Epigenetic modulation of BRCA-1 and MGMT genes,and histones H4 and H3 are associated with breast tumors

Aberrant patterns in promoter methylation of tumor-suppressor genes and posttranslational modifications of histone proteins are considered as major features of malignancy. In this study, we aimed to investigate promoter methylation of three tumor-suppressor genes (BRCA-1, MGMT, and P16) and three histone marks (H3K9ac, H3K18ac, and H4K20me3) in patients with breast tumors. This case-control study included 27 patients with malignant breast tumors (MBT) and 31 patients with benign breast tumors (BBT). The methylation-specific PCR was used for determining promoter methylation of BRCA-1, MGMT, and P16 genes. Western blot analysis was performed to detect histone lysine acetylation (H3K9ac and H3K18ac) and lysine methylation (H4K20me3). BRCA-1 promoter methylation was detected in 44.4% of the MBT whereas this alteration was found in 9.7% of BBT (P = 0.005). The Kaplan-Meier analysis indicated that hypermethylation in BRCA-1 promoter was significantly associated with poor overall survival of patients with breast cancer (P = 0.039). MGMT promoter methylation was identified in 18.5% of MBT and 0.0% of the BBT (P = 0.01). The frequency of P16 promoter methylation was 25.8% in BBT and 11.1% in MBT (P = 0.12). As compared with BBT, MBT samples displayed the aberrant patterns of histones marks with hypomethylation of H4K20 and hypoacetylation of H3K18 (P = 0.03 and P = 0.04, respectively). There was a negative significant correlation between H3K9ac levels and tumor size in MBT group (r = −0.672; P = 0.008). The present findings suggest that promoter hypermethylation of MGMT and BRCA-1 genes along with alterations in H3K18ac and H4K20me3 levels may have prognostic values in patients with breast cancer. Moreover, the detection of these epigenetic modifications in breast tumors could be helpful in finding new methods for breast cancer therapy.     

<Emphasis Type="Italic">FHIT</Emphasis> Gene Sequence Variants and Reduced Fhit Protein Expression in Glioblastoma Multiforme

Molecular studies have an important role in the elucidation of the mechanisms involved in Glioblastoma multiforme (GBM) development. The occurrence of FHIT gene alterations, which has an important role in different cancers, has not yet been studied well in GBM. We aimed to investigate the occurrence of alterations of FHIT gene sequence and protein expression in the GBMs. Sequence alterations in exons 5–9 of the FHIT gene were screened in 63 GBMs using the single-strand conformational polymorphism method, followed by DNA sequencing. Additionally, the level of Fhit protein expression in tissues of 48 tumors was assessed by immunohistochemistry (IHC). In our investigation, FHIT gene alterations in the coding region were detected in 11 of the 63 GBM cases (17.5%). Two different sequence variants were determined: one novel missense variant (G→C transition at codon 49) and one previously described silent alteration (C→T transition at codon 88). Using web-based programs, such as SIFT and ESEfinder, it was determined that both alterations might have caused significant modification on protein function. In addition, we identified a previously reported an intronic polymorphism (T→A transition at IVS8-17) in 47.5% of cases as a similar rate (45%) in the control group. Moreover, it was observed that Fhit protein expression was reduced in 87.5% of tumors. In conclusion, the reduction or loss of Fhit protein expression by genetic alterations or epigenetic mechanisms in GBM might be associated with brain tumorigenesis.     

The global DNA methylation surrogate LINE-1 methylation is correlated with MGMT promoter methylation and is a better prognostic factor for glioma

