Aberrant CpG methylation of the TFAP2A gene constitutes a mechanism for loss of TFAP2A expression in human metastatic melanoma

Metastatic melanoma is a deadly treatment-resistant form of skin cancer whose global incidence is on the rise. During melanocyte transformation and melanoma progression the expression profile of many genes changes. Among these, a gene implicated in several steps of melanocyte development, TFAP2A, is frequently silenced; however, the molecular mechanism of TFAP2A silencing in human melanoma remains unknown. In this study, we measured TFAP2A mRNA expression in primary human melanocytes compared to 11 human melanoma samples by quantitative real-time RT-PCR. In addition, we assessed CpG DNA methylation of the TFAP2A promoter in these samples using bisulfite sequencing. Compared to primary melanocytes, which showed high TFAP2A mRNA expression and no promoter methylation, human melanoma samples showed decreased TFAP2A mRNA expression and increased promoter methylation. We further show that increased CpG methylation correlates with decreased TFAP2A mRNA expression. Using The Cancer Genome Atlas, we further identified TFAP2A as a gene displaying among the most decreased expression in stage 4 melanomas vs. non-stage 4 melanomas, and whose CpG methylation was frequently associated with lack of mRNA expression. Based on our data, we conclude that TFAP2A expression in human melanomas can be silenced by aberrant CpG methylation of the TFAP2A promoter. We have identified aberrant CpG DNA methylation as an epigenetic mark associated with TFAP2A silencing in human melanoma that could have significant implications for the therapy of human melanoma using epigenetic modifying drugs.     

Aberrant CpG methylation of the TFAP2A gene constitutes a mechanism for loss of TFAP2A expression in human metastatic melanoma

Metastatic melanoma is a deadly treatment-resistant form of skin cancer whose global incidence is on the rise. During melanocyte transformation and melanoma progression the expression profile of many genes changes. Among these, a gene implicated in several steps of melanocyte development, TFAP2A, is frequently silenced; however, the molecular mechanism of TFAP2A silencing in human melanoma remains unknown. In this study, we measured TFAP2A mRNA expression in primary human melanocytes compared to 11 human melanoma samples by quantitative real-time RT-PCR. In addition, we assessed CpG DNA methylation of the TFAP2A promoter in these samples using bisulfite sequencing. Compared to primary melanocytes, which showed high TFAP2A mRNA expression and no promoter methylation, human melanoma samples showed decreased TFAP2A mRNA expression and increased promoter methylation. We further show that increased CpG methylation correlates with decreased TFAP2A mRNA expression. Using The Cancer Genome Atlas, we further identified TFAP2A as a gene displaying among the most decreased expression in stage 4 melanomas vs. non-stage 4 melanomas, and whose CpG methylation was frequently associated with lack of mRNA expression. Based on our data, we conclude that TFAP2A expression in human melanomas can be silenced by aberrant CpG methylation of the TFAP2A promoter. We have identified aberrant CpG DNA methylation as an epigenetic mark associated with TFAP2A silencing in human melanoma that could have significant implications for the therapy of human melanoma using epigenetic modifying drugs.     

Promoter hypomethylation contributes to the expression of MUC3A in cancer cells

MUC3A is a membrane-bound glycoprotein that is aberrantly expressed in carcinomas and is a risk factor for a poor prognosis. However, the exact mechanism of MUC3A expression has yet to be clarified. Here, we provide the first evidence that MUC3A gene expression is controlled by the CpG methylation status of the proximal promoter region. We show that the DNA methylation pattern is intimately correlated with MUC3A expression in breast, lung, pancreas and colon cancer cell lines. The DNA methylation status of 30 CpG sites from −660 to +273 was mapped using MassARRAY analysis. MUC3A-negative cancer cell lines and those with low MUC3A expression (e.g., MCF-7) were highly methylated in the proximal promoter region, corresponding to 9 CpG sites (−345 to −75 bp), whereas MUC3A-positive cell lines (e.g., LS174T) had low methylation levels. Moreover, 5-aza-2′-deoxycytidine and trichostatin A treatment of MUC3A-negative cells or those with low MUC3A expression caused elevation of MUC3A mRNA. Our results suggest that DNA hypomethylation in the 5′-flanking region of the MUC3A gene plays an important role in MUC3A expression in carcinomas of various organs. An understanding of epigenetic changes in MUC3A may contribute to the diagnosis of carcinogenic risk and to prediction of outcome in patients with cancer.     

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.     

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

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

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

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

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.     

Methylation differences of the neuronatin gene promoter region in liver between normal and cloned pigs

Abnormal phenotypes in cloned pigs can be partly due to changes in epigenetic modifications such as methylation levels of promoter CpG islands. Neuronatin is an imprinted gene, conserved in human, pig, cattle and mouse, which is expressed exclusively from the paternal allele. Three CpG islands located in the promoter region of the porcine neuronatin gene have the potential to regulate the gene expression by cytosine methylation. To illustrate whether neuronatin was differentially expressed among nuclear transfer macroglossia–positive and nuclear transfer macroglossia–negative pigs and in vitro‐fertilized pigs, we detected its expression level by qRT‐PCR and further quantified methylation levels by pyrosequencing DNA from the liver. The results showed that neuronatin was expressed at a significantly higher level in livers of nuclear transfer macroglossia‐positive pigs compared with normal cloned and in vitro‐fertilized pigs. Livers of nuclear transfer macroglossia‐positive pigs also had a significantly lower methylation level at CpG island 2 and CpG island 3 in the promoter region.     

