Higher Promoter Methylation Levels Research Articles

DNA methylation can alter CRISPR/Cas9 editing frequency and DNA repair outcome in a target-specific manner.

Summary The impact of epigenetic modifications on the efficacy of CRISPR/Cas9‐mediated double‐stranded DNA breaks and subsequent DNA repair is poorly understood, especially in plants.In this study, we investigated the effect of the level of cytosine methylation on the outcome of CRISPR/Cas9‐induced mutations at multiple Cas9 target sites in Nicotiana benthamiana leaf cells using next‐generation sequencing.We found that high levels of promoter methylation, but not gene‐body methylation, decreased the frequency of Cas9‐mediated mutations. DNA methylation also influenced the ratio of insertions and deletions and potentially the type of Cas9 cleavage in a target‐specific manner. In addition, we detected an over‐representation of deletion events governed by a single 5′‐terminal nucleotide at Cas9‐induced DNA breaks.Our findings suggest that DNA methylation can indirectly impair Cas9 activity and subsequent DNA repair, probably through changes in the local chromatin structure. In addition to the well described Cas9‐induced blunt‐end double‐stranded DNA breaks, we provide evidence for Cas9‐mediated staggered DNA cuts in plant cells. Both types of cut may direct microhomology‐mediated DNA repair by a novel, as yet undescribed, mechanism.

Copy number variations and young duplicate genes have high methylation levels in sticklebacks.

Gene duplication is an important driver of genomic diversity that can promote adaptive evolution. However, like most mutations, a newly duplicated gene is often deleterious and removed from the genome by drift or natural selection. The early molecular changes that occur soon after duplication therefore may influence the long-term survival of gene duplicates, but relatively little empirical data exist on the events near the onset of duplication before mutations have time to accumulate. In this study, we contrast gene expression and DNA methylation levels of duplicate genes in the threespine stickleback, Gasterosteus aculeatus, including recently emerged duplications that segregate as copy number variations (CNVs). We find that younger duplicate genes have higher levels of promoter methylation than older genes, and that gene CNVs have higher promoter methylation than non-CNVs. These results suggest preferential duplication of highly methylated genes or rapid methylation changes soon after duplication. We also find a negative association between methylation and expression, providing a putative role for methylation in suppressing transcription that compensates for increases in gene copy numbers and promoting paralog retention. We propose that methylation contributes to the longevity of young duplicate genes, extending the window of opportunity for functional divergence via mutation.

DAMEfinder: a method to detect differential allele-specific methylation

BackgroundDNA methylation is a highly studied epigenetic signature that is associated with regulation of gene expression, whereby genes with high levels of promoter methylation are generally repressed. Genomic imprinting occurs when one of the parental alleles is methylated, i.e., when there is inherited allele-specific methylation (ASM). A special case of imprinting occurs during X chromosome inactivation in females, where one of the two X chromosomes is silenced, to achieve dosage compensation between the sexes. Another more widespread form of ASM is sequence dependent (SD-ASM), where ASM is linked to a nearby heterozygous single nucleotide polymorphism (SNP).ResultsWe developed a method to screen for genomic regions that exhibit loss or gain of ASM in samples from two conditions (treatments, diseases, etc.). The method relies on the availability of bisulfite sequencing data from multiple samples of the two conditions. We leverage other established computational methods to screen for these regions within a new R package called DAMEfinder. It calculates an ASM score for all CpG sites or pairs in the genome of each sample, and then quantifies the change in ASM between conditions. It then clusters nearby CpG sites with consistent change into regions. In the absence of SNP information, our method relies only on reads to quantify ASM. This novel ASM score compares favorably to current methods that also screen for ASM. Not only does it easily discern between imprinted and non-imprinted regions, but also females from males based on X chromosome inactivation. We also applied DAMEfinder to a colorectal cancer dataset and observed that colorectal cancer subtypes are distinguishable according to their ASM signature. We also re-discover known cases of loss of imprinting.ConclusionWe have designed DAMEfinder to detect regions of differential ASM (DAMEs), which is a more refined definition of differential methylation, and can therefore help in breaking down the complexity of DNA methylation and its influence in development and disease.

