteristic curve (AUC) values 0.667, 0.745, and 0.725, respectively. The functional analysis suggested that miRNA methylation is involved in various biological processes such as WNT, MAPK, and mTOR signaling pathways. The accuracy of the vascular infiltration-related model was consistent with our previous bioinformatics assay. And upregulation of miR-199a-3p decreased migration and invasion abilities. The screened miRNA promoter methylation sites can be served as biomarkers for judging OS, vascular infiltration, pathology grade, and clinical stage. It can also provide new targets for improving the treatment and prognosis of HCC patients.     

Decreased expression of EZH2 reactivates RASSF2A by reversal of promoter methylation in breast cancer cells

EZH2, the catalytic subunit of polycomb repressor complex 2, has oncogenic properties, whereas RASSF2A, a Ras association domain family protein, has a tumor suppressor role in many types of human cancer. However, the interrelationship between these two genes remains unclear. Here, we showed that the downregulation of EZH2 reduces CpG island methylation of the RASSF2A promoter, thereby leading to increased RASSF2A expression. Our findings also showed that knockdown of EZH2 increased RASSF2A expression in the human breast cancer cell line MCF‐7 in cooperation with DNMT1. This was similar to the effect of 5‐Aza‐CdR, a DNA methylation inhibitor that reactivates tumor suppressor genes and activated RASSF2A expression in our study. The EZH2 inhibitor DZNep markedly suppressed the proliferation, migration, and invasion of MCF‐7 cells treated with ADR and TAM. EZH2 inhibits the expression of tumor suppressor gene RASSF2A via promoter hypermethylation. Thus, it plays an important role in tumorigenesis and is a potential therapeutic target for the treatment of breast cancer.     

Superantigen-induced apoptotic death of tumor cells is mediated by cytotoxic lymphocytes, cytokines, and nitric oxide.

The bacterial superantigen staphylococcal enterotoxin A (SEA) is a potent inducer of CTL activity and cytokine production in vivo. Protein A (PA) of Staphylococcal aureus has been found to have diverse biological response modifying properties and to possess antitumor, antitoxic and antiparasitic effects. In this study we examined the anti-tumor effect of these two superantigens used separately as well as in combination in mice carrying the Ehrlich ascites tumor. With combined treatment, DNA cell cycle analysis of tumor cells showed a significant (P < 0.05) percentage of tumor cell death. Levels of the soluble mediators TNF-alpha, IFN-gamma and IL-1 as well as NO were elevated. Additionally, CD4(+) and CD8(+) specific T cells in spleen, thymus and PBMC in tumor carrying mice were increased (P < 0.01). Our data altogether suggests that enhanced tumor cell death is caused by the increased CTL activity, cytokine and nitric oxide levels, in response to the combined effect of SEA + PA.     

Cytokines Released by Human Peripheral Blood Mononuclear Cells Inhibit the Production of Early and Late Cytomegalovirus Proteins

Cytomegalovirus-infected human fibroblasts are susceptible to lysis by natural killer cells and cytotoxic T cells. The purpose of this study was to determine whether non-lytic mechanisms might also contribute to the control of cytomegalovirus infection. The appearance of cytomegalovirus proteins in infected fibroblasts was determined by flow cytometry. Infected fibroblasts incubated with peripheral blood mononuclear cells for 3 days expressed less early and late proteins than fibroblasts incubated without peripheral blood mononuclear cells. Supernatants generated by the cocultivation of peripheral blood mononuclear cells with cytomegalovirus-infected fibroblasts inhibited the production of cytomegalovirus early and late proteins. The soluble factors in supernatants which contributed to the inhibitory effect were identified as interferons α, β and γ, and tumor necrosis factors α and β. The ability of supernatants to inhibit the production of cytomegalovirus early protein was mimicked by combinations of corresponding recombinant cytokines. The inhibition of cytomegalovirus protein production by cytokines produced by peripheral blood mononuclear cells may contribute to early containment of cytomegalovirus infection.