Gliomas are the most frequently occurring primary brain tumor in the central nervous system of adults. Glioblastoma multiformes (GBMs, WHO grade 4) have a dismal prognosis despite the use of the alkylating agent, temozolomide (TMZ), and even low grade gliomas (LGGs, WHO grade 2) eventually transform to malignant secondary GBMs. Although GBM patients benefit from promoter hypermethylation of the O(6)-methylguanine-DNA methyltransferase (MGMT) that is the main determinant of resistance to TMZ, recent studies suggested that MGMT promoter methylation is of prognostic as well as predictive significance for the efficacy of TMZ. Glioma-CpG island methylator phenotype (G-CIMP) in the global genome was shown to be a significant predictor of improved survival in patients with GBM. Collectively, we hypothesized that MGMT promoter methylation might reflect global DNA methylation. Additionally in LGGs, the significance of MGMT promoter methylation is still undetermined. In the current study, we aimed to determine the correlation between clinical, genetic, and epigenetic profiles including LINE-1 and different cancer-related genes and the clinical outcome in newly diagnosed 57 LGG and 54 GBM patients. Here, we demonstrated that (1) IDH1/2 mutation is closely correlated with MGMT promoter methylation and 1p/19q codeletion in LGGs, (2) LINE-1 methylation levels in primary and secondary GBMs are lower than those in LGGs and normal brain tissues, (3) LINE-1 methylation is proportional to MGMT promoter methylation in gliomas, and (4) higher LINE-1 methylation is a favorable prognostic factor in primary GBMs, even compared to MGMT promoter methylation. As a global DNA methylation marker, LINE-1 may be a promising marker in gliomas.     

Effect of aberrant promoter methylation of FHIT and RASSF1A genes on susceptibility to cervical cancer in a North Indian population

As current evidence suggests the involvement of epigenetic modification of tumour suppressor genes in human cancer, we investigated the aberrant promoter methylation of FHIT and RASSF1A genes in human papillomavirus (HPV)-mediated cervical cancer in Indian women. We analysed 60 cervical cancer tissue biopsies of different clinical stage and histological grading and 23 healthy control samples with normal cervical cytology. Methylation-specific polymerase chain reaction (MSP) was performed to analyse the methylation status of FHIT and RASSF1A genes and confirmed by sequencing. Both patients and controls were screened for HPV infection and 98% of the HPV-infected cases showed positivity for HPV type 16. Aberrant promoter methylation of the FHIT gene was found in 28.3% (17/60) of cases and of the RASSF1A gene in 35.0% (21/60) of cases; promoter methylation of both the genes was found in 13.3% (8/60) of cervical cancer cases. Methylation was significantly (p<0.01) associated with the cervical cancer cases compared with controls. None of the 23 controls was found to be methylated in either of these genes. This is the first study indicating a correlation between the promoter methylation of FHIT and RASSF1A genes and the clinical stage and histological grading of cervical carcinoma in Indian women. Future studies are underway to examine the practical implications of these findings for use as a biomarker.     

DNA methylation changes in murine breast adenocarcinomas allow the identification of candidate genes for human breast carcinogenesis

Epigenetic inactivation due to aberrant promoter methylation is a key process in breast tumorigenesis. Murine models for human breast cancer have been established for nearly every important human oncogene or tumor suppressor gene. Mouse-to-human comparative gene expression and cytogenetic profiling have been widely investigated for these models; however, little is known about the conservation of epigenetic alterations during tumorigenesis. To determine if this key process in human breast tumorigenesis is also mirrored in a murine breast cancer model, we mapped cytosine methylation changes in primary adenocarcinomas and paired lung metastases derived from the polyomavirus middle T antigen mouse model. Global changes in methylcytosine levels were observed in all tumors when compared to the normal mammary gland. Aberrant methylation and associated gene silencing was observed for Hoxa7, a gene that is differentially methylated in human breast tumors, and Gata2, a novel candidate gene. Analysis of HOXA7 and GATA2 expression in a bank of human primary tumors confirms that the expression of these genes is also reduced in human breast cancer. In addition, HOXA7 hypermethylation is observed in breast cancer tissues when compared to adjacent tumor-free tissue. Based on these studies, we present a model in which comparative epigenetic techniques can be used to identify novel candidate genes important for human breast tumorigenesis, in both primary and metastatic tumors.     