Hearing loss risk and DNA methylation signatures in preschool children following lead and cadmium exposure from an electronic waste recycling area

Experimental studies have uncovered chemical exposure-induced ototoxicity, but population-based hearing risk assessment especially for early-life exposure to heavy metals and relevant biological mechanism remains unclear. We aimed to measure lead (Pb) and cadmium (Cd) levels, blood DNA methylations of Rb1, CASP8 and MeCP2 and hearing in 116 preschool children 3- to 7-years of age from an e-waste and a reference area, and to evaluate the association of exposures with hearing loss potentially affected by epigenetic modifications. A higher median Pb level but not Cd was found in the exposed group than the reference group. Average hearing thresholds in either ear of the exposed children were higher. Higher promoter methylation levels at cg02978827 and position +14, and lower at position +4 of Rb1 were found in the exposed group. Pb was positively correlated with chewing pencil habit while negatively correlated with washing hands before dinner. Slightly negative trends of promoter methylations in Rb1 and CASP8, while a strong positive trend of MeCP2 promoter methylation, were found along with increasing Pb and Cd levels. Logistic analyses showed the adjusted OR of Pb for hearing loss in the left ear and both ears was 1.46 (95% CI: 1.12, 1.91) and 1.40 (95% CI: 1.06, 1.84), respectively. Our results show an elevated Pb level, altered promoter DNA methylations and hearing ability in children of e-waste areas, suggesting that epigenetic changes of specific genes involves in the development of the auditory system during early exposure to environmental chemicals.

Down-regulation of TROP-2 Predicts Poor Prognosis of Hepatocellular Carcinoma Patients.

Hepatocellular carcinoma (HCC) is one of the most common and lethal cancer types worldwide, especially in Asian countries. Genetic alterations, including hyperactivation of oncogenes and loss of expression of tumor suppressor genes, greatly contribute to the initiation and progression of HCC. Here we report that down‐regulation of trophoblast cell surface antigen 2 (TROP‐2) was frequently detected in HCC. Transcriptome sequencing of non‐tumor and HCC patient samples revealed down‐regulation of TROP‐2 in tumor tissues. Immunohistochemical staining showed nearly undetectable levels of TROP‐2 in HCC tissues but distinct and strong staining of TROP‐2 in adjacent non‐tumor tissues. The frequent down‐regulation of TROP‐2 expression was further confirmed in an in‐house cohort of 205 pairs of HCC patient samples and in the Cancer Genome Atlas (TCGA) databases. Furthermore, the down‐regulation of TROP‐2 was associated with poor overall survival of HCC patients, severe adjacent organ invasion, and poor differentiation of HCC. Using bisulfite genomic sequencing and methylation‐specific polymerase chain reaction analyses, we show that higher levels of promoter methylation were detected in the DNA samples of HCC tissues (low TROP‐2 expression) than that of the non‐tumor tissues (high TROP‐2 expression). Conclusion: Taken together, our data suggest that promoter hypermethylation contributes to the frequent down‐regulation of TROP‐2 in HCC, and that TROP‐2 down‐regulation predicts poor prognosis of HCC patients.

Wnt signaling pathway upregulates DNMT1 to trigger NHERF1 promoter hypermethylation in colon cancer.

NHERF1 is downregulated and has been identified as a new marker of colorectal cancer. However, the molecular mechanism of NHERF1 downregulation in colon cancer is not well understood. In the present study, we demonstrated that the NHERF1 mRNA level was downregulated and correlated with outcomes in colon cancer patients. NHERF1 expression was associated with EMT phenotype. High levels of promoter methylation of NHERF1 in colon cancer were observed. NHERF1 expression was restored by demethylation treatment. In addition, a negative correlation between methylation and mRNA expression of NHERF1 was observed in the TCGA dataset. Moreover, DNMT1 inhibited NHERF1 expression invivo. DNMT1 expression was found to be negatively correlated with NHERF1 and NHERF1 expression was also restored by a DNMT1 inhibitor. DNMT1 and NHERF1 were regulated by the Wnt signaling pathway. In addition, among DNMT1 high expression patients, the Wnt signaling pathway was negatively correlated with NHERF1 expression. In contrast, among DNMT1 low expression patients, the Wnt signaling pathway was not correlated with NHERF1 expression. In conclusion, Wnt signaling increases DNMT1 expression. DNMT1 contributes to promoter hypermethylation and epigenetic NHERF1 silencing in colon cancer. In addition, we provide novel insight into the mechanisms underlying the regulation of NHERF1 expression and occurrence/progression of colon cancer.

