Promoter Methylation Profile Research Articles

Intragenic long interspersed element-1 sequences promote promoter hypermethylation in lung adenocarcinoma, multiple myeloma and prostate cancer

In cancers, although the methylation of long interspersed element- 1 sequences (LINE-1s) and tumor suppressor gene promoters are modified in the opposite direction, LINE-1 hypomethylation and promoter hypermethylation of some loci are directly associated. During carcinogenesis, the reduction in LINE-1 methylation occurs. Intragenic LINE-1s produces antisense RNA in introns and reduces mRNA transcription levels. Several antisense RNAs have been reported to mediate methylation of the associated CpG islands. Here we compared genome-wide promoter methylation and expression profiles of LINE-1-hypomethylated malignancies, reported in the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo), including lung adenocarcinoma, multiple myeloma and prostate cancer. Then we analysed a microarray experiment if promoters of a set of genes containing LINE-1s or Alu are commonly methylated. Finally, the differences in structural characteristics of LINE-1s were compared between LINE-1 groups. Here we found that genes that contained LINE-1s were frequently repressed (p < 0.01) and possessed promoter hypermethylation (p < 1.0E-4). The expression levels of genes containing LINE-1s with promoter hypermethylation were the lowest. Finally, the genomic distributions of gene-repressing LINE-1s and promoter-hypermethylating LINE-1s were neither co-segregated nor randomly segregated. In conclusion, cancer-associated intragenic LINE-1 epigenetic change promotes promoter hypermethylation and represses gene expression. These two mechanisms are independently influenced by genomic locations but synergistically down-regulate genes.

Aberrant Promoter Methylation and Expression of UTF1 during Cervical Carcinogenesis

Promoter methylation profiles are proposed as potential prognosis and/or diagnosis biomarkers in cervical cancer. Up to now, little is known about the promoter methylation profile and expression pattern of stem cell (SC) markers during tumor development. In this study, we were interested to identify SC genes methylation profiles during cervical carcinogenesis. A genome-wide promoter methylation screening revealed a strong hypermethylation of Undifferentiated cell Transcription Factor 1 (UTF1) promoter in cervical cancer in comparison with normal ectocervix. By direct bisulfite pyrosequencing of DNA isolated from liquid-based cytological samples, we showed that UTF1 promoter methylation increases with lesion severity, the highest level of methylation being found in carcinoma. This hypermethylation was associated with increased UTF1 mRNA and protein expression. By using quantitative RT-PCR and Western Blot, we showed that both UTF1 mRNA and protein are present in epithelial cancer cell lines, even in the absence of its two main described regulators Oct4A and Sox2. Moreover, by immunofluorescence, we confirmed the nuclear localisation of UTF1 in cell lines. Surprisingly, direct bisulfite pyrosequencing revealed that the inhibition of DNA methyltransferase by 5-aza-2′-deoxycytidine was associated with decreased UTF1 gene methylation and expression in two cervical cancer cell lines of the four tested. These findings strongly suggest that UTF1 promoter methylation profile might be a useful biomarker for cervical cancer diagnosis and raise the questions of its role during epithelial carcinogenesis and of the mechanisms regulating its expression.

Genome‐wide identification of Epstein‐Barr virus–driven promoter methylation profiles of human genes in gastric cancer cells

Aberrant methylation of tumor-related genes has been reported in Epstein-Barr virus (EBV)-associated gastric cancers. This study sought to profile EBV-driven hypermethylation in EBV-infected cells. The EBV-positive AGS gastric cancer cell line (AGS-EBV) and EBV-negative AGS cells were used in this study. DNA methyltransferase-3b (DNMT3b) activity was assessed by EpiQuick activity assay, and genome-wide DNA methylation profiles were assessed by methyl-DNA immunoprecipitation microarray assay. EBV infection was confirmed in AGS-EBV cells by EBV-encoded RNA in situ hybridization. Expression and activity of DNA methyltransferase-3b (DNMT3b) was significantly increased in AGS-EBV compared to AGS. Ectopic expression of LMP2A (latent membrane protein 2A) in AGS increased activity of DNMT3b. A total of 1065 genes were differentially methylated by EBV infection (fold-changes ≥ 2, P < .05) in AGS-EBV compared to AGS cells. The majority of the differentially methylated genes (83.2%, 886 of 1065 genes) had cytosine-guanine dinucleotide (CpG) hypermethylation in AGS-EBV (fold-changes 2.43∼65.2) versus that found in AGS cells. Gene ontology analysis revealed that hypermethylated genes were enriched in the important cancer pathways (≥ 10 genes each, P ≤ .05) including mitogen-activated protein kinase signaling, cell adhesion molecules, wnt signaling pathway, and so forth. Six novel hypermethylated candidates (IL15RA, REC8, SSTR1, EPHB6, MDGA2, and SCARF2) were further validated. Higher levels of DNA methylation were confirmed for all these genes in AGS-EBV cells by bisulfite genomic sequencing. Furthermore, these candidates were silenced or down-regulated in AGS-EBV cells, but can be restored by demethylation treatment. EBV infection in AGS cells induced aberrant CpG hypermethylation of 886 genes involving in important cancer-related pathways. Induction of promoter methylation by EBV is regulated by up-regulation of DNMT3b through LMP2A.

