Gene Promoter CpG Island Research Articles

Occurrence of HHIP gene CpG island methylation in gastric cancer.

The present study aimed to observe the methylation status of the CpG islands at the human hedgehog interacting protein (HHIP) gene in gastric cancer tissues, peritumoral tissues and the AGS cell line, to analyze the association between the methylation status of the CpG islands and the tumorigenesis of gastric cancer. The HHIP mRNA expression in 60 human gastric carcinnoma tissues, peritumoral tissues and the gastric carcinoma AGS cell line were detected by reverse transcription polymerase chain reaction (RT-PCR). The HHIP methylation status of the promoter region in the gastric carcinnoma tissues and peritumoral tissues was detected by methylation-specific PCR (MSP). Prior to and following treatment with methyl transferase inhibitor 5-aza-2′-deoxycitydine (5-aza-dc), the HHIP mRNA expression level, the methylation status of the promoter region and the methylation site loci on the CpG islands in the AGS cells were detected by RT-PCR, MSP and bisulfite sequencing PCR (BSP), respectively. The correlation between the methylation status of the CpG islands at the HHIP promoter region and the HHIP mRNA expression level were analyzed. It was found that the expression level of the HHIP mRNA in the gastric carcinoma tissues was significantly lower than that in the adjacent tissues (0.82±0.38 vs. 1.60±0.26, respectively; P<0.001). No significant correlations were observed between the expression of HHIP mRNA and age, gender, tumor-node-metastasis stage, differentiation degree and presence of lymph node metastasis (P>0.05). The degree of methylation of the HHIP gene promotor in the peritumoral tissues (17.7±3.59%) was significantly lower than that in the gastric cancer tissues (62.9±6.14%) and in the AGS cells (99.7±0.67%) (P<0.05). Compared with prior to 5-aza-dc intervention, the HHIP mRNA expression level in the AGS cells was significantly increased subsequent to intervention (0.21±0.12 vs. 4.68±0.22; P<0.01), while the degree of methylation in the AGS cells was significantly decreased (90.2±0.67 vs. 10.1±0.21%; P<0.01), and the methylation sites in CpG islands were significantly reduced. The degree of HHIP methylation showed a negative correlation with the level of mRNA expression (r=−0.693; P<0.01). It can be hypothesized that a high degree of methylation of the HHIP gene promoter CpG islands in gastric cancer tissues and cells causes a decrease in HHIP mRNA expression, which may be involved in the carcinogenesis of gastric cancer.

MP66-10 IDENTIFICATION OF DIFFERENTIALLY AFFECTED PROCESSES, PATHWAYS, CELLULAR DISTRIBUTION, AND PROTEIN-PROTEIN INTERACTIONS AMONGST SEMINAL PLASMA PROTEINS FROM MEN WITH OXIDATIVE STRESS

Following the identification of H4K12ac binding promoters in sperm of healthy donors and subfertile patients, in the present study, we aimed to investigate whether loss of H4K12ac is associated with altered DNA methylation in subfertile patients. METHODS: Chromatin immunoprecipitation with monoclonal antibody against H4K12ac on ejaculates from 20 subfertile patients with impaired chromatin condensation. Ejaculates from 7 healthy donors served as control. H4K12ac immunoprecipitates were analyzed by hybridization on HG18 promoter array (NimbleGen). Subsequently, DNA methylation of 10 developmentally important gene promoters has been analysed applying pyrosequencing. RESULTS: More than 500 H4K12ac-binding gene promoters have been identified in healthy donors, whereas only 149 gene promoters were associated with H4K12ac in subfertile patients. Loss of H4K12ac-binding could be demonstrated for TRIP13, AFF4, AXIN1, EP300, LRP5, RUVBL1, USP9X, NCOA6, POU2F1 and NSD1. The latter is a methyltransferase, mutation of which causes Sotos syndrome and incidence of which is slightly increased in children conceived with intracytoplasmic sperm injection. Pyrosequencing revealed hypomethylation (5-10%) within CpG islands of H4K12ac-binding gene promoters in both healthy donors and subfertile patients. For NSD1, a significant decrease of DNA methylation in all of the 12 CpG islands could be observed (p1⁄40.004). CONCLUSIONS: Data demonstrate that accessibility of histone binding is not limited by DNA methylation in normozoospermic men. Loss of H4K12ac in selected developmentally important gene promoters in subfertile patients was not accompanied by a change in the methylation status except for NSD1. We therefore suggest that aberrant histone acetylation within developmentally important gene promoters in subfertile men e and not DNA methylation may reflect insufficient chromatin compaction and aberrant transfer of epigenetic marks to the oocyte.