MiR-193b promoter methylation accurately detects prostate cancer in urine sediments and miR-34b/c or miR-129-2 promoter methylation define subsets of clinically aggressive tumors

BackgroundContemporary challenges of prostate cancer (PCa) include overdiagnosis and overtreatment, entailing the need for novel clinical tools to improve risk stratification and therapy selection. PCa diagnosis and prognostication might be perfected using epigenetic biomarkers, among which aberrant DNA methylation of microRNA promoters has not been systematically explored. Herein, we identified aberrantly methylated microRNAs promoters in PCa and assessed its diagnostic and prognostic biomarker potential.MethodsUsing HumanMethylation450 BeadChip-based analysis differentially methylated CpGs in microRNA promoters were identified. Promoter methylation of six microRNAs (miR-34b/c, miR-129-2, miR-152, miR-193b, miR-663a and miR-1258) was analyzed by qMSP in three sets (180 prostatectomies, 95 urine sediments and 74 prostate biopsies). Biomarkers’ diagnostic (validity estimates) and prognostic [disease-free (DFS) and disease-specific survival (DSS)] performance was assessed.ResultsSignificantly higher promoter methylation levels in PCa were confirmed for six candidate microRNAs. Except for miR-152, all displayed AUC values higher than 0.90, with miR-1258 and miR-193b disclosing the best performance (AUC = 0.99 and AUC = 0.96, respectively). In urine samples, miR-193b showed the best performance (91.6% sensitivity, 95.7% specificity, AUC = 0.96). Moreover, higher miR-129-2 independently predicted for shorter DSS and miR−34b/c methylation levels independently predicted for shorter DFS and DSS.ConclusionsQuantitative miR-193b, miR-129-2 and miR-34b/c promoter methylation might be clinically useful PCa biomarkers for non-invasive detection/diagnosis and prognostication, both in tissue and urine samples.

High promoter methylation levels of glutathione-S-transferase M3 predict poor prognosis of acute-on-chronic hepatitis B liver failure.

This study aimed to evaluate the prognostic value of glutathione-S-transferase M3 (GSTM3) gene promoter methylation in patients with acute-on-chronic hepatitis B liver failure (ACHBLF). A total of 119 patients with ACHBLF, 60 patients with chronic hepatitis B and 30 healthy controls were enrolled. We used a quantitative methylation detection technique, MethyLight, to examine the methylation levels of GSTM3 in peripheral blood mononuclear cells. The GSTM3 methylation level was significantly higher in patients with ACHBLF than those in patients with chronic hepatitis B and healthy controls (both P <0.05). In patients with ACHBLF, GSTM3 methylation level percentage of methylated reference (PMR) positively correlated with total bilirubin, international normalized ratio, and Model for End-stage Liver Disease (MELD) score, and negatively correlated with prothrombin activity and albumin (all P <0.05). The PMR for GSTM3 of non-survivors was significantly increased compared to that of survivors (P < 0.05). Multivariate analysis indicated that GSTM3 methylation level was one of the independent prognostic factors for 3-month mortality of ACHBLF (P = 0.000). The area under the receiver-operator characteristic curve of PMR for GSTM3 in predicting 3-month mortality of ACHBLF was not statistically different from that of MELD score (0.798 vs. 0.716, P = 0.152). However, the area under the curve of PMR for GSTM3 was significantly higher than that of MELD score in predicting 1-month mortality (0.887 vs. 0.737, P = 0.020). Promoter methylation levels of GSTM3 in peripheral blood mononuclear cells closely correlated with disease severity and could be used to predict prognosis of patients with ACHBLF.

Abstract 5155: Integrated genomic analysis of colorectal cancer progression reveals activation of EGFR through demethylation of the EREG promoter

Abstract The molecular drivers that underlie transformation of colonic epithelium into colorectal adenocarcinoma (CRC) have been well described. However, the mechanisms through which some of the clinically targeted pathways are activated during CRC progression have yet to be elucidated. Here, we employed an integrative genomics approach to examine CRC progression. Transcriptional profiling of laser capture microdissected colonic crypt cells, differentiated surface epithelium, adenomas, carcinomas, and metastases, showed distinctive patterns in the activation of developmental and oncogenic pathways, including the clinically important EGFR axis. We observed a dramatic up-regulation of the EGFR ligand EREG in primary and metastatic cancer cells as compared to normal and adenomatous tissues that was indicative of autocrine tumor production, and confirmed this pattern of gene expression by in situ hybridization. Global methylation analysis indicated that up-regulation of EREG during the adenoma-carcinoma transition was associated with de-methylation of two key sites within the EREG promoter and this was accompanied by an increase in the levels of EGFR phosphorylation, as assessed by reverse phase protein analysis. In a clinical trial setting, we observed that low levels of EREG methylation in patients who received cetuximab as part of a Phase II study were associated with high expression of the ligand and a favorable response to therapy. Conversely, high levels of promoter methylation and low levels of EREG expression were observed in tumors that progressed after treatment. We also noted an inverse correlation between EREG methylation and expression levels in several other cancers from the TCGA datasets, including those of the head and neck, lung, and bladder. We propose that up-regulation of EREG expression through promoter de-methylation might be an important means of activating the EGFR pathway during the genesis of CRC and, potentially, other types of cancer. Citation Format: Xueping Qu, Thomas Sandmann, Henry Frierson, Ling Fu, Eloisa Fuentes, Kimberly Walter, Kwame Okrah, Craig Rumpel, Christopher Moskaluk, Shan Lu, Yulei Wang, Richard Bourgon, Elicia Penuel, Andrea Pirzkall, Lukas Amler, Mark Lackner, Josep Tabenero, Garret Hampton, Omar Kabbarah. Integrated genomic analysis of colorectal cancer progression reveals activation of EGFR through demethylation of the EREG promoter. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5155.