Correlation between BRAF mutation and promoter methylation of TIMP3, RARβ2 and RASSF1A in thyroid cancer

Our aim was to comprehensively analyze promoter hypermethylation of a panel of novel and known methylation markers for thyroid neoplasms and to establish their relationship with BRAF mutation and clinicopathologic parameters of thyroid cancer. A cohort of thyroid tumors, consisting of 44 cancers and 44 benign thyroid lesions, as well as 15 samples of adjacent normal thyroid tissue, was evaluated for BRAF mutation and promoter hypermethylation. Genes for quantitative methylation specific PCR (QMSP) were selected by a candidate gene approach. Twenty-two genes were tested: TSHR, RASSF1A, RARβ2, DAPK, hMLH1, ATM, S100, p16, CTNNB1, GSTP1, CALCA, TIMP3, TGFßR2, THBS1, MINT1, CTNNB1, MT1G, PAK3, NISCH, DCC, AIM1 and KIF1A. The PCR-based “mutector assay” was used to detect BRAF mutation. All p values reported are two sided. Considerable overlap was seen in the methylation markers among the different tissue groups. Significantly higher methylation frequency and level were observed for KIF1A and RARß2 in cancer samples compared with benign tumors. A negative correlation between BRAF mutation and RASSF1A methylation, and a positive correlation with RARß2 methylation were observed in accordance with previous results. In addition, positive correlation with TIMP3 and a marginal correlation with DCC methylation were observed. The present study constitutes a comprehensive promoter methylation profile of thyroid neoplasia and shows that results must be analyzed in a tissue-specific manner to identify clinically useful methylation markers. Integration of genetic and epigenetic changes in thyroid cancer will help identify relevant biologic pathways that drive its development.

Abstract 3127: Cigarette smoke condensate (CSC) induces differential expression and promoter methylation profiles of critical genes involved in lung cancer in NL-20 lung cells in vitro: Short-term and chronic exposure

Abstract Lung cancer is the leading cause of cancer deaths in the United States. Smoking accounts for a large number of those deaths. Recently, the Food and Drug Administration has been given authority to regulate any new cigarette products coming to market and further demonstrate the harm caused by constituents in cigarettes or those chemicals created from smoking. Establishing early diagnostic markers are critical to effective prevention programs. This study examined the effects of cigarette smoke condensate on expression and promoter methylation profile of critical genes involved in various stages of lung cancer development, such as E-cadherin (ECAD), O6-mehyl-guanine-DNA methyltransferase (MGMT), and RASSF1A. NL-20 cells were treated with 0.1, 1, 10 or 100 µg/ml of CSC for 24, 48 and 72 hrs for short-term exposures. ECAD showed a significant decrease in expression after 72 hrs with both 10 and 100 µg/ml CSC. This decrease in expression correlated to hypermethylation of the ECAD promoter at both concentrations using MSP analysis. MGMT expression increased only with the concentration of 100 µg/ml CSC at all time points. This correlated to an unmethylated profile of the promoter at this concentration. RASSF1A promoter profile was methylated in the control samples. However, when treated with 10 or 100 µg/ml CSC, an unmethylated promoter profile was noted. This correlated to increased expression of RASSF1A at both the 10 and 100 µg/ml concentrations. No changes were noted at 0.1 or 1 µg/ml of CSC in NL-20 lung cells. For chronic studies, the cells were exposed for 30 days with 10 or 100 µg/ml CSC, with media changes every three days adding new CSC. After 30 days, cells showed morphological changes associated with transformation. Foci were noted. These cells were further analyzed for further changes in critical genes involved in lung cancer. This study provides critical data showing epigenetic regulation of critical genes involved in DNA repair, invasion and tumor suppressor with treatment of lung cells with CSC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3127. doi:1538-7445.AM2012-3127

Promoter methylation of serotonin transporter gene is associated with obesity measures: a monozygotic twin study

