Hypomethylation Of Gene Promoters Research Articles

Identification of NLRP3 Inflammation-Related Gene Promoter Hypomethylation in Diabetic Retinopathy.

PurposeTo identify and validate key genes that could provide a new perspective for genetic marker screening of diabetic retinopathy (DR).MethodsThe gene expression and DNA methylation profiles were obtained from the Gene Expression Omnibus. Differential expression analysis was conducted using the limma package, and then the functions of the differentially expressed genes (DEGs) were analyzed using the DAVID database, followed by protein–protein interaction (PPI) networks using Cytoscape software. We employed the Sequenom MassARRAY system to detect the promoter methylation levels of the candidate genes in peripheral blood mononuclear cells from 32 healthy individuals and 94 patients with type 2 diabetes mellitus (T2D; 64 with DR and 30 without DR) and in fibrovascular membranes (FVMs) from three proliferative DR patients and three controls with idiopathic epiretinal membranes. The mRNA levels of candidate genes were further confirmed via real-time polymerase chain reaction.ResultsA significant enrichment of 5906 DEGs was found in immune and inflammatory responses. TGFB1, CCL2, and TNFSF2 were identified as the top three core genes associated with NLRP3 inflammation in PPI networks. These genes have relatively low levels of promoter methylation, which have been validated in peripheral blood mononuclear cells and FVMs from DR patients, and the methylation levels were found to be negative correlated with the mRNA levels and HbA1c levels in T2D patients.ConclusionsOverall, these data indicate that promoter hypomethylation of NLRP3, TGFB1, CCL2, and TNFSF2 may increase the risk of DR in the Chinese Han population, indicating that these genes might serve as potential targets for the detection and treatment of DR.

Serotonin Transporter Gene Promoter Hypomethylation as a Predictor of Antidepressant Treatment Response in Major Depression: A Replication Study.

BackgroundThe serotonin transporter gene (SLC6A4; 5-HTT; SERT) is considered a prime candidate in pharmacogenetic research in major depressive disorder (MDD). Besides genetic variation, recent advances have spotlighted the involvement of epigenetic mechanisms such as DNA methylation in predicting antidepressant treatment response in “pharmaco-epigenetic” approaches. In MDD, lower SLC6A4 promoter methylation has been suggested to predict impaired response to serotonergic antidepressants. The present study sought to replicate and extend this finding in a large, independent sample of MDD patients.MethodsThe sample comprised n = 236 Caucasian patients with MDD receiving antidepressant medication in a naturalistic treatment setting. Functional DNA methylation of 9 CpG sites located in the SLC6A4 promoter region was analyzed via direct sequencing of sodium bisulfite– treated DNA extracted from blood cells. Patients were assessed over the course of a 6-week in-patient treatment using the Hamilton Depression Scale (HAM-D).ResultsResults confirm relative SLC6A4 hypomethylation to predict impaired antidepressant response both dimensionally and categorically (HAM-D reductions < 50%) and to furthermore be indicative of nonremission (HAM-D > 7). This also held true in a homogenous subgroup of patients continuously treated with selective serotonin reuptake inhibitors or serotonin/noradrenaline reuptake inhibitors (n = 110).ConclusionsImpaired response to serotonergic antidepressants via SLC6A4 hypomethylation may be conveyed by increased gene expression and consequently decreased serotonin availability, which may counteract the effects of serotonergic antidepressants. The present results could in the future inform clinical decision-making towards a more personalized treatment of MDD.

Serotonin transporter gene promoter hypomethylation in obsessive-compulsive disorder – Predictor of impaired response to exposure treatment?

Treatment resistance is common in obsessive-compulsive disorder (OCD) and associated with a significant burden for the individual patient. Accordingly, the identification of biomarkers as early predictors of the clinical response has become a central goal in the search for more efficacious and personalized treatments. Epigenetic mechanisms such as DNA methylation of the serotonin transporter gene (SLC6A4) have been suggested to predict therapy outcome in mental disorders closely related to OCD, but have not yet been investigated as such in OCD. The present therapy-epigenetic study therefore sought to address the potential role of SLC6A4 promoter methylation in the prediction of treatment response for the first time in OCD.Overall, 112 patients with primary OCD were investigated over the course of 8–10-week OCD-specific, cognitive behavioral therapy (CBT) comprising exposure and response prevention/management (phase I) and in vivo exposure exercises (‘flooding’, phase II). OCD symptoms were measured using the Yale-Brown Obsessive Compulsive Scale (Y-BOCS) at baseline as well as before and after the in vivo exposure phase. SLC6A4 promoter methylation at baseline was analyzed via pyrosequencing of sodium bisulfite-treated DNA extracted from blood cells.Lower baseline SLC6A4 promoter methylation predicted impaired treatment response (defined as reduction in Y-BOCS scores) in phase II (but not phase I) of CBT (β = −0.359, p = .002).SLC6A4 methylation may thus constitute a potential early biomarker predicting biologically mediated clinical changes elicited specifically by exposure treatment. These results carry promise for clinical application and in the future could aid in early treatment modification and personalized treatment efforts.