Epigenetics of human myometrium: DNA methylation of genes encoding contraction-associated proteins in term and preterm labor.

Preterm birth involves the interaction of societal and environmental factors potentially modulating the length of gestation via the epigenome. An established form of epigenetic regulation is DNA methylation where promoter hypermethylation is associated with gene repression. We hypothesized we would find differences in DNA methylation in the myometrium of women with preterm labor of different phenotypes versus normal term labor. Myometrial tissue was obtained at cesarean section at term with or without labor, preterm without labor, idiopathic preterm labor, and twin gestations with labor. Genomic DNA was isolated, and samples in each group were combined and analyzed on a NimbleGen 2.1M human DNA methylation array. Differences in methylation from -8 to +3 kb of transcription start sites of 22 contraction-associated genes were determined. Cytosine methylation was not present in CpG islands of any gene but was present outside of CpG islands in shores and shelves in 19 genes. No differential methylation was found across the tissue groups for six genes (PTGES3L, PTGER2, PTGER4, PTGFRN, ESR2, and GJA1). For 13 genes, differential methylation occurred in several patterns between tissue groups. We find a correlation between hypomethylation and increased mRNA expression of PTGES/mPGES-1, indicating potential functional relevance of methylation, but no such correlation for PTGS2/COX-2, suggesting other regulatory mechanisms for PTGS2 at labor. The majority of differential DNA methylation of myometrial contraction-associated genes with different labor phenotypes occurs outside of CpG islands in gene promoters, suggesting that the entirety of DNA methylation across the genome should be considered.

Promoter CpG island methylation of RET predicts poor prognosis in stage II colorectal cancer patients

Improved prognostic stratification of patients with TNM stage II colorectal cancer (CRC) is desired, since 20–30% of high-risk stage II patients may die within five years of diagnosis. This study was conducted to investigate REarranged during Transfection (RET) gene promoter CpG island methylation as a possible prognostic marker for TNM stage II CRC patients.The utility of RET promoter CpG island methylation in tumors of stage II CRC patients as a prognostic biomarker for CRC related death was studied in three independent series (including 233, 231, and 294 TNM stage II patients, respectively) by using MSP and pyrosequencing. The prognostic value of RET promoter CpG island methylation was analyzed by using Cox regression analysis.In the first series, analyzed by MSP, CRC stage II patients (n = 233) with RET methylated tumors had a significantly worse overall survival as compared to those with unmethylated tumors (HRmultivariable = 2.51, 95%-CI: 1.42–4.43). Despite a significant prognostic effect of RET methylation in stage III patients of a second series, analyzed by MSP, the prognostic effect in stage II patients (n = 231) was not statistically significant (HRmultivariable = 1.16, 95%-CI 0.71–1.92). The third series (n = 294), analyzed by pyrosequencing, confirmed a statistically significant association between RET methylation and poor overall survival in stage II patients (HRmultivariable = 1.91, 95%-CI: 1.04–3.53). Our results show that RET promoter CpG island methylation, analyzed by two different techniques, is associated with a poor prognosis in stage II CRC in two independent series and a poor prognosis in stage III CRC in one series. RET methylation may serve as a useful and robust tool for clinical practice to identify high-risk stage II CRC patients with a poor prognosis. This merits further investigation.

Methylation of serum insulin‐like growth factor‐binding protein 7 promoter in hepatitis B virus‐associated hepatocellular carcinoma

Methylation of gene promoter CpG islands is an important early event in hepatocellular carcinoma (HCC), and detection of cell-free tumor-specific DNA methylation is becoming a useful noninvasive method for HCC. This study was aimed at determining the diagnostic value of serum insulin-like growth factor-binding protein 7 (IGFBP7) promoter methylation in hepatitis B virus-associated HCC. A total of 217 subjects, including 136 HCC patients, 46 patients with chronic hepatitis B (CHB), and 35 healthy controls (HCs), were included. The methylation status of the serum IGFBP7 gene promoter was determined using methylation-specific PCR. The frequency of serum IGFBP7 promoter methylation in HCC patients (89/136, 65%) was significantly higher than that in CHB patients (8/46, 17%; X(2) = 31.883, P < 0.001) and HCs (5/35, 14%; X(2) = 29.429, P < 0.001). Moreover, elevated IGFBP7 methylation frequency was also observed in HCC patients with vascular invasion compared with those without vascular invasion (84 versus 60%, X(2) = 6.633, P = 0.010). The sensitivities of serum IGFBP7 methylation and alpha-fetoprotein (AFP) in detecting HCC were 65 and 57%, respectively. Of note, the combination of IGFBP7 methylation and AFP showed 85% for sensitivity. These results suggest that methylation of the serum IGFBP7 gene promoter may serve as a useful noninvasive biomarker for HCC diagnosis.