Objective:Epigenetic mechanisms are increasingly being recognized as an important factor for obesity. The serotonin transporter gene (SLC6A4) has a critical role in regulating food intake, body weight and energy balance. This study examines the potential association between SLC6A4 promoter methylation and obesity measures in a monozygotic (MZ) twin sample.Methods:We studied 84 MZ twin pairs drawn from the Vietnam Era Twin Registry. Obesity measures include body mass index (BMI), body weight, waist circumference (WC) and waist-hip ratio (WHR). The SLC6A4 promoter methylation profile in peripheral blood leukocytes was quantified by bisulfite pyrosequencing. The association between methylation variation and obesity parameters was examined by mixed-model regression and matched pair analysis, adjusting for age, smoking, alcohol consumption, physical activity and total daily energy intake. Multiple testing was controlled using the adjusted false discovery rate (q-value).Results:Mean methylation level was positively correlated with BMI (r=0.29; P=0.0002), body weight (r=0.31; P<0.0001) and WC (r=0.20; P=0.009), but not WHR. Intra-pair differences in mean methylation were significantly correlated with intra-pair differences in BMI, body weight and WC, but not WHR. On average, a 1% increase in mean methylation was associated with 0.33 kg m−2 increase in BMI (95% CI: 0.02–0.65; P=0.03), 1.16 kg increase in body weight (95% CI, 0.16–2.16; P=0.02) and 0.78 cm increase in WC (95% CI, 0.05–1.50; P=0.03) after controlling for potential confounders.Conclusions:SLC6A4 promoter hypermethylation is significantly associated with an increased prevalence of obesity within a MZ twin study.

Epigenetic screen of human DNA repair genes identifies aberrant promoter methylation of NEIL1 in head and neck squamous cell carcinoma

Aberrant promoter methylation of different DNA repair genes has a critical role in the development and progression of various cancer types, including head and neck squamous cell carcinomas (HNSCCs). A systematic analysis of known human repair genes for promoter methylation is however missing. We generated quantitative promoter methylation profiles in single CpG units of 160 human DNA repair genes in a set of DNAs isolated from fresh frozen HNSCC and normal tissues using MassARRAY technology. Ninety-eight percent of these genes contained CpG islands (CGIs) in their promoter region; thus, DNA methylation is a potential regulatory mechanism. Methylation data were obtained for 145 genes, from which 15 genes exhibited more than a 20% difference in methylation levels between tumor and normal tissues, manifested either as hypermethylation or as hypomethylation. Analyses of promoter methylation with mRNA expression identified the DNA glycosylase NEIL1 (nei endonuclease VIII-like 1) as the most prominent candidate gene. NEIL1 promoter hypermethylation was confirmed in additional fresh frozen HNSCC samples, normal mucosa, HNSCC cell lines and primary human skin keratinocytes. The investigation of laser-microdissected tissues further substantiated increased methylation levels in tumor versus matched non-tumor cells. Immunohistological analysis revealed significantly less NEIL1 protein expression in tumor tissues. 5-Aza-2'-deoxycytidine treatment and DNMT1 knockdown resulted in the re-expression of NEIL1 in HNSCC cell lines, which initially carried hypermethylated promoter regions. In conclusion, our results suggest that DNA methylation contributes to the downregulation of NEIL1 expression and might thus have a role in modulating the response to therapies of HNSCC.

Richter Syndrome (RS): Genome-Wide Promoter Methylation Profile Differs From De Novo Diffuse Large B-Cell Lymphoma (DLBCL) and Affects Genes Involved in Stem-Cell Maintenance and TP53 Pathway