CpG dinucleotide-specific hypermethylation of the TNS3 gene promoter in human renal cell carcinoma

Tensin3 is a cytoskeletal regulatory protein that inhibits cell motility. Downregulation of the gene encoding Tensin3 (TNS3) in human renal cell carcinoma (RCC) may contribute to cancer cell metastatic behavior. We speculated that epigenetic mechanisms, e.g., gene promoter hypermethylation, might account for TNS3 downregulation. In this study, we identified and validated a TNS3 gene promoter containing a CpG island, and quantified the methylation level within this region in RCC. Using a luciferase reporter assay we demonstrated a functional minimal promoter activity for a 500-bp sequence within the TNS3 CpG island. Pyrosequencing enabled quantitative determination of DNA methylation of each CpG dinucleotide (a total of 43) in the TNS3 gene promoter. Across the entire analyzed CpG stretch, RCC DNA showed a higher methylation level than both non-tumor kidney DNA and normal control DNA. Out of all the CpGs analyzed, two CpG dinucleotides, specifically position 2 and 8, showed the most pronounced increases in methylation levels in tumor samples. Furthermore, CpG-specific higher methylation levels were correlated with lower TNS3 gene expression levels in RCC samples. In addition, pharmacological demethylation treatment of cultured kidney cells caused a 3-fold upregulation of Tensin3 expression. In conclusion, these results reveal a differential methylation pattern in the TNS3 promoter occurring in human RCC, suggesting an epigenetic mechanism for aberrant Tensin downregulation in human kidney cancer.     

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

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

Hypermethylation of growth Arrest DNA-damage-inducible gene 45 in non-small cell lung cancer and its relationship with clinicopathologic features

The growth arrest DNA-damage-inducible protein 45 (GADD45) can serve as a key coordinator of the stress response by regulating cell cycle progression, genomic stability, DNA repair, and other stress-related responses. Although deregulation of GADD45 expression has been reported in several types of human tumors, its role in lung cancer is still unknown. DNA hypermethylation of promoter CpG islands is known to be a major mechanism for epigenetic inactivation of tumor suppressor genes. We investigated the methylation status of GADD45 family genes (GADD45A, B, and G) in 139 patients with non-small cell lung cancer (NSCLC) using methylation-specific PCR (MSP) and correlated the results with clinicopathologic features of the patients. Methylation frequencies in tumors were 1.4% for GADD45A, 7.2% for GADD45B, and 31.6% for GADD45G. RT-PCR and MSP analysis showed that promoter methylation of the GADD45G gene resulted in downregulation of its mRNA expression. GADD45G methylation was significantly more frequent in female patients than male patients (P = 0.035). This finding suggests that methylation-associated down-regulation of the GADD45G gene may be involved in lung tumorigenesis.     

DNA methylation regulates gabrb2 mRNA expression: developmental variations and disruptions in l‐methionine‐induced zebrafish with schizophrenia‐like symptoms

Single nucleotide polymorphisms (SNPs) in the human type A gamma‐aminobutyric acid (GABA) receptor β₂ subunit gene (GABRB2) have been associated with schizophrenia and quantitatively correlated with mRNA expression in the postmortem brain tissue of patients with schizophrenia. l ‐Methionine (MET) administration has been reported to cause a recrudescence of psychotic symptoms in patients with schizophrenia, and similar symptoms have been generated in MET‐induced mice. In this study, a zebrafish animal model was used to evaluate the relationship between the gabrb2 mRNA expression and its promoter DNA methylation in developmental and MET‐induced schizophrenia‐like zebrafish. The results indicated developmental increases in global DNA methylation and decreases in gabrb2 promoter methylation in zebrafish. A significant increase in gabrb2 mRNA levels was observed after GABA was synthesized. Additionally, the MET‐triggered schizophrenia‐like symptoms in adult zebrafish, involving social withdrawal and cognitive dysfunction analyzed with social interaction and T‐maze behavioral tests, were accompanied by significantly increased DNA methylation levels in the global genome and the gabrb2 promoter. Furthermore, the significant correlation between gabrb2 mRNA expression and gabrb2 promoter methylation observed in the developmental stages became non‐significant in MET‐triggered adult zebrafish. These findings demonstrate that gabrb2 mRNA expression is associated with DNA methylation varies by developmental stage and show that these epigenetic association mechanisms are disrupted in MET‐triggered adult zebrafish with schizophrenia‐like symptoms. In conclusion, these results provide plausible epigenetic evidence of the GABA_A receptor β₂ subunit involvement in the schizophrenia‐like behaviors and demonstrate the potential use of zebrafish models in neuropsychiatric research.