Advances of CpG island methylation phenotypes in gastric cancer

CpG island methylator phenotype (CIMP) refers to a set of multiple gene promoter CpG island methylation phenotype which exists in tumor at the same time.Many studies show that CIMP is ubiquitous in gastric cancer,which is related to the pathogenesis,diagnosis,patient's condition,prognosis and curative effect of gastric cancer. Key words: Stomach neoplasms; Methylation; CIMP

OP010: The role of epigenetic alteration in oral cancer and in oral mucosa under alcohol and cigarette smoking. A study of a panel of eleven genes by methylation specific PCR (MSP)

Hyper-methylation in CpG islands of gene promoters, an epigenetic phenomenon down-regulating gene expression, is directly linked with carcinogenesis. In our laboratory we have analysed, in a series of primary OSCCs with matched normal oral mucosa under alcohol-smoking chronic exposure, the methylation status of a panel of genes, including hMLH1, CDH1, CDKN2a, MGMT, Rar-β2, SFRP-1, SFRP-2, SFRP-4, SFRP-5, Wif-1 and DKK-3 in order to define an epigenetic fingerprint for the precancer lesions and oral cancers. Thirty-seven FFPE OSCCs with relative controls of normal oral epithelium were analysed by methylation specific PCR (MSP). We have observed different frequencies of gene methylation. For all genes, the Wald Test and the logistic multiple regression were performed, in order to verify the association between methylation status of gene promoter (covariates) and presence of cancer (response variable). Interestingly also the apparently health oral mucosa shows a methylated background. The most frequently methylated gene in OSCC were WNT-related genes (SFRP2, 45%; SFRP4, 78%; SFRP5, 59%), CDKn2a (74%) and hMLH1(53%). RAR-beta-2 and MGMT promoter hyper-methylation was found in both cases only in 13.5% of OSCCs, but more frequently in healthy oral mucosa (respectively, 28.5% and 23% of studied cases). The Wald test confirmed the statistical significance for RAR-beta-2 de-methylation in cancer (p = 0.044; CI at 95%). This work, highlighting the importance of epigenetic silencing, have shown that a panel of genes may be useful in clinical practice separating normal oral epithelia from the cancerous. All these results suggest that employing of an epigenetic fingerprint may improve the current diagnostic tools, but also contribute indirectly to therapeutics for the correct clinical management of oral neoplastic and pre-neoplastic lesions.

Abstract 1770: Frequent BRCA1 promoter methylation and low 53BP1 status in sporadic triple-negative breast cancer.