Abstract 1359RS represents the development of DLBCL in the context of chronic lymphocytic leukemia (CLL). The pathogenesis of RS is still largely unknown. We have previously shown that RS lacks many of the typical genetic lesions recurrently observed in de novo DLBCL (Rossi et al, Blood 2011). Here, we report genome-wide promoter methylation profiling of RS and compare it with the methylation profile of de novo DLBCL, and non-transformed CLL. Methods.The study included 21 RS, 10 clonally related CLL phases (seven paired with RS samples), 9 de novo non-GC DLBCL, 6 non-transformed CLL and 6 normal peripheral blood CD19+ B-cells. All RS were classified as DLBCL, showed a non-germinal center (GC) phenotype, and lacked EBV infection. Both RS and DLBCL DNA was extracted from diagnostic frozen lymph node biopsies. DNA samples underwent bisulfite treatment with the EZ DNA methylation kit and were hybridized on Illumina Infinium HumanMethylation27 arrays, that enables the interrogation of more than 27,000 CpG island over the entire human genome. Probes were discarded if they: i) mapped outside CpG islands; ii) presented a standard deviation <0.10 across all the samples; or iii) were always non-methylated, always fully methylated, or always partially methylated. For clustering and for unpaired t-tests, probes mapped on sex chromosomes were excluded. Supervised analysis of differential methylation between groups was performed using a t-test on the continuous beta values, followed by multiple test correction (MTC). A paired t-test was utilized for comparing the methylation profile between the RS phase and the matched CLL-phase. Probes showing a q-value <0.1 were defined differentially methylated. Results.By unsupervised clustering, RS samples separated from both de novo DLBCL, that instead clustered in a single group, and non-transformed CLL.There were over 3,000 probes differentially methylated between RS and de novo DLBCL. The 443 probes showing higher methylation in RS were significantly enriched of promoter regions of genes involved in TP53 signaling (P=6E-03) and cell cycle regulation (P=5E-02) pathways. The 2,687 probes showing higher methylation in de novo DLBCL were significantly enriched of promoter regions of genes involved in histone modification, including genes harboring the H3K27me3 mark (P < 1.0E–16), and of genes that are targets of the EED and SUZ12 Polycomb proteins (P < 1.0E–16).Analysis of paired CLL/RS sequential samples revealed an extensive overlap between the methylation profile of the CLL phase and the transformed RS phase, suggesting that changes in the methylation pattern are an early event in RS pathogenesis that occurred already at the time of the initial CLL clone. Two single probes in the promoter regions of the OSM and S1PR4/EDG6 genes were differentially methylated in CLL/RS sequential samples and, therefore, might have contributed to the acquisition of the aggressive clinico-pathologic phenotype of RS. OSM codes for oncostatin M, which inhibits cell proliferation and induces cell differentiation and apoptosis. S1PR4/EDG6 is a gene specifically expressed in the lymphoid tissue and involved in cell migration. Both OSM and S1PR4/EDG6 promoter regions were unmethylated in the chronic CLL phase, and became methylated in the paired aggressive RS phase, suggesting the acquisition of methylation during transformation.CLL that subsequently transformed to RS differed from CLL that never transformed to RS for 2 probes showing a higher and 2 a lower degree of methylation. Since changes in the methylation pattern are an early event in RS, this observation prompts the investigation of the methylation status of DLX5 and GBGT1, the two genes with promoter regions showing a higher degree of methylation, as biomarkers allowing the early identification of CLL that will transform to RS. Conclusions.RS clearly differ from de novo DLBCL in terms of methylation profile. The overrepresentation of epigenetic changes affecting genes of the TP53 pathway, along with the significantly higher prevalence of TP53 structural abnormalities, might explain the differences in chemosensitivity between RS and de novo DLBCL. Epigenetic changes are an early events in RS pathogenesis, suggesting that, similarly to what shown in transformed follicular lymphoma, lesions initiating transformation are acquired by a cell belonging to the initial tumor clone, rather being progressively accumulated during disease course. Disclosures:No relevant conflicts of interest to declare.

Evidence for widespread changes in promoter methylation profile in human placenta in response to increasing gestational age and environmental/stochastic factors

BackgroundThe human placenta facilitates the exchange of nutrients, gas and waste between the fetal and maternal circulations. It also protects the fetus from the maternal immune response. Due to its role at the feto-maternal interface, the placenta is subject to many environmental exposures that can potentially alter its epigenetic profile. Previous studies have reported gene expression differences in placenta over gestation, as well as inter-individual variation in expression of some genes. However, the factors contributing to this variation in gene expression remain poorly understood.ResultsIn this study, we performed a genome-wide DNA methylation analysis of gene promoters in placenta tissue from three pregnancy trimesters. We identified large-scale differences in DNA methylation levels between first, second and third trimesters, with an overall progressive increase in average methylation from first to third trimester. The most differentially methylated genes included many immune regulators, reflecting the change in placental immuno-modulation as pregnancy progresses. We also detected increased inter-individual variation in the third trimester relative to first and second, supporting an accumulation of environmentally induced (or stochastic) changes in DNA methylation pattern. These highly variable genes were enriched for those involved in amino acid and other metabolic pathways, potentially reflecting the adaptation of the human placenta to different environments.ConclusionsThe identification of cellular pathways subject to drift in response to environmental influences provide a basis for future studies examining the role of specific environmental factors on DNA methylation pattern and placenta-associated adverse pregnancy outcomes.