Integrated genomic analysis of colorectal cancer progression reveals activation of EGFR through demethylation of the EREG promoter.

Key molecular drivers that underlie transformation of colonic epithelium into colorectal adenocarcinoma (CRC) are well described. However, the mechanisms through which clinically targeted pathways are activated during CRC progression have yet to be elucidated. Here, we used an integrative genomics approach to examine CRC progression. We used laser capture microdissection to isolate colonic crypt cells, differentiated surface epithelium, adenomas, carcinomas and metastases, and used gene expression profiling to identify pathways that were differentially expressed between the different cell types. We identified a number of potentially important transcriptional changes in developmental and oncogenic pathways, and noted a marked upregulation of EREG in primary and metastatic cancer cells. We confirmed this pattern of gene expression by in situ hybridization and observed staining consistent with autocrine expression in the tumor cells. Upregulation of EREG during the adenoma–carcinoma transition was associated with demethylation of two key sites within its promoter, and this was accompanied by an increase in the levels of epidermal growth factor receptor (EGFR) phosphorylation, as assessed by reverse-phase protein analysis. In CRC cell lines, we demonstrated that EREG demethylation led to its transcriptional upregulation, higher levels of EGFR phosphorylation, and sensitization to EGFR inhibitors. Low levels of EREG methylation in patients who received cetuximab as part of a phase II study were associated with high expression of the ligand and a favorable response to therapy. Conversely, high levels of promoter methylation and low levels of EREG expression were observed in tumors that progressed after treatment. We also noted an inverse correlation between EREG methylation and expression levels in several other cancers, including those of the head and neck, lung and bladder. Therefore, we propose that upregulation of EREG expression through promoter demethylation might be an important means of activating the EGFR pathway during the genesis of CRC and potentially other cancers.

Aberrant Promoter Methylation of the Tumour Suppressor RASSF10 and Its Growth Inhibitory Function in Breast Cancer.

Breast cancer is the most common cancer in women, with 1.7 million new cases each year. As early diagnosis and prognosis are crucial factors in cancer treatment, we investigated potential DNA methylation biomarkers of the tumour suppressor family Ras-association domain family (RASSF). Promoter hypermethylation of tumour suppressors leads to their inactivation and thereby promotes cancer development and progression. In this study we analysed the tumour suppressors RASSF1A and RASSF10. Our study shows that RASSF10 is expressed in normal breast but inactivated by methylation in breast cancer. We observed a significant inactivating promoter methylation of RASSF10 in primary breast tumours. RASSF10 is inactivated in 63% of primary breast cancer samples but only 4% of normal control breast tissue is methylated (p < 0.005). RASSF1A also shows high promoter methylation levels in breast cancer of 56% vs. 8% of normal tissue (p < 0.005). Interestingly more than 80% of breast cancer samples harboured a hypermethylation of RASSF10 and/or RASSF1A promoter. Matching samples exhibited a strong tumour specific promoter methylation of RASSF10 in comparison to the normal control breast tissue. Demethylation treatment of breast cancer cell lines MCF7 and T47D reversed RASSF10 promoter hypermethylation and re-established RASSF10 expression. In addition, we could show the growth inhibitory potential of RASSF10 in breast cancer cell lines MCF7 and T47D upon exogenous expression of RASSF10 by colony formation. We could further show, that RASSF10 induced apoptotic changes in MCF7 and T47D cells, which was verified by a significant increase in the apoptotic sub G1 fraction by 50% using flow cytometry for MCF7 cells. In summary, our study shows the breast tumour specific inactivation of RASSF10 and RASSF1A due to DNA methylation of their CpG island promoters. Furthermore RASSF10 was characterised by the ability to block growth of breast cancer cell lines by apoptosis induction.