Epigenetic regulation of TLR2‐mediated periapical inflammation

To determinethe methylation pattern of TLR2 gene promoter and its association with the transcriptional regulation of periapical inflammatory and angiogenic responses in symptomatic and asymptomatic forms of apical periodontitis. In this cross-sectional study, apical lesions were obtained from volunteers with asymptomatic apical periodontitis (AAP) (n=17) and symptomatic apical periodontitis (SAP) (n=17) scheduled for tooth extraction, and both total RNA and DNA were extracted. DNA was bisulfite-treated, a region of CpG island within the TLR2 gene was amplified by qPCR and the products were sequenced. Additionally, the mRNA expression of TLR2, TLR4, IL-6, IL-12, TNFalpha, IL-23, IL-10, TGFbeta, VEGFA and CDH5 was analysed by qPCR. The data were analysed with chi-square tests, Mann-Whitney or unpaired t-tests, and Spearman´s correlation; variable adjustments were performed using multiple linear regression (P<0.05). TLR2 depicted a hypomethylated DNA profile at the CpG island in SAPwhen compared with AAP, along with upregulated expression of TLR2, with pro-inflammatory cytokines IL-6 and IL-23, and the angiogenesis marker CDH5 (P<0.05). TLR2 methylation percentage negatively correlated with mRNA levels of IL-23 and CDH5 in apical periodontitis. Lower methylation frequencies of single CpG dinucleotides -8 and -10 localized in close proximity to nuclear factor κB (NFκB) binding within the TLR2 promoter were identified in SAP versus AAP (P<0.05). Finally, unmethylated -10 and -8 single sites demonstrated up-regulation of IL-23, IL-10 and CDH5 transcripts compared to their methylated counterparts (P<0.05). TLR2 gene promoter hypomethylation was linked to transcriptional activity of pro-inflammatory cytokines and angiogenic markers in exacerbated periapical inflammation. Moreover, unmethylated single sites in close proximity to NFκB binding were involved in active transcription of IL-23, IL-10 and CDH5.

Curcumin stimulates angiogenesis through VEGF and expression of HLA‐G in first‐trimester human placental trophoblasts

Curcumin has a protective role in placental diseases like preeclampsia and preterm birth. Very little is known about its functional effects on growth, angiogenesis, and epigenetic activities of human first trimester placenta. HTR8/SVneo trophoblasts cells were used as model for human first trimester placenta. Effects of curcumin (≥80%) in these cells were investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), radioactive thymidine uptake, quantitative real-time polymerase chain reaction (qRT-PCR), promoter DNA methylation, qRT-PCR array, tube formation, wound healing, and immunoblot assays. PC3 (prostate cancer), JEG-3 (trophoblast), and HMEC-1 (endothelial) cells were used as control in various experiments. Unlike in PC3 cells, curcumin stimulated growth, proliferation, and viability in HTR8/SVneo cells. Curcumin increased tube formation, and messenger RNA (mRNA) expression of angiogenic factors such as vascular endothelial growth factor A (VEGFA) and protein expression of proangiogenic factor VEGF receptor-2 and fatty acid-binding protein-4 (FABP4) in these cells. Curcumin-stimulated tube formation was associated with an increased expression of VEGFR2 and FABP4. The stimulatory effects of curcumin were inhibited by VEGFR2 (SU5416) and FABP4 (BMS309403) inhibitors. Curcumin also significantly increased both mRNA and protein expression of HLA-G in HTR8/SVneo cells. Curcumin increased mRNA expression of DNMT3A and NOTCH signaling system whereas down-regulated mRNA expression of HSD11β2. Curcumin enhanced hypomethylation of gene promoters against oxidative stress and DNA damage pathway mediators. Curcumin promotes cell growth, migration, and thus angiogenic potential of these cells. Increased expression of HLA-G by curcumin, hitherto unknown, is a novel finding since HLA-G not only favors the immune environment for invasive trophoblasts but also positively modulates angiogenesis.