Association of PPARGC1A and CAPNS1 gene polymorphisms and expression with meat quality traits in pigs

This study aimed to investigate the genes PPARGC1A (peroxisome proliferator-activated receptor gamma-coactivator 1A) and CAPNS1 (calpain small subunit 1) as candidate genes affecting meat quality traits in pigs. Four polymorphisms were identified in PPARCG1A and three in CAPNS1. The PPARGC1A polymorphism c.1288T>A was associated with pH and cooking loss in a F2 Duroc×Pietrain experimental cross (DuPi, n=313) and with pH values in Italian Large White (ILW, n=380) and Italian Landrace (ILA, n=158) populations (P<0.05). The CAPNS1 polymorphism c.429A>C was associated with pH and conductivity in DuPi and with meat color in ILA (P<0.05). PPARGC1A mRNA expression associated with drip loss (P<0.01) and the same tendency was found for CAPNS1 (P=0.06). The promoter methylation profiling suggested that methylation is not involved in CAPNS1 expression regulation. In conclusion, porcine PPARGC1A and CAPNS1 genes may affect meat quality traits, with breed-specific differences, and they could be used as markers for the improvement of meat quality in pigs.

Abstract 2005: Modulation of tumor suppressor gene DNA-methylation by quercetin and dietary indoles

Abstract Epidemiological and preclinical research provide strong evidence for the anticancer properties of bioactive dietary compounds abundantly found in fruits, vegetables and other fiber-rich food stuff. However, the mechanisms by which nutritional intake of such dietary compounds prevent oncogenesis is largely unknown. We hypothesize that synergistic activities of different dietary compounds through epigenetic mechanisms underlie some of the chemopreventive functions of the dietary compounds. We undertook a preliminary study to examine the effects of Quercetin, Indole-3-Carbinol (I3C) and diindolylmethane (DIM) on promoter methylation profiles of selected genes known to be suppressed by methylation in colorectal cancer. Quercetin is a polyphenolic flavonoid, while I3C and DIM are dietary indoles, DIM being a derivative of I3C. The genes queried in this study were p16INK4a, E-cadherin (CDH1), and IGFBP7. p16INK4a is a cell cycle regulator silenced in up to 40-50% of colorectal carcinomas. E-cadherin is a tumor suppressor and metastasis regulator protein the loss of which promotes wnt-signaling, which is dysregulated in the majority of colorectal cancers. IGFBP7 has recently been identified as a p53-responsive gene frequently silenced in colorectal and gastric cancers. In this study, RKO colorectal cancer cells were treated with the three dietary compounds, vehicle (DMSO), or the demethylating compound 5-azacytidine. DNA was harvested and bisulfite converted. Methylation of the gene promoters was examined by methylation-specific PCR using primer sets that amplify only methylated DNA. Gel electrophoresis and Real-time PCR were used to assess the relative abundance of methylated DNA in control and treated samples. Preliminary results show that the dietary compounds can reverse the methylated-promoter phenotype, with the indole compounds having higher potency compared to quercetin especially in demethylating the CDH1 gene promoter. Further experiments will elucidate the molecular mechanisms through which the compounds modulate gene methylation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2005. doi:10.1158/1538-7445.AM2011-2005

Abstract 488: Reprogramming cancer cells into terminal differentiation via induced pluripotency

Abstract Induced pluripotent stem cells (iPSCs) are defined as somatic cells that have been “reprogrammed” (using either the four transcriptional factors Oct4, Sox2, Klf4 and c-Myc or Oct4, Sox2, Nanog, and Lin28) to exhibit embryonic stem cell-like pluripotent differentiation properties. The generation of iPSCs was a great achievement for the field of cell transplantation and tissue engineering. More recently multiple cancer cell lines (e.g., leukemic cells, melanoma cells &amp; gastric cancer cells) have been successfully reprogrammed to the iPSC-state, at least as defined by (1) expression of genes specific to undifferentiated embryonic stem cells; and (2) pluripotency as defined by embryoid body formation in vitro and early markers of commitment to various differentiation lineages. However, the question that remains unanswered is: can the cancer derived iPSCs complete maturation along a given lineage, be it the lineage from which it was derived (i.e., originally prematurely blocked) or an alternative lineage? If the former were demonstrated it would in essence answer the question of whether global changes in chromatin architecture or ‘epigenetics’ (which are believed to underlie the mechanism of nuclear iPSC reprogramming) can supersede the specific genetic changes (i.e., mutations) in DNA that resulted in the lineage specific differentiation block and subsequent tumorigenesis. To attempt to answer these questions we generated iPSC-cancer cells from sarcoma cell lines representing undifferentiated sarcomas, osteosarcoma cells, liposarcoma cells, and sarcomas of unknown lineage. While the original sarcoma cell lines could not be differentiated in vitro into mature connective tissue cells, their iPSC-counterparts readily differentiated into multiple mature connective tissue lineages. Furthermore, assessment of proliferation indicated that acquisition of the mature phenotype was concurrent with cessation of proliferation; and thus abrogation of tumorigenicity. We are currently (1) performing gene expression, miRNA, and global DNA promoter methylation profiling in order to assess the mechanism by which iPSC-based reprogramming of cancer cells over-rides the inherent genetic mutations in these cells and restores the capacity for terminal differentiation; and (2) extending our work to carcinomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 488. doi:10.1158/1538-7445.AM2011-488