The tumor suppressing effects of QKI-5 in prostate cancer

In recent years, the RNA-binding protein quaking 5 (QKI-5) has been recognized as a novel tumor suppressor in many cancers. To date, no studies have examined the role of QKI-5 in prostate cancer. The present study was designed to elucidate the correlation of QKI-5 expression with the clinical pathological features and prognosis of prostate cancer. In an overwhelming majority of the 184 cases of prostate cancer samples analyzed, the QKI-5 expression was significantly decreased, which was largely due to the high promoter methylation levels. Using lentiviral vectors, we established two stable prostate cancer cell lines with altered QKI-5 expression, including a QKI-5 overexpressing PC3 cell line and a DU145 cell line with knocked-down QKI-5 expression. The effects of the lentiviral-mediated QKI-5 knockdown on the PC3 cells and DU145 cells were assessed by cell growth curves, flow cytometry (FCM), and an invasion assay. The PC3 cells were transplanted into nude mice, and then, the tumor growth curves and TUNEL staining were determined. These results demonstrated that QKI-5 was highly expressed in benign prostatic hyperplasia (BPH) tissues but not in carcinomatous tissues and that QKI-5 effectively inhibited prostate cancer cell proliferation in vitro and in vivo. In addition, the decrease in QKI-5 expression was closely correlated with the prostate cancer Gleason score, poor differentiation, degree of invasion, lymph node metastasis, distant metastasis, TNM grading, and poor survival. These results indicate that the QKI-5 expression may be a novel, independent factor in the prognosis of prostate cancer patients.

Abstract 68: Beyond GWAS: The prognostic significance of promoter DNA methylation in patients with neuroblastoma.

Abstract Background: Neuroblastoma is a genetically heterogenic tumor diagnosed in childhood which exhibits broad clinical diversity ranging from rapid disease progression to spontaneous regression. Although amplification of the MYCN oncogene is one of the most prominent prognostic markers of this disease it is only present in around 25% of patients. Hence, additional molecular markers are needed for patients lacking MYCN amplification to enable better stratification of patient risk groups. To date, genome wide association studies have identified few gene candidates in neuroblastoma patients that can be further developed for prognostic use in the clinic. DNA methylation is a major epigenetic mechanism for gene silencing in a wide range of human cancers and methylation sites may reveal new prognostic targets in neuroblastoma. Recent studies have suggested that the methylation status of specific gene promoters may be useful for clinically discriminating subgroups of children diagnosed with neuroblastoma. To date, these investigations have assessed DNA methylation using variable techniques; focused on small numbers of candidate genes and; examined few primary tumor samples. The aims of our study were to characterize the clinical significance of promoter methylation in a large cohort of primary neuroblastoma tumors and investigate the association between DNA methylation and clinical outcome. Methods: A customized Illumina GoldenGate methylation assay was used to assess methylation status of 96 CpG sites within 48 candidate genes in primary neuroblastoma tumors obtained from 131 children diagnosed in Australia between 1985 and 2000. Candidate genes were selected on the basis of previous reports of altered DNA methylation in embryonal cancers. Levels of DNA methylation were quantified and confirmed in a subset of 48 patient samples using combined bisulphite restriction analysis (CoBRA). A Cox proportional hazards model was used to investigate the association between promoter hypermethylation and the risk of relapse or death within 5 years of diagnosis, while adjusting for known prognostic cofactors such as MYCN amplification, age and stage at diagnosis. Results: The level of promoter methylation of DNAJC15, NTRK1 and TNFRSF10D were significantly higher in older patients at diagnosis (p&amp;lt;0.01), while higher levels of promoter methylation of DNAJC15, NTRK1 and PYCARD were observed in patients with MYCN amplification (p&amp;lt;0.001). In multivariate analysis, promoter hypermethylation of FOLH1, MYOD1 and THBS1 remained significant independent predictors of poorer clinical outcome, after adjusting for MYCN amplification, age at diagnosis and tumor stage (p≤0.017). Around a third of patients displayed promoter hypermethylation in 2 or more of these genes and were almost 3 times more likely to relapse or die than those who did not display promoter hypermethylation (HR=2.72; 95%C1=1.55-4.78; p=0.001). Conclusion: DNA methylation of FOLH1, MYOD1 and THBS1 appears to have independent prognostic value in children diagnosed with neuroblastoma. While the functional consequences of promoter hypermethylation in individual neuroblastoma patients remains to be fully determined, these investigations show how epigenetic profiling of tumor tissue can provide additional genetic markers for predicting treatment response in the clinical setting and has the potential to reveal important new therapeutic targets for selected patient subsets. Citation Format: Diana T. Lau, Luke B. Hesson, Murray D. Norris, Glenn Marshall, Michelle Haber, Lesley J. Ashton. Beyond GWAS: The prognostic significance of promoter DNA methylation in patients with neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Post-GWAS Horizons in Molecular Epidemiology: Digging Deeper into the Environment; 2012 Nov 11-14; Hollywood, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(11 Suppl):Abstract nr 68.