Lifelong physical activity is associated with promoter hypomethylation of genes involved in metabolism, myogenesis, contractile properties and oxidative stress resistance in aged human skeletal muscle

Lifelong regular physical activity is associated with reduced risk of type 2 diabetes (T2D), maintenance of muscle mass and increased metabolic capacity. However, little is known about epigenetic mechanisms that might contribute to these beneficial effects in aged individuals. We investigated the effect of lifelong physical activity on global DNA methylation patterns in skeletal muscle of healthy aged men, who had either performed regular exercise or remained sedentary their entire lives (average age 62 years). DNA methylation was significantly lower in 714 promoters of the physically active than inactive men while methylation of introns, exons and CpG islands was similar in the two groups. Promoters for genes encoding critical insulin-responsive enzymes in glycogen metabolism, glycolysis and TCA cycle were hypomethylated in active relative to inactive men. Hypomethylation was also found in promoters of myosin light chain, dystrophin, actin polymerization, PAK regulatory genes and oxidative stress response genes. A cluster of genes regulated by GSK3β-TCF7L2 also displayed promoter hypomethylation. Together, our results suggest that lifelong physical activity is associated with DNA methylation patterns that potentially allow for increased insulin sensitivity and a higher expression of genes in energy metabolism, myogenesis, contractile properties and oxidative stress resistance in skeletal muscle of aged individuals.

Correlation between RAGE gene promoter methylation and diabetic retinal inflammation.

The methylation status of the receptor for advanced glycation end products (RAGE) gene promoter in peripheral blood mononuclear cells (PBMCs) of type 2 diabetic retinopathy (DR) patients was evaluated to investigate the correlation between RAGE gene promoter methylation and diabetic retinal inflammation. Eighty patients admitted and diagnosed as type 2 DR in Qilu Hospital, Shandong University during the period from October, 2013 to October, 2015 were enrolled in this study. They were the observation group and 40 healthy subjects were enrolled in the control group. PBMCs were collected from patients using density gradient centrifugation, and the methylation status of RAGE gene promoters was detected using methylation-specific PCP (MSP). Interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α) levels of in the serum were measured using enzyme-linked immunosorbent assay (ELISA). PBMCs in patients with positive RAGE gene promoter methylation were isolated and cultured and RAGE gene promoter methylation was inhibited using the demethylating agent, 5′-aza-2′-deoxycytidine (5-aza-dC). The methylation status of RAGE gene promoters in PBMCs was detected via MSP. IL-1β, IL-6 and TNF-α levels in the supernatant of PBMC culture solution were evaluated using ELISA. MSP results showed that there were 26 cases (32.50%) of RAGE gene promoter methylation in PBMCs in DR patients. RAGE gene promoters were methylated in all normal healthy subjects. IL-1β, IL-6 and TNF-α levels in serum for positive RAGE gene promoter methylation group were significantly lower than those in negative RAGE gene promoter methylation group (p<0.01). 5-aza-dC inhibited the RAGE gene promoter methylation of PBMCs in patients with positive RAGE gene promoter methylation. The inhibition of methylation in RAGE gene promoter increased the levels of IL-1β, IL-6 and TNF-α in supernatant of culture solution. In conclusion, RAGE gene promoter hypomethylation was detected in DR patients, indicating that RAGE gene promoter methylation could inhibit the diabetic retinal inflammation.

Aberrant KLK4 gene promoter hypomethylation in pediatric hepatoblastomas

DNA methylation has a crucial role in cancer biology and has been recognized as an activator of oncogenes and inactivator of tumor suppressor genes, both of which are mechanisms for tumorigenesis. Kallikrein-related peptidase 4 (KLK4), has been suggested to be an oncogene in various types of cancer. The aim of the present study was to assess the DNA methylation patterns of the KLK4 gene in cancerous samples harvested from patients with hepatoblastoma (HB). KLK4 mRNA expression levels were detected using reverse transcription-quantitative polymerase chain reaction and assessed its DNA methylation patterns using high-throughput mass spectrometry on a matrix-assisted laser desorption/ionization time-of-flight mass array. A total of 10 HB and 10 normal liver tissue samples were obtained from patients with HB. The results of the present study showed that a significantly higher level of KLK4 mRNA expression levels were detected in HB tissues, as compared with the matched controls. Furthermore, the KLK4 gene promoter region was distinctively less methylated in the HB samples compared with the controls and negatively correlated with KLK4 mRNA expression levels. These findings indicate that aberrant methylation of KLK4 may contribute to its upregulated mRNA expression in HB.