Downregulation of ASPP1 in gestational trophoblastic disease: correlation with hypermethylation, apoptotic activity and clinical outcome

Gestational trophoblastic disease encompasses a spectrum of trophoblastic lesions including true neoplasms such as choriocarcinomas and the potentially malignant hydatidiform moles, which may develop persistent disease requiring chemotherapy. ASPP1, a member of apoptosis-stimulating proteins of p53 (ASPPs), is a proapoptotic protein that can stimulate apoptosis through its interaction with p53. We evaluated the promoter methylation and expression profiles of ASPP1 in different trophoblastic tissues and its in vitro functional effect on two choriocarcinoma cell lines, namely JEG-3 and JAR. Significant downregulation of ASPP1 mRNA and protein levels was demonstrated in hydatidiform moles and choriocarcinomas, when compared with normal placentas by quantitative-PCR and immunohistochemistry. The ASPP1 mRNA level was significantly correlated with its hypermethylation status, evaluated with methylation-specific PCR, in placenta and gestational trophoblastic disease samples (P=0.024). Moreover, lower ASPP1 immunoreactivity was shown in hydatidiform moles that progressed to persistent gestational trophoblastic neoplasms than in those that regressed (P=0.045). A significant correlation was also found between expression of ASPP1 and proliferative indices (assessed by Ki67 and MCM7), apoptotic activity (M30 CytoDeath antibody), p53 and caspase-8 immunoreactivities. An in vitro study showed that ectopic expression of ASPP1 could trigger apoptosis through intrinsic and extrinsic pathways as indicated by an increase in cleaved caspase-9 and Fas ligand protein expression. The latter suggests a hitherto unreported novel link between ASPP1 and the extrinsic pathway of apoptosis. Our findings suggest that downregulation of ASPP1 by hypermethylation may be involved in the pathogenesis and progress of gestational trophoblastic disease, probably through its effect on apoptosis.

Methylome analysis reveals Jak-STAT pathway deregulation in putative breast cancer stem cells

Growing evidence supports the existence of a subpopulation of cancer cells with stem cell characteristics within breast tumors. In spite of its potential clinical implications, an understanding of the mechanisms responsible for retaining the stem cell characteristics in these cells is still lacking. Here, we used the mammosphere model combined with DNA methylation bead arrays and quantitative gene expression to characterize the epigenetic mechanisms involved in the regulation of developmental pathways in putative breast cancer stem cells. Our results revealed that MCF7-derived mammospheres exhibit distinct CpG promoter methylation profiles in a specific set of genes, including those involved in Jak-STAT signaling pathway. Hypomethylation of several gene components of the Jak-STAT pathway was correlated with an increased expression in mammospheres relative to parental cells. Remarkably, cell sorting of the cells with a putative cancer stem cell phenotype (CD44+/CD24 low) suggests a constitutive activation of Jak-STAT pathway in these cells. These results show that Jak-STAT activation may represent a characteristic of putative breast cancer stem cells. In addition, they favor the concept that the expression of cancer stem-like pathways and the establishment and maintenance of defining properties of cancer stem cells are orchestrated by epigenetic mechanisms.

Hypermethylation of multiple tumor-related genes associated with DMNT3b up-regulation served as a biomarker for early diagnosis of esophageal squamous cell carcinoma