Abstract Purpose: BRCA1 plays a key role in DNA repair and cellular stress response. Inhibitors of PARP show promising clinical activity in metastatic, triple-negative BRCA-mutated breast cancer. P53 binding protein 1 (53BP1) is a protein involved in DNA damage checkpoint activation and DNA repair that is involved in both non homologous end-joining and homology-mediated repair of double-strand DNA breaks. When both BRCA1 and 53BP1 are lost, homology-mediated repair appears to be restored and the sensitivity to DNA damaging agents is reduced. Loss of 53BP1 leads to resistance to cis-platinum and PARP inhibitors in BRCA1-deficient cells, suggesting that 53BP1 is a critical modulator of repair choice in BRCA1 mutant, and possibly epigenetically inactivated cells. Reduced 53BP1 expression has been reported in sporadic basal-like and triple-negative and BRCA-associated breast cancers. It thus appears important to simultaneously evaluate 53BP1 status altogether with BRCA1 mutation/methylation in order to estimate the homology-mediated functionality in breast cancer tumors. Methods: We investigated cytosolic 53BP1 levels and BRCA1 gene promoter hypermethylation profile in 151 surgical tumor samples from patients treated in our institution between January 2006 and November 2009 and evaluated their statistical association with classical breast cancer predictive and prognostic factors. DNA methylation patterns in the CpG islands of BRCA1 gene promoter were determined by a methylation specific PCR distinguishing unmethylated from methylated alleles in a given gene on the basis of sequence changes produced following bisulfite treatment of DNA. Cytosolic 53BP1 level was evaluated using a commercially available assay kit. Results: Median age was 54 years (range 29-75 years). Median 53BP1 protein level (pg/mg of cytosolic protein) was 9.5. Using this cut-off, low 53BP1 levels were significantly associated with negative ER and PR status, triple-negative profile, low cytosolic PARP activity, BRCA1 promoter hypermethylation and absence of nodal metastases. The hypermethylation status was significantly associated with UPA and PAI-1 levels, SBR grade, SBR Mitotic count score, ER, PR and HER-2 negative status, and then conversely triple negative (ER, PR and HER-2 negative) profile. Twenty nine percent of the triple negative tumors presented a hypermethylated status, comparing to 5 and 2% for HR+/HER-2- and HER-2+ tumors respectively. In the triple negative population (N=48), only 2 of the 13 tumors with BRCA1 promoter hypermethylation, presented high (&amp;gt;9.5 pg/mg) 53BP1 protein levels Conclusion: Triple negative breast cancers harboring simultaneously a high level of 53BP1 and BRCA1 promoter hypermethylation are a putative target population for development of drugs targeting DNA repair. However, this population appears to be restricted to a small subgroup of triple negative breast cancers. Citation Format: William Jacot, Simon Thezenas, Anne-Claire Laberenne, Romain Senal, Cathy Viglianti, Frédéric Bibeau, Jean-Pierre Bleuse, Gilles Romieu, Pierre-Jean Lamy. Frequent BRCA1 promoter methylation and low 53BP1 status in sporadic triple-negative breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1770. doi:10.1158/1538-7445.AM2013-1770

Abstract 2970: Acute depletion of DNA methyltransferases reveals unique and overlapping target sites in cancer.

Abstract DNA methyltransferases (DNMTs) are responsible for establishing (DNMT3A, DNMT3B, DNMT3L) and maintaining (DNMT1) DNA methylation to regulate gene transcription and promote overall genome stability. Global changes in DNA methylation, such as hypomethylation of repetitive sequences and hypermethylation of tumor supressor gene promoters, are commonly observed in various types of cancer; however, the DNMTs’ contribution to this aberrant methylation remains largely unknown. In this study, we aim to identify unique and overlapping target sites for each of the DNMTs in order to better understand aberrant DNA methylation in cancer that will ultimately permit development of new therapeutic strategies. We utilize siRNA-mediated knockdown technology to acutely deplete the mRNA for each of the DNMTs in both individual and combinatorial fashion in NCCIT embryonal carcinoma cells. Subsequently, DNA methylation is observed genome-wide in each DNA sample using two different methodologies: (1) Methyl-CpG Binding Domain (MBD)-seq, which identifies regions of the genome that are enriched for DNA methylation, and (2) Illumina Infinium HumanMethylation450 BeadChip (450K array) allowing for specific CpG site methylation status determination. Aligned MBD-seq sequences for individual DNMT knockdowns are analyzed by read coverage nomalization within 100Kbp windows and using various peak-calling algorithms (e.g. MACS, BALM). For the 450K array, DNA samples for both individual and combination knockdowns undergo bisulfite conversion and array processing; β-values for each CpG site are derived from the array signal intensities using GenomeStudio and R/Bioconductor (minfi). Genome-wide DNA methylation analysis reveals that DNMT1 depletion, both individual and in combination with knockdown of other DNMTs, results in global demethylation among all genomic features. Interestingly, a small population of genes exhibit hypermethylation in gene-promoter CpG islands for DNMT1 (individual only) depletion. In contrast, de novo methyltransferase depletion (individual and combination) shows more specific demethylation effects. In particular, hypomethylation events resulting from DNMT3B depletion (individual and combination (not 3B+3L)) occur within gene bodies and largely outside of CpG islands and flanking regions (shores, shelves). Additionally, a number of hypermethylation events occurring within the 3’UTR region of genes are observed in de novo methyltransferase depleted samples. We anticipate further analysis will reveal unique and overlapping target sites for each of the DNMTs that will lay the ground-work necessary to characterize and understand DNMT recruitment both in normal and cancerous tissues. Citation Format: Rochelle L. Tiedemann, Jeong-Hyeon Choi, Keith D. Robertson. Acute depletion of DNA methyltransferases reveals unique and overlapping target sites in cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2970. doi:10.1158/1538-7445.AM2013-2970