Extra-coding RNAs regulate neuronal DNA methylation dynamics.

Epigenetic mechanisms such as DNA methylation are essential regulators of the function and information storage capacity of neurons. DNA methylation is highly dynamic in the developing and adult brain, and is actively regulated by neuronal activity and behavioural experiences. However, it is presently unclear how methylation status at individual genes is targeted for modification. Here, we report that extra-coding RNAs (ecRNAs) interact with DNA methyltransferases and regulate neuronal DNA methylation. Expression of ecRNA species is associated with gene promoter hypomethylation, is altered by neuronal activity, and is overrepresented at genes involved in neuronal function. Knockdown of the Fos ecRNA locus results in gene hypermethylation and mRNA silencing, and hippocampal expression of Fos ecRNA is required for long-term fear memory formation in rats. These results suggest that ecRNAs are fundamental regulators of DNA methylation patterns in neuronal systems, and reveal a promising avenue for therapeutic targeting in neuropsychiatric disease states.

Ectopic Expression of Testis Germ Cell Proteins in Cancer and Its Potential Role in Genomic Instability.

Genomic instability is a hallmark of human cancer and an enabling factor for the genetic alterations that drive cancer development. The processes involved in genomic instability resemble those of meiosis, where genetic material is interchanged between homologous chromosomes. In most types of human cancer, epigenetic changes, including hypomethylation of gene promoters, lead to the ectopic expression of a large number of proteins normally restricted to the germ cells of the testis. Due to the similarities between meiosis and genomic instability, it has been proposed that activation of meiotic programs may drive genomic instability in cancer cells. Some germ cell proteins with ectopic expression in cancer cells indeed seem to promote genomic instability, while others reduce polyploidy and maintain mitotic fidelity. Furthermore, oncogenic germ cell proteins may indirectly contribute to genomic instability through induction of replication stress, similar to classic oncogenes. Thus, current evidence suggests that testis germ cell proteins are implicated in cancer development by regulating genomic instability during tumorigenesis, and these proteins therefore represent promising targets for novel therapeutic strategies.

Subtype-specific overexpression of the Rac-GEF P-REX1 in breast cancer is associated with promoter hypomethylation.

IntroductionThe Rac-GEF P-REX1 is a key mediator of ErbB signaling in breast cancer recently implicated in mammary tumorigenesis and metastatic dissemination. Although P-REX1 is essentially undetectable in normal human mammary epithelial tissue, this Rac-GEF is markedly upregulated in human breast carcinomas, particularly of the luminal subtype. The mechanisms underlying P-REX1 upregulation in breast cancer are unknown. Toward the goal of dissecting the mechanistic basis of P-REX1 overexpression in breast cancer, in this study we focused on the analysis of methylation of the PREX1 gene promoter.MethodsTo determine the methylation status of the PREX1 promoter region, we used bisulfite genomic sequencing and pyrosequencing approaches. Re-expression studies in cell lines were carried out by treatment of breast cancer cells with the demethylating agent 5-aza-2′-deoxycitidine. PREX1 gene methylation in different human breast cancer subtypes was analyzed from the TCGA database.ResultsWe found that the human PREX1 gene promoter has a CpG island located between -1.2 kb and +1.4 kb, and that DNA methylation in this region inversely correlates with P-REX1 expression in human breast cancer cell lines. A comprehensive analysis of human breast cancer cell lines and tumors revealed significant hypomethylation of the PREX1 promoter in ER-positive, luminal subtype, whereas hypermethylation occurs in basal-like breast cancer. Treatment of normal MCF-10A or basal-like cancer cells, MDA-MB-231 with the demethylating agent 5-aza-2′-deoxycitidine in combination with the histone deacetylase inhibitor trichostatin A restores P-REX1 levels to those observed in luminal breast cancer cell lines, suggesting that aberrant expression of P-REX1 in luminal breast cancer is a consequence of PREX1 promoter demethylation. Unlike PREX1, the pro-metastatic Rho/Rac-GEF, VAV3, is not regulated by methylation. Notably, PREX1 gene promoter hypomethylation is a prognostic marker of poor patient survival.ConclusionsOur study identified for the first time gene promoter hypomethylation as a distinctive subtype-specific mechanism for controlling the expression of a key regulator of Rac-mediated motility and metastasis in breast cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0441-7) contains supplementary material, which is available to authorized users.

TET2 Inhibits Differentiation of Embryonic Stem Cells but Does Not Overcome Methylation-Induced Gene Silencing.