This study was designed to determine the significance of DNA methyltransferases (DNMTs) in DNA hypermethylation in esophageal squamous cell carcinoma (ESCC) and to identify DNA methylation markers in serum for the early diagnosis of ESCC. A promoter methylation profile of 12 tumor-related genes was assessed using methylation-specific PCR in ESCC and paired non-tumor tissue samples from 47 patients. Expression levels of DNMTs were examined by real-time reverse transcription-PCR and immunohistochemistry. Using MethyLight, the methylation status of 5 genes was analyzed in serum samples from 45 patients and 15 healthy individuals. A total of 46 (97.9%) of 47 ESCC samples showed methylation in at least one of the examined genes, and methylation was most frequent for RAR-β (46.8%), DAPK (46.8%), p16 (44.7%), and CDH1 (42.6%). Methylation of RASSF1A was significantly correlated with the poorly differentiated tumors and the early pathologic tumor classification (P=0.035 and P=0.046, respectively). Tumoral DNMT3b mRNA up-regulation was significantly correlated with hypermethylation of multiple tumor-related genes (P=0.021). In addition, hypermethylation of cell-free serum DNA was common in ESCC patients, and diagnostic accuracy was increased when methylation of multiple genes (RAR-β, DAPK, CDH1, p16 and RASSF1A) were analyzed in combination (ROC AUC 0.911, 82.2% sensitivity and 100% specificity). The present study suggests that hypermethylation of multiple tumor-related genes may be involved in the pathogenesis of ESCC and mediated by the increase of DNMT3b expression. A cluster of multiple methylated genes in serum DNA has the potential as a novel biomarker for ESCC diagnosis.

The effects of EBV transformation on gene expression levels and methylation profiles

Epstein-Barr virus (EBV) transformed lymphoblastoid cell lines (LCLs) provide a conveniently accessible and renewable resource for functional genomic studies in humans. The ability to accumulate multidimensional data pertaining to the same individual cell lines, from complete genomic sequences to detailed gene regulatory profiles, further enhances the utility of LCLs as a model system. A lingering concern, however, is that the changes associated with EBV transformation of B cells reduce the usefulness of LCLs as a surrogate model for primary tissues. To evaluate the validity of this concern, we compared global gene expression and methylation profiles between CD20+ primary B cells sampled from six individuals and six independent replicates of transformed LCLs derived from each sample. These data allowed us to obtain a detailed catalog of the genes and pathways whose regulation is affected by EBV transformation. We found that the expression levels and promoter methylation profiles of more than half of the studied genes were affected by the EBV transformation, including enrichments of genes involved in transcription regulation, cell cycle and immune response. However, we show that most of the differences in gene expression levels between LCLs and B cells are of small magnitude, and that LCLs can often recapitulate the naturally occurring gene expression variation in primary B cells. Thus, our observations suggest that inference of the genetic architecture that underlies regulatory variation in LCLs can typically be generalized to primary B cells. In contrast, inference based on functional studies in LCLs may be more limited to the cell lines.

The bovine genome contains three differentially methylated paralogous copies of the P450c17 encoding gene (CYP17A1)

CYP17A1 encodes the key enzyme of androgen biosynthesis, P450c17. The gene is expressed in a number of steroidogenic tissues among them testis, ovary, placenta and adrenal gland. The proper analysis of CYP17A1 expression and of epigenetic parameters however, is hampered by the presence of more than one copy of the gene within the bovine genome. Therefore, as a prerequisite for future studies we characterized these copies and analyzed their promoter methylation and expression profiles in different tissues. DNA methylation levels were determined by bisulfite modification, amplification, cloning and sequencing. Transcription was analyzed by RT-PCR. From bovine genomic DNA three different CYP17A1 promoter sequences could be amplified with a sequence similarity of 94.8%, 95.6% and 98.7%. Based on these sequences we could reconstruct, by in silico analysis, the promoter regions and eight potentially coding exons of two loci, CYP17A1a and CYP17A1b, and the promoter region and truncated first exon of a third locus, CYP17A1x. By using locus-specific primers, only transcripts of CYP17A1a, but not of CYP17A1b could be detected in testis, epididymis, theca, corpus luteum, placental cotyledons, adrenal gland and preoptic brain area. Methylation analysis revealed that only the CYP17A1a promoter was hypo-methylated in the tested P450c17 active tissues, whereas both other copies showed higher levels of methylation. From these data we conclude that the bovine genome contains three paralogous copies of the CYP17A1 gene, of which two (CYP17A1b and CYP17A1x) might be silenced by epigenetic modification (promoter methylation).