Identification of Global DNA Methylation Signatures in Glioblastoma-Derived Cancer Stem Cells

Glioblastoma(GBM)is the most common and most aggressive primary brain tumor in adults.The existence of a small population of stem-like tumor cells that efficiently propagate tumors and resist cytotoxic therapy is one proposed mechanism leading to the resilient behavior of tumor cells and poor prognosis.In this study,we performed an in-depth analysis of the DNA methylation landscape in GBMderived cancer stem cells(GSCs).Parallel comparisons of primary tumors and GSC lines derived from these tumors with normal controls(a neural stem cell(NSC)line and normal brain tissue)identified groups of hyper- and hypomethylated genes that display a trend of either increasing or decreasing methylation levels in the order of controls,primary GBMs,and their counterpart GSC lines,respectively.Interestingly,concurrent promoter hypermethylation and gene body hypomethylation were observed in a subset of genes including MGMT,AJAP1 and PTPRN2.These unique DNA methylation signatures were also found in primary GBM-derived xenograft tumors indicating that they are not tissue culture-related epigenetic changes.Integration of GSC-specific epigenetic signatures with gene expression analysis further identified candidate tumor suppressor genes that are frequently down-regulated in GBMs such as SPINT2,NEFM and PENK.Forced re-expression of SPINT2 reduced glioma cell proliferative capacity,anchorage independent growth,cell motility,and tumor sphere formation in vitro.The results from this study demonstrate that GSCs possess unique epigenetic signatures that may play important roles in the pathogenesis of GBM.     

Epigenetic Impact of Long-Term Shiftwork: Pilot Evidence From Circadian Genes and Whole-Genome Methylation Analysis

Epigenetic association studies have demonstrated differential promoter methylation in the core circadian genes in breast cancer cases relative to cancer-free controls. The current pilot study aims to investigate whether epigenetic changes affecting breast cancer risk could be caused by circadian disruption through exposure to light at night. Archived DNA samples extracted from whole blood of 117 female subjects from a prospective cohort conducted in Denmark were included in this study. A polymerase chain reaction (PCR)-based method was used for detection of gene-promoter methylation, whereas genome-wide methylation analysis was performed using the Illumina Infinium Methylation Chip. Long-term shiftwork resulted in the same promoter hypomethylation of CLOCK and hypermethylation of CRY2, as was previously observed in breast cancer case-control studies. Genome-wide methylation analysis further discovered widespread methylation alterations in shiftworkers, including changes in many methylation- and cancer-relevant genes. Pathway analysis of the genes with altered methylation patterns revealed several cancer-related pathways. One of the top three networks generated was designated as “DNA replication, recombination, and repair, gene expression, behavior” with ESR1 (estrogen receptor α) featured most prominently in the network, underscoring the potential breast cancer relevance of the genes differentially methylated in long-term shiftworkers. These results, although exploratory, demonstrate the first evidence of the cancer-relevant epigenetic effects of night shiftwork, which warrant further investigation. Considering there are millions of shiftworkers worldwide, understanding the effects of this exposure may lead to novel strategies for cancer prevention and new policies regulating shiftwork. (Author correspondence: yong.zhu@yale.edu)     

Epigenetic,Genetic and Environmental Interactions in Esophageal Squamous Cell Carcinoma from Northeast India

Background

Esophageal squamous cell carcinoma (ESCC) develops as a result of complex epigenetic, genetic and environmental interactions. Epigenetic changes like, promoter hypermethylation of multiple tumour suppressor genes are frequent events in cancer, and certain habit-related carcinogens are thought to be capable of inducing aberrant methylation. However, the effects of environmental carcinogens depend upon the level of metabolism by carcinogen metabolizing enzymes. As such key interactions between habits related factors and carcinogen metabolizing gene polymorphisms towards modulating promoter methylation of genes are likely. However, this remains largely unexplored in ESCC. Here, we studied the interaction of various habits related factors and polymorphism of GSTM1/GSTT1 genes towards inducing promoter hypermethylation of multiple tumour suppressor genes.