TET2 is a methylcytosine dioxygenase that is frequently mutated in myeloid malignancies, notably myelodysplasia and acute myeloid leukemia. TET2 catalyses the conversion of 5′-methylcytosine to 5′-hydroxymethylcytosine within DNA and has been implicated in the process of genomic demethylation. However, the mechanism by which TET2 loss of function results in hematopoietic dysplasia and leukemogenesis is poorly understood. Here, we show that TET2 is expressed in undifferentiated embryonic stem cells and that its knockdown results in reduction of 5′-hydroxymethylcytosine in genomic DNA. We also present DNA methylation data from bone marrow samples obtained from patients with TET2-mutated myelodysplasia. Based on these findings, we sought to identify the role of TET2 in regulating pluripotency and differentiation. We show that overexpression of TET2 in a stably integrated transgene leads to increased alkaline phosphatase expression in differentiating ES cells and impaired differentiation in methylcellulose culture. We speculate that this effect is due to TET2-mediated expression of stem cell genes in ES cells via hydroxylation of 5′-methylcytosines at key promoter sequences within genomic DNA. This leads to relative hypomethylation of gene promoters as 5′-hydroxymethylcytosine is not a substrate for DNMT1-mediated maintenance methylation. We sought to test this hypothesis by cotransfecting the TET2 gene with methylated reporter genes. The results of these experiments are presented.

Genome Wide Promoter Methylation Patterns Predict AML Subtype Outcomes and Identify Novel Pathways Characterizing Diagnostic and Relapsed Disease in Children

AbstractAbstract 1287The NCI-directed TARGET AML initiative has provided an opportunity to evaluate the genomic, transcriptomic and epigenomic patterns of large numbers of pediatric patients with newly diagnosed and relapsed AML with the goal of identifying distinctive changes in pathways that are prognostic or can be therapeutically targeted. Genomic sequencing has revealed an important, but limited number of gene mutations, suggesting that epigenetic alterations may be required to explain the development and biological behavior of AML. We have analyzed genome-wide promoter methylation patterns using Illumina Infinium Methylation 27 arrays on 200 paired diagnostic (Dx) and remission (Rm) cases plus 47 case-matched relapsed (Rl) samples.Marked differences in DNA methylation were observed in the AML compared to remission bone marrows. DNA methylation patterns could discriminate specific types of cytogenetically characterized AML, but no subset of specific methylation changes were identified that predicted outcome across the entire cohort irrespective of AML subtype. Due to the strong correlations of methylation pattern with cytogenetically defined subtypes, such a global signature for outcome would be expected to require either a very strong signal or very large sample size to detect a prognostic, methylation signature that was able to validated in heterogeneous cohorts. We therefore evaluated prognostic methylation features within each cytogenetic group. Using a linear model and empirical Bayes statistic, selective groups of high quality methylation probe signals revealed an association with survival that independently predicted outcome within each cytogenetic subtype. Using a top-scoring pair (TSP) algorithm for each karyotype subtype, five different TSPs each for Inv16, MLL, t(8;21) and normal karyotype subgroups were identified that predicted outcome with an accuracy of 100% except for the t(8;21) subtype in which accuracy of predicting outcome was 87%.We used an alternative analytical approach to determine whether specific epigenetic promoter patterns were associated with diagnosis and/or relapse AML compared to remission bone marrows across all subtypes. These patterns could potentially be considered to be essential for AML development and/or progression. We divided patient data into balanced training and test sets. Using a novel pathway analysis based on outlier statistics, we identified methylation changes independent of cytogenetic subtype in gene promoters associated with specific cellular pathways. Cellular pathways of potential interest characterized by preferentially hypermethylated promoters of genes in the AML diagnostic samples compared to remission samples included Hedgehog, extracellular matrix receptor interactions, cell adhesion molecules, (NCAM/CAM) and phospholipase pathways. Those gene sets that showed hypomethylation in the AML diagnostic samples were selectively associated with cytochrome P450, tyrosine, and arachidonic acid metabolism. We generated a 10 gene signature from genes for two of the most significant pathways, i.e., the Hedgehog and cytochrome P450 metabolism pathways, using TSP and applied this TSP signature using a Fisher's exact test for both Dx vs Rm and Rl vs Rm samples (Table).Test Set Dx v RmCall DxCall RmTest Rl v RmCall RlCall RmTrue Dx933True Rl241True Rm1977True Rm718OR 120, p < 2.2 × 10−16OR 55, p < 8.1 × 10−7The confirmation of the signature in the independent test set validated the significance of the Hedgehog and P450 pathways in newly diagnosed and relapsed pediatric AML.These data demonstrate the unique contribution of epigenetic changes as a prognostic indicator in subgroups of AML. Importantly, outlier analysis followed by pathway-based interrogation identified several key gene sets that are selectively epigenetically altered in an otherwise uncharacterized subset of pediatric AML. Of note, the hypermethylation and hypomethylation of gene promoters characterizing the Hedgehog and of promoters of cytochrome P450 pathways respectively was highly associated with AML diagnosis and relapse samples compared to remission marrows, suggesting a specific role for these pathways in the physiology and maintenance of pediatric AML. Work in progress is examining samples for mutations, gene copy number, expression and functional consequences of the pathways that are selectively altered by epigenetic mechanisms. Disclosures:No relevant conflicts of interest to declare.