The promoter methylation of the Syk gene in nasopharyngeal carcinoma cell lines

The aim of this study was to investigate the mRNA and protein expression levels of the Syk gene as well as its promoter methylation in nasopharyngeal carcinoma (NPC) cell lines. The CNE-1 (highly differentiated), CNE-2 (poorly differentiated) and NP69 (non-cancerous human immortalized nasopharyngeal epithelial cells) cell lines were used in the present study. The MS-PCR, Q-RT-PCR and western blotting methods were used to examine the Syk gene promoter methylation levels and mRNA and protein expression in the three cell lines. The promoter methylation levels in CNE-1, CNE-2 and NP69 cells were 36%, 62% and 0, respectively. The mRNA levels in CNE-1 and CNE-2 cells were 42±3.5 and 28±2% of that in NP69, respectively; the protein levels in CNE-1 and CNE-2 cells were 36±4.5 and 16±2.5 of that in NP69, respectively; the statistical differences between groups were significant. The lower differentiation levels of the NPC cell lines correlate with lower levels of mRNA and protein expression of the Syk gene, as well as higher promoter methylation levels.

Effector CD4+ T Cell Expression Signatures and Immune-Mediated Disease Associated Genes

Genome-wide association studies (GWAS) in immune-mediated diseases have identified over 150 associated genomic loci. Many of these loci play a role in T cell responses, and regulation of T cell differentiation plays a critical role in immune-mediated diseases; however, the relationship between implicated disease loci and T cell differentiation is incompletely understood. To further address this relationship, we examined differential gene expression in naïve human CD4+ T cells, as well as in in vitro differentiated Th1, memory Th17-negative and Th17-enriched CD4+ T cells subsets using microarray and RNASeq. We observed a marked enrichment for increased expression in memory CD4+ compared to naïve CD4+ T cells of genes contained among immune–mediated disease loci. Within memory T cells, expression of disease-associated genes was typically increased in Th17-enriched compared to Th17-negative cells. Utilizing RNASeq and promoter methylation studies, we identified a differential regulation pattern for genes solely expressed in Th17 cells (IL17A and CCL20) compared to genes expressed in both Th17 and Th1 cells (IL23R and IL12RB2), where high levels of promoter methylation are correlated to near zero RNASeq levels for IL17A and CCL20. These findings have implications for human Th17 celI plasticity and for the regulation of Th17-Th1 expression signatures. Importantly, utilizing RNASeq we found an abundant isoform of IL23R terminating before the transmembrane domain that was enriched in Th17 cells. In addition to molecular resolution, we find that RNASeq provides significantly improved power to define differential gene expression and identify alternative gene variants relative to microarray analysis. The comprehensive integration of differential gene expression between cell subsets with disease-association signals, and functional pathways provides insight into disease pathogenesis.

Abstract 4095: Stimulation of blood based biomarker release in primary tumor models of the head and neck