Methodology/Principal Findings

The study included 112 ESCC cases and 130 age and gender matched controls. Conditional logistic regression was used to calculate odds ratios (OR) and multifactor dimensionality reduction (MDR) was used to explore high order interactions. Tobacco chewing and smoking were the major individual risk factors of ESCC after adjusting for all potential confounding factors. With regards to methylation status, significantly higher methylation frequencies were observed in tobacco chewers than non chewers for all the four genes under study (p<0.01). In logistic regression analysis, betel quid chewing, alcohol consumption and null GSTT1 genotypes imparted maximum risk for ESCC without promoter hypermethylation. Whereas, tobacco chewing, smoking and GSTT1 null variants were the most important risk factors for ESCC with promoter hypermethylation. MDR analysis revealed two predictor models for ESCC with promoter hypermethylation (Tobacco chewing/Smoking/Betel quid chewing/GSTT1 null) and ESCC without promoter hypermethylation (Betel quid chewing/Alcohol/GSTT1) with TBA of 0.69 and 0.75 respectively and CVC of 10/10 in both models.

Conclusion

Our study identified a possible interaction between tobacco consumption and carcinogen metabolizing gene polymorphisms towards modulating promoter methylation of tumour suppressor genes in ESCC.     

Prognosis of Glioblastoma With Oligodendroglioma Component is Associated With the IDH1 Mutation and MGMT Methylation Status

Glioblastoma (GBM) with oligodendroglioma component (GBMO) is a newly described GBM subtype in the 2007 World Health Organization classification. However, its biological and genetic characteristics are largely unknown. We investigated the clinicopathological and molecular features of 34 GBMOs and compared the survival rate of these patients with those of patients with astrocytoma, oligodendroglioma, anaplastic oligoastrocytoma (AOA), and conventional GBMs in our hospital. GBMO could be divided into two groups based on the presence of an IDH1 mutation. The IDH1 mutation was more frequently found in secondary GBMO, which had lower frequencies of EGFR amplification but higher MGMT methylation than the wild type IDH1 group, and patients with mutant IDH1 GBMO were on average younger than those with wild-type IDH1. Therefore, GBMO is a clinically and molecularly heterogeneous subtype, largely belonging to a proneural and classical subtype of GBM. The survival rate of GBMO patients itself was worse than that of AOA patients but not significantly better than that of conventional GBM patients. GBMO survival was independent of the dominant histopathological subtype i.e., astrocyte-dominant or oligodendroglioma -dominant, but it was significantly associated with the IDH1 mutation and MGMT methylation status. Therefore, GBMO should be regarded as a separate entity from AOA and must be classified as a subtype of GBM. However, further study is needed to determine whether it is a pathologic variant or a pattern of GBM because GBMO has a similar prognosis to conventional GBMs.     

Genome-wide DNA methylation studies suggest distinct DNA methylation patterns in pediatric embryonal and alveolar rhabdomyosarcomas

Rhabdomyosarcoma is the most common soft-tissue sarcoma in children. While cytogenetic abnormalities have been well characterized in this disease, aberrant epigenetic events such as DNA hypermethylation have not been described in genome-wide studies. We have analyzed the methylation status of 25,500 promoters in normal skeletal muscle, and in cell lines and tumor samples of embryonal and alveolar rhabdomyosarcoma from pediatric patients. We identified over 1,900 CpG islands that are hypermethylated in rhabdomyosarcomas relative to skeletal muscle. Genes involved in tissue development, differentiation, and oncogenesis such as DNAJA4, HES5, IRX1, BMP8A, GATA4, GATA6, ALX3, and P4HTM were hypermethylated in both RMS cell lines and primary samples, implicating aberrant DNA methylation in the pathogenesis of rhabdomyosarcoma. Furthermore, cluster analysis revealed embryonal and alveolar subtypes had distinct DNA methylation patterns, with the alveolar subtype being enriched in DNA hypermethylation of polycomb target genes. These results suggest that DNA methylation signatures may aid in the diagnosis and risk stratification of pediatric rhabdomyosarcoma and help identify new targets for therapy.     

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.     