Distinct Functional Patterns of Gene Promoter Hypomethylation and Hypermethylation in Cancer Genomes

BackgroundAberrant DNA methylation plays important roles in carcinogenesis. However, the functional significance of genome-wide hypermethylation and hypomethylation of gene promoters in carcinogenesis currently remain unclear.Principal FindingsBased on genome-wide methylation data for five cancer types, we showed that genes with promoter hypermethylation were highly consistent in function across different cancer types, and so were genes with promoter hypomethylation. Functions related to “developmental processes” and “regulation of biology processes” were significantly enriched with hypermethylated genes but were depleted of hypomethylated genes. In contrast, functions related to “cell killing” and “response to stimulus”, including immune and inflammatory response, were associated with an enrichment of hypomethylated genes and depletion of hypermethylated genes. We also observed that some families of cytokines secreted by immune cells, such as IL10 family cytokines and chemokines, tended to be hypomethylated in various cancer types. These results provide new hints for understanding the distinct functional roles of genome-wide hypermethylation and hypomethylation of gene promoters in carcinogenesis.ConclusionsGenes with promoter hypermethylation and hypomethylation are highly consistent in function across different cancer types, respectively, but these two groups of genes tend to be enriched in different functions associated with cancer. Especially, we speculate that hypomethylation of gene promoters may play roles in inducing immunity and inflammation disorders in precancerous conditions, which may provide hints for improving epigenetic therapy and immunotherapy of cancer.

Aberrant Stratifin Overexpression Is Regulated by Tumor-Associated CpG Demethylation in Lung Adenocarcinoma

We previously have shown the aberrant overexpression of stratifin (SFN, 14-3-3 ς) in lung adenocarcinoma. Although SFN is known to facilitate tumor cell proliferation, the mechanism that underlies its aberrant expression has remained unclear. SFN, the downstream target of p53, often has been reported to be hypermethylated and subsequently silenced in certain cancers; however, its hypomethylation-linked reactivation has not yet been validated. In this study, we investigated the DNA methylation status of the SFN promoter region using 8 lung cancer cell lines and 32 specimens of adenocarcinoma tissue. Real-time methylation-specific PCR analysis showed that although both normal lung tissue and adenocarcinoma in situ bore a completely methylated SFN promoter, the promoter region in almost all invasive adenocarcinomas was at least partially methylated. The expression of SFN and its level of methylation were correlated strongly. Furthermore, statistical analysis revealed that the level of methylation became reduced with progression of the pathologic stage, although no clear relationship between methylation level and p53 abnormality was found. These results suggest that methylation-related silencing of SFN occurs in both normal lung tissues and adenocarcinoma in situ, and that demethylation of the SFN promoter participates in the aberrant expression of SFN in invasive adenocarcinoma cells, independently of p53 alteration. This novel finding might be informative for clarifying the mechanism that underlies the progression of early lung adenocarcinoma.

A DNA methylation signature associated with aberrant promoter DNA hypermethylation of DNMT3B in human colorectal cancer

Altered promoter DNA methylation, one of the most important molecular alterations in cancer, is proposed to correlate with deregulation of DNA methyltransferases, although the molecular mechanisms implicated are still poorly understood. Here we show that the de novo DNA methyltransferase DNMT3B is frequently repressed in human colorectal cancer cell lines (CCL) and primary tumours by aberrant DNA hypermethylation of its distal promoter. At the epigenome level, DNMT3B promoter hypermethylation was associated with the hypomethylation of gene promoters usually hypermethylated in the healthy colon. Forced DNMT3B overexpression in cancer cells restored the methylation levels of these promoters in the healthy colon. Our results show a new molecular mechanism of aberrant DNMT3B regulation in colon cancer and suggest that its expression is associated with the methylation of constitutively hypermethylated promoters in the healthy colon.