Abstract Approximately 50,000 head and neck cancers (HNC) occur in the United States per year and are responsible for 11,000 deaths. Identification of early detection tumor biomarkers in bodily fluids has been the goal of many studies in recent years, however none have gained wide clinical acceptance. New approaches are needed to improve detection of biomarkers with higher sensitivity and specificity for HNC. Epigenetic changes, such as DNA methylation, are hallmarks of cancer and are potential biomarker candidates. Promoter methylation of DCC, KIF1A and EDNRB were shown by our group to be cancer specific in HNC. Proof of principal of techniques enhancing detection sensitivity in an animal model may allow us, in the future, to translate our results to human clinical trials. We aim to study methods to enhance the sensitivity of blood based biomarker detection in a head and neck tumorgraft model. The model proposed is based on acquisition of human HNC tumor tissue and implantation into immunodeficient mice. We exposed these mice to various stimulations that consist of low dose treatments, which would not be expected to cause much cell death or damage, nor considerable adverse effects. They comprise Radiation (1Gy), Cisplatin (2.5μg/g), Taxol (25μg/g), Cetuximab (15μg/g), Erlotinib (10μg/g). Every group consisted of 7 mice implanted with the same tumor that was grown until the same size: one mouse for each treatment and two control mice. Blood from a mouse without tumor was first analyzed to confirm that our detection was restricted to human DNA sequences. Thus far we have analyzed 5 tumors (5 groups). Following the exposures, promoter methylation levels of DCC, KIF1A and EDNRB were evaluated by quantitative methylation specific PCR (QMSP) in the tumor as well as in the serum. Although, high levels of methylation were observed in the tumors, we were not able to detect hypermethylation in the bloodstream of non-stimulated animals. In one of our groups, the mouse stimulated with Taxol showed sufficient biomarker release into the bloodstream to provide signal amplification of all 3 genes analyzed, under very stringent conditions of qPCR analysis. The corresponding tumor showed high levels of promoter methylation of DCC, KIF1A and EDNRB, however these were undetectable in the non-stimulated group, or those mice stimulated with any condition other than Taxol. We did not observe any changes in any of the other four groups analyzed; no epigenetic alteration was detectable in any serum samples, although observed in the tumor tissue. These preliminary data suggest that low dose of Taxol was able to increase the concentration of biomarkers circulating in the blood. Clinical applications of biomarker release include monitoring patients that are at high risk for disease recurrence, and detecting the presence of cancer cells before clinical symptoms appear. We will now expand our study to explore other experimental variables that facilitate biomarker release. 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 4095. doi:1538-7445.AM2012-4095

Abstract 1425: Prognostic significance of promoter DNA methylation in patients with neuroblastoma

Abstract Background: Neuroblastoma is a genetically heterogenic tumor diagnosed in childhood which exhibits broad clinical diversity ranging from rapid disease progression to spontaneous regression. One of the most prominent prognostic markers of this disease is the amplification of the MYCN oncogene, which occurs in approximately 25% of patients. However, additional molecular markers are needed for patients lacking MYCN amplification to enable better stratification of patient risk groups. DNA methylation is a major epigenetic mechanism for gene silencing in a wide range of human cancers, including neuroblastoma. Recent studies have suggested that the methylation status of specific gene promoters may be useful for clinically discriminating subgroups of children diagnosed with neuroblastoma. To date, these investigations have assessed DNA methylation using variable techniques; focused on small numbers of candidate genes and; examined few primary tumor samples. The aims of our study were to characterize the clinical significance of promoter methylation in a large cohort of primary neuroblastoma tumors and investigate the association between DNA methylation and clinical outcome. Methods: A customized Illumina GoldenGate methylation assay was used to assess methylation status of 96 CpG sites within 48 candidate genes in primary neuroblastoma tumors obtained from 131 children diagnosed in Australia between 1985 and 2000. Candidate genes were selected on the basis of previous reports of altered DNA methylation in embryonal cancers. Levels of DNA methylation were quantified and confirmed in a subset of 48 patient samples using combined bisulphite restriction analysis (CoBRA). A Cox proportional hazards model was used to investigate the association between promoter hypermethylation and the risk of relapse or death within 5 years of diagnosis, while adjusting for known prognostic cofactors such as MYCN amplification, age and stage at diagnosis. Results: The level of promoter methylation of DNAJC15, NTRK1 and TNFRSF10D were significantly higher in older patients at diagnosis (p&amp;lt;0.01), while higher levels of promoter methylation of DNAJC15, NTRK1 and PYCARD were observed in patients with MYCN amplification (p&amp;lt;0.001). In multivariate analysis, promoter hypermethylation of FOLH1, MTRR, MYOD1 and THBS1 remained significant independent predictors of poorer clinical outcome, after adjusting for MYCN amplification, age at diagnosis, and tumor stage (p≤0.009). Moreover, over 40% of patients displayed promoter hypermethylation in 2 or more of these genes and were 3 times more likely to relapse or die than those who did not display promoter hypermethylation (HR=3.08; 95%C1=1.71-5.54; p&amp;lt;0.001). Conclusion: DNA methylation of FOLH1, MTRR, MYOD1 and THBS1 appears to have independent prognostic value in children diagnosed with neuroblastoma and highlights important new therapeutic targets for selected patient subsets. 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 1425. doi:1538-7445.AM2012-1425