Epigenetics and Neural Developmental Disorders

Neural developmental disorders, such as autism, Rett Syndrome, Fragile X syndrome, and Angelman syndrome manifest during early postnatal neural development. Although the genes responsible for some of these disorders have been identified, how the mutations of these genes affect neural development is currently unclear. Emerging evidence suggest that these disorders share common underlying defects in neuronal morphology, synaptic connectivity and brain plasticity. In particular, alterations in dendritic branching and spine morphology play a central role in the pathophysiology of most mental retardation disorders, suggesting that common pathways regulating neuronal function may be affected. Epigenetic modulations, mediated by DNA methylation, RNA-associated silencing, and histone modification, can serve as an intermediate process that imprints dynamic environmental experiences on the “fixed” genome, resulting in stable alterations in phenotypes. Disturbance in epigenetic regulations can lead to inappropriate expression or silencing of genes, causing an array of multi-system disorders and neoplasias. Rett syndrome, the most common form of mental retardation in young girls, is due to germline mutation of MECP2, encoding a methylated DNA binding protein that translates DNA methylation into gene repression. Angelman syndrome is due to faulty genomic imprinting or maternal mutations in UBE3A. Fragile X Syndrome, in most cases, results from the hypermethylation of FMR1 promoter, hence the loss of expression of functional FMRP protein. Autism, with its complex etiology, may have strong epigenetic link. Together, these observations strongly suggest that epigenetic mechanisms may play a critical role in brain development and etiology of related disorders. This report summarizes the scientific discussions and major conclusions from a recent conference that aimed to gain insight into the common molecular pathways affected among these disorders and discover potential therapeutic targets that have been missed by looking at one disorder at a time.     

DNA methylation profile in chronic myelomonocytic leukemia associates with distinct clinical,biological and genetic features

Chromosomal abnormalities are detected in 20–30% of patients with chronic myelomonocytic leukemia (CMML) and correlate with prognosis. On the mutation level, disruptive alterations are particularly frequent in chromatin regulatory genes. However, little is known about the consequential alterations in the epigenetic marking of the genome. Here, we report the analysis of genomic DNA methylation patterns of 64 CMML patients and 10 healthy controls, using a DNA methylation microarray focused on promoter regions. Differential methylation analysis between patients and controls allowed us to identify abnormalities in DNA methylation, including hypermethylation of specific genes and large genome regions with aberrant DNA methylation. Unsupervised hierarchical cluster analysis identified two main clusters that associated with the clinical, biological, and genetic features of patients. Group 1 was enriched in patients with adverse clinical and biological characteristics and poorer overall and progression-free survival. In addition, significant differences in DNA methylation were observed between patients with low risk and intermediate/high risk karyotypes and between TET2 mutant and wild type patients. Taken together, our results demonstrate that altered DNA methylation patterns reflect the CMML disease state and allow to identify patient groups with distinct clinical features.     

The influence of one-carbon metabolism on gene promoter methylation in a population-based breast cancer study

Abnormal methylation in gene promoters is a hallmark of the cancer genome; however, factors that may influence promoter methylation have not been well elucidated. As the one-carbon metabolism pathway provides the universal methyl donor for methylation reactions, perturbation of this pathway might influence DNA methylation and, ultimately, affect gene functions. Utilizing approximately 800 breast cancer tumor tissues from a large population-based study, we investigated the relationships between dietary and genetic factors involved in the one-carbon metabolism pathway and promoter methylation of a panel of 13 breast cancer-related genes. We found that CCND2, HIN1 and CHD1 were the most “dietary sensitive” genes, as methylation of their promoters was associated with intakes of at least two out of the eight dietary methyl factors examined. On the other hand, some micronutrients (i.e., B₂ and B₆) were more “epigenetically active” as their intake levels correlated with promoter methylation status in 3 out of the 13 breast cancer genes evaluated. Both positive (hypermethylation) and inverse (hypomethylation) associations with high micronutrient intake were observed. Unlike what we saw for dietary factors, we did not observe any clear patterns between one-carbon genetic polymorphisms and the promoter methylation status of the genes examined. Our results provide preliminary evidence that one-carbon metabolism may have the capacity to influence the breast cancer epigenome. Given that epigenetic alterations are thought to occur early in cancer development and are potentially reversible, dietary modifications may offer promising venues for cancer intervention and prevention.