Genome-Wide Methylation Analysis of Primary Mantle Cell Lymphomas Identifies Novel Gene Targets for Epigenetic Drug Therapy.

Abstract 673Mantle Cell Lymphoma (MCL) is an aggressive and incurable malignancy arising from naïve B cells (NBC) in the mantle zone of lymph node follicles. Murine models over-expressing Cyclin D1, the putative oncogene implicated in the majority of MCL, do not fully recapitulate the disease phenotype. We therefore hypothesize that there are additional mechanisms contributing to MCL pathogenesis and undertook an integrative approach by studying genome-wide DNA methylation and gene expression in primary MCL to uncover additional genes and pathways involved in MCL pathogenesis.We therefore compared and contrasted the DNA methylation levels of 14,000 gene promoters in MCL patients and normal tonsillar NBC controls using the HELP (HPA II tiny fragment Enrichment by Ligation mediated PCR) assay. All patient samples were obtained prior to any treatment from peripheral blood or apheresis specimens from newly diagnosed patients with histologically confirmed MCL. We found significant hypo-methylation of gene promoters in the MCL patients as compared to normal NBCs. Integrating genomic methylation data from HELP and gene expression data from Affymetrix U133 arrays, we determined a signature of differentially methylated genes with reciprocal changes in mRNA levels. Using pathway analysis and gene ontogeny, we selected genes for validation by choosing loci that were differentially methylated and fulfilling the following characteristics (i) demonstrating a clear methylation state change (from hypomethylated in normal B cells to methylated in MCL or vice-versa) using a threshold of 0.0 on the log ratio scale, (ii) Genes that function as tumor suppressors and were hypermethylated and suppressed in MCLs in our data (iii) Overexpressed/ hypomethylated genes with existing therapeutic options available or in clinical trial (iv) involved in pathways controlling biological processes with known relevance to MCL i.e. cell cycle control, apoptosis. Our panel included four differentially hypermethylated genes CDKN2B, MLF-1, PCDH8, HOXD8 and four differentially hypomethylated genes CD37, HDAC1, NOTCH1 and CDK5. MassArray Epityper analysis confirmed the presence of differential methylation at the promoter region of these genes, which was consistent between MCL patients and cell lines in all 8 genes studied. Remarkably, PCDH8 and CDKN2B have previously been shown to be silenced by methylation at their gene promoters and transfection of PCDH8 and CDKN2B have been shown to reduce tumor growth in breast cancer and MCL cell line models respectively.Based on these data, we hypothesized that the aberrantly hypermethylated and thereby silenced tumor suppressor genes in MCL could be pharmacologically induced by DNA hypomethylating agents and HDAC inhibitors for therapeutic benefit. We therefore next treated MCL cell lines MINO and Z138 with the DNA methyltransferase inhibitor Decitabine alone and in combination with the HDAC inhibitor SAHA. HELP analysis of MINO and Z138 cells treated with hypomethylating doses of decitabine (0.5uM × 3days) showed widespread reversal of aberrant gene promoter hypermethylation. Hypomethylation in these cell lines was accompanied with 3-7 fold increase in mRNA levels of tumor suppressor genes CDKN2B, MLF-1, PCDH8 and HOXD8. Concurrent treatment with SAHA (1 uM x 1 dose) synergized with Decitabine leading to 5-15 fold increase in mRNA of these tumor suppressor genes in Z138 cells. Importantly, treatment with Decitabine and SAHA as single agents decreased MCL cell viability by 60% and 40% respectively and the combination synergised in anti-MCL cytotoxicity with > 90% decrease in cell viability.In conclusion, our analysis shows prominent aberrant gene promoter methylation patterns in MCL genome and identifies novel differentially methylated and expressed genes in MCL cell lines and primary MCLs. Furthermore, we demonstrate that reversal of aberrant hypermethylated and silenced genes can be targeted for therapeutic benefit using epigenetic drugs in MCL. We are currently modeling our combination epigenetic drug therapy in primary MCL cells in culture and murine xenograft systems. We expect these results to support the development of clinical trials investigating the prospective use of combination epigenetic therapy in MCL. Disclosures:No relevant conflicts of interest to declare.