Promoter methylation profile of GSTP1 and RASSF1A in benign hyperplasia and metastatic prostate cancer patients in a Kashmiri population

Promoter hypermethylation is a marginal approach to inactivating tumor suppressor genes in cancer. DNA hypermethylation is a well-recognized epigenetic malfunction observed in several malignancies, most predominantly in prostate cancer. Aberrant DNA methylation patterns are considered to be the earliest somatic genome changes in prostate cancer. The function of promoter hypermethylation in malignant transformation of the prostate has been widely studied, from its presence in benign hyperplasia (BHP) to development and to the advanced stages of tumor formation. In the present study, we examined the promoter hypermethylation status of the glutathione S-transferase P1 (GSTP1) and RASSF1A genes in 45 BHP samples, 50proven prostate tumor samples and 80normal samples. Hypermethylated GSTP1 was found in 29/50(58.0%) prostate carcinoma cases and 12/45(26.6%) BHP cases. The RASSF1A gene was methylated in 17/50(34.0%) prostate cancer samples and 7/45(15.5%) BHP samples. On the basis of these findings, we propose that the epigenetic regulation of the GSTP1 and RASSF1A genes through promoter hypermethylation may play a crucial role in the progression of prostate cancer, and has probable involvement in BHP.

DNA methylation profiling of phyllodes and fibroadenoma tumours of the breast

Phyllodes tumours and cellular fibroadenomas are both fibroepithelial tumours of the breast. Phyllodes tumours, unlike fibroadenomas, have the ability to recur and metastasise. Although these lesions can be distinguished by their stromal cellularity, mitotic index, presence or absence of stromal overgrowth and cellular atypia, there is overlap and not infrequently a definitive diagnosis cannot be made, particularly on biopsy. We sought to evaluate whether DNA promoter methylation profiling using selected genes known to be methylated in cancer would allow us to learn more about the biology of these tumours, and whether it could identify methylation markers that could differentiate phyllodes tumours from fibroadenomas and/or distinguish phyllodes tumours of different grades. Methylation-sensitive high resolution melting (MS-HRM) was used to screen promoter DNA methylation changes in 86 phyllodes tumours (15 benign, 28 borderline, 43 malignant) and 26 fibroadenomas. A panel of 11 genes (RASSF1A, TWIST1, APC, WIF1, MGMT, MAL, RARβ, CDKN2A, CDH1, TP73 and MLH1) was tested. Methylation status was correlated with histology and with clinicopathological parameters. Five of the gene promoters showed some methylation in a proportion of phyllodes tumours; RASSF1A, 45.3%; TWIST1, 10.7%; APC, 4.1%; WIF1, 2.9% and MGMT, 1.3%. Only two genes showed any methylation in fibroadenomas usually at background levels; RASSF1A, 53.8% and MGMT, 8.3%. No CDKN2A methylation was observed in either tumour type, contrary to previous reports. Overall, the methylation patterns differed little from that which might be seen in normal cells. However, significant levels of methylation of RASSF1A (24.4%) and TWIST1 (7.1%) was observed in some phyllodes tumours. Elevated RASSF1A and/or TWIST1 methylation was significantly associated with phyllodes tumours compared with fibroadenomas (P = 0.02), TWIST1 methylation correlated with increasing malignancy in phyllodes tumours (P < 0.001). In conclusion, assessment of methylation of RASSF1A and TWIST1 may aid in the diagnosis of phyllodes tumours. The absence of frequent methylation in fibroadenomas supports a non-neoplastic origin.

Quantitative Methylation Profiles for Multiple Tumor Suppressor Gene Promoters in Salivary Gland Tumors

BackgroundMethylation profiling of tumor suppressor gene (TSGs) promoters is quickly becoming a powerful diagnostic tool for the early detection, prognosis, and even prediction of clinical response to treatment. Few studies address this in salivary gland tumors (SGTs); hence the promoter methylation profile of various TSGs was quantitatively assessed in primary SGT tissue to determine if tumor-specific alterations could be detected.MethodologyDNA isolated from 78 tumor and 17 normal parotid gland specimens was assayed for promoter methylation status of 19 TSGs by fluorescence-based, quantitative methylation-specific PCR (qMSP). The data were utilized in a binary fashion as well as quantitatively (using a methylation quotient) allowing for better profiling and interpretation of results.Principal FindingsThe average number of methylation events across the studied genes was highest in salivary duct carcinoma (SDC), with a methylation value of 9.6, compared to the normal 4.5 (p<0.0003). There was a variable frequency and individual methylation quotient detected, depending on the TSG and the tumor type. When comparing normal, benign, and malignant SGTs, there was a statistically significant trend for increasing methylation in APC, Mint 1, PGP9.5, RAR-β, and Timp3.Conclusions/SignificanceScreening promoter methylation profiles in SGTs showed considerable heterogeneity. The methylation status of certain markers was surprisingly high in even normal salivary tissue, confirming the need for such controls. Several TSGs were found to be associated with malignant SGTs, especially SDC. Further study is needed to evaluate the potential use of these associations in the detection, prognosis, and therapeutic outcome of these rare tumors.