Abstract LB-183: Association of aberrant promoter methylation of novel genes with clinicalpathology and prognosis of urothelial carcinoma

Abstract DNA methylation profiles are one of valuable biomarkers to molecularly define cancer subtypes that may serve as better indicators for disease progression, treatment and prognosis. In the present study, we investigated the aberrant promoter methylation of 5 tumor-related genes, CCND1, CHFR, FAM51A1, LGALS4, and RBBP7, which were selected from pooled DNA methylation profiles of urothelial carcinoma (UC) with various clinicopathological parameters. DNA was extracted from 90 patients with confirmed UC and 10 adjacent non-tumor controls. Promoter aberrant methylation of these 5 genes was determined by quantitative methylation-specific PCR (qMSP). Our results showed that methylation levels were higher in UC as compared with controls in promoter of CHFR, FAM51A1, LGALS4, and RBBP7 (6.24 vs. 4.27; 0.66 vs. 0.01; 7.47 vs. 2.14; 0.45 vs. 0.01, respectively). Among UC patients, the levels of promoter methylation of CCND1, CHFR and LGALS4 were significantly associated with tumor stages (p&amp;lt; 0.01), whereas that of FAM51A1 was inversely associated with tumor stages (p=0.012). Tumors with poor differentiation showed higher levels of promoter methylation on CCND1, CHFR and LGALS4 genes than tumors with moderate differentiation (p&amp;lt;0.05). UC patients with highly methylated CCND1, CHFR, LGALS4 or RBBP7 tended to have a poor survival than patients with low levels of methylated gene (p&amp;lt;0.02). This is the first report to show the association of promoter methylation of these 5 genes with UC tissues. We anticipate that the methylation status of these novel marker genes may have great clinical implications for further classifying UC patients. 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 LB-183. doi:10.1158/1538-7445.AM2011-LB-183

CpG methylation attenuates Sp1 and Sp3 binding to the human extracellular superoxide dismutase promoter and regulates its cell-specific expression

Extracellular superoxide dismutase (EC-SOD) plays an important role in maintaining normal redox homeostasis in the lung. It is expressed at very high levels in pulmonary fibroblasts, alveolar type II epithelial cells, and smooth muscle cells. The molecular mechanisms governing this cell-specific expression of EC-SOD are mostly unknown. In our previous studies we showed that EC-SOD cell-specific expression was not attributable to differential transcriptional regulation, suggesting that other, possibly epigenetic, mechanisms are involved in regulation of its expression. In this paper, we show high levels of promoter methylation in A549 cells and correspondingly low levels of methylation in MRC5 cells. Inhibition of DNA methyltransferase activity by 5-azacytidine in A549 cells reactivated EC-SOD transcription (2.75+/-0.16-fold, P<0.001), demonstrating the importance of methylation in the repression of EC-SOD expression. Furthermore, methylation of cytosines in the promoter markedly decreased Sp1/Sp3-driven promoter activity to 30.09+/-2.85% (P<0.001) compared to unmethylated promoter. This attenuation of transcription of the promoter/reporter construct was, at least in part, attributable to the binding of the methyl-binding protein MeCP2 in the insect cells. However, no binding of MeCP2 or MBD2 protein to the EC-SOD promoter was detected in mammalian cells in vivo. We also found marked differences in the chromatin organization of the EC-SOD promoter between these two cell lines, further supporting the important role epigenetic modifications play in the regulation of EC-SOD expression.

Methylation in p14ARF is Frequently Observed in Colorectal Cancer with Low-level Microsatellite Instability

Colorectal cancer (CRC) can be classified as high-level microsatellite instability (MSI-H), low-level MSI (MSI-L) and microsatellite stable (MSS) depending on levels of MSI. MSI-H CRC relies on a distinct molecular pathway due to the mismatch repair (MMR) deficiency and shows methylation in multiple gene promoters. The genetic pathway leading to MSI-L is unknown, although higher levels of promoter methylation are observed in this group compared with MSS CRCs. This study explored how promoter methylation affects MSI phenotype, by analysing the methylation status of eight CRC-related promoters, MSI phenotype and KRAS/BRAF mutations in a series of 234 CRCs. Promoter methylation of p14(ARF) was significantly related to MSI-L CRC with KRAS mutation. The MSI-H phenotype was related to methylation of MLH1 as expected, while the MSS phenotype was related to methylation of p16(INK4a) and O(6)-methylguanine-DNA methyltransferase, although this was not statistically significant. Thus, promoter methylation of p14(ARF) could be a significant alteration leading to CRC with MSI-L.