S-Adenosylhomocysteine increases β-amyloid formation in BV-2 microglial cells by increased expressions of β-amyloid precursor protein and presenilin 1 and by hypomethylation of these gene promoters

S-Adenosylhomocysteine (SAH) has been implicated as a risk factor for neurodegenerative diseases such as Alzheimer's disease. As SAH is a potent inhibitor of all cellular methyltransferases, we herein examined the hypothesis that SAH may increase the formation of amyloid β-peptide (Aβ) in BV-2 mouse microglial cells through hypomethylation of presenilin 1 protein (PS1) and β-site amyloid precursor protein cleaving enzyme 1 (BACE1), both of which cleave Aβ precursor protein (APP) to form Aβ. The results showed that SAH increased Aβ protein formation in a concentration-dependent manner (10–500 nM), and this effect of SAH was accompanied by significantly increased expression of APP and PS1 proteins, although SAH only significantly increased the expression of BACE1 at the highest concentration used (500 nM). SAH (500 nM) markedly induced hypomethylation of APP and PS1 gene promoters. Incubation of cells with 5′-azc (20 μM), also an inhibitor of DNA methyltransferases enhanced Aβ protein expression and APP and PS1 gene promoters hypomethylation. By contrast, pre-incubation of cells with betaine (1.0 mM), 30 min followed by incubation with SAH (500 nM) or 5′-azc (20 μM) for 24 h markedly prevented the expression of Aβ protein (by 50%, P < 0.05) and the gene promoter hypomethylation of APP and PS1. Taken together, this study demonstrates that SAH increases the production of Aβ in BV-2 cells possibly by increased expression of APP and induction of hypomethylation of APP and PS1 gene promoters.

Identification of interactive networks of gene expression associated with osteosarcoma oncogenesis by integrated molecular profiling

Altered gene expression in tumors can be caused by copy number alterations to DNA or mutation affecting coding or regulatory regions of genes. However, epigenetic events may also influence gene expression. Malignant cells can show major disruptions in DNA methylation profiles, which are manifested as aberrant hypermethylation or as hypomethylation of gene promoters, as well as global genomic hypomethylation. In this study we performed integrative whole-genome analysis of DNA copy number, promoter methylation and gene expression using 10 osteosarcomas. We identified significant changes including: hypomethylation, gain, and overexpression of histone cluster 2 genes at chromosome 1q21.1-q21.3; loss of chromosome 8p21.2-p21.3 and underexpression of DOCK5 and TNFRSF10A/D genes; and amplification-related overexpression of RUNX2 at chromosome 6p12.3-p21.1. Amplification and overexpression of RUNX2 could disrupt G2/M cell cycle checkpoints, and downstream osteosarcoma-specific changes, such as failure of bone differentiation and genomic polyploidization. Failure of DOCK5-signaling, together with p53 and TNFRSF10A/D-related cell cycle and death pathways, may play a critical role in abrogating apoptosis. Our analyses show that the RUNX2 interactome may be constitutively activated in osteosarcoma, and that the downstream intracellular pathways are strongly associated with the regulation of osteoblast differentiation and control of cell cycle and apoptosis in osteosarcoma.

Maternal embryonic leucine zipper kinase transcript abundance correlates with malignancy grade in human astrocytomas

We have performed cDNA microarray analyses to identify gene expression differences between highly invasive glioblastoma multiforme (GBM) and typically benign pilocytic astrocytomas (PA). Despite the significant clinical and pathological differences between the 2 tumor types, only 63 genes were found to exhibit 2-fold or greater overexpression in GBM as compared to PA. Forty percent of these genes are related to the regulation of the cell cycle and mitosis. QT-PCR validation of 6 overexpressed genes: MELK, AUKB, ASPM, PRC1, IL13RA2 and KIAA0101 confirmed at least a 5-fold increase in the average expression levels in GBM. Maternal embryonic leucine zipper kinase (MELK) exhibited the most statistically significant difference. A more detailed investigation of MELK expression was undertaken to study its oncogenic relevance. In the examination of more than 100 tumors of the central nervous system, we found progressively higher expression of MELK with astrocytoma grade and a noteworthy uniformity of high level expression in GBM. Similar level of overexpression was also observed in medulloblastoma. We found neither gene promoter hypomethylation nor amplification to be a factor in MELK expression, but were able to demonstrate that MELK knockdown in malignant astrocytoma cell lines caused a reduction in proliferation and anchorage-independent growth in in vitro assays. Our results indicate that GBM and PA differ by the expression of surprisingly few genes. Among them, MELK correlated with malignancy grade in astrocytomas and represents a therapeutic target for the management of the most frequent brain tumors in adult and children.