Methylation status of WWOX gene promoter CpG islands in epithelial ovarian cancer and its clinical significance

WW domain-containing oxidoreductase (WWOX) is a newly identified tumor suppressor gene that is associated with abnormal DNA methylation. The aim of this study was to evaluate the methylation status of CpG islands in the WWOX gene promoter region in cases of epithelial ovarian cancer and explore the correlation between the methylation status of the WWOX gene CpG islands and clinicopathological indices in patients with epithelial ovarian cancer. The methylation status of the WWOX gene CpG island was evaluated by methylation-specific polymerase chain reaction (MSP) in 48 patients with epithelial ovarian cancer, 18 patients with borderline epithelial ovarian tumors, 26 patients with epithelial benign tumors and 33 patients with normal ovarian tissues. Results showed that the rates of CpG island methylation in the WWOX gene promoter region in epithelial ovarian cancer tissues, borderline ovarian tumor tissues and benign ovarian tumor tissues were 43.75, 26.32 and 3.84%, respectively. The WWOX gene CpG islands were not methylated in normal ovarian tissues. The rate of CpG island methylation in epithelial ovarian cancer tissues was higher than that of other ovarian tissues and these differences were found to be statistically significant (P<0.01). The rate of CpG island methylation in the WWOX gene promoter region in late-stage (stage III and IV) epithelial ovarian cancer tissues was higher than that of early-stage (stage I and II) epithelial ovarian cancer tissues, and these differences were found to be statistically significant (P<0.05). In conclusion, epithelial ovarian cancer tissues showed CpG island hypermethylation in the WWOX gene promoter region, which may be an important mechanism leading to WWOX gene inactivation. Atypical methylation of WWOX gene is associated with the formation and progression of epithelial ovarian cancer, rendering it a potentially important indicator in the early diagnosis and prognosis of epithelial ovarian cancer.

Methylation State of WWOX Gene Promoter CpG Islands in Epithelial Ovarian Cancer and Its Clinical Significance

Methylation State of WWOX Gene Promoter CpG Islands in Epithelial Ovarian Cancer and Its Clinical Significance

Abstract 679: Retrospective evaluation of the BRCA1 - PARP pathway in breast cancer tissues

Abstract Purpose: Poly(adenosine diphosphate-ribose) polymerase (PARP) and BRCA1 plays a key role in DNA repair and cellular stress response. Inhibitors of PARP show promising clinical activity in metastatic, triple-negative or BRCA-mutated breast cancer. However, a comprehensive analysis of the PARP activity / BRCA1 methylation profile in non-BRCA1 mutated tumors has not yet been assessed. Methods: We investigated cytosolic PARP activity and BRCA1 methylation profile in 155 surgical tumour samples from patients treated in our institution between January 2006 and November 2009 and evaluated their statistical association with classical breast cancer predictive and prognostic factors. DNA methylation patterns in the CpG islands of BRCA1 gene promoter were determined by a methylation specific PCR distinguishing unmethylated from methylated alleles in a given gene on the basis of sequence changes produced following bisulfite treatment of DNA. Cytosolic PARP activity was evaluated using a commercially available assay kit. Results: A total of 155 BC patients (50 HER-2 positive, 57 hormone receptors positive (HR+) / HER-2 negative and 48 triple negative tumors) were considered eligible for this study. Median age was 54 years (range 29-75 years). Only one tumor was classified as SBR score I and SBR Tubule formation score 1. No tumor was SBR Nuclear pleomorphism score 1. Considering these results, SBR scores I and II and SBR Tubule formation scores 1 and 2 were grouped for statistical analyses. Mean PARP activity (U/mg of cytosolic protein) was 12.2 (standard deviation 17.02), with a median of 7.0 (range 1.0 to 114.2). The only clearly associated parameter was SBR Mitotic count score. Bisulfite treatment was successfully performed for the whole 155 specimen. Eighteen tumors presented an hypermethylation of the BRCA1 gene promoter. The hypermethylation status was significantly associated with UPA and PAI-1 levels, SBR grade, SBR Mitotic count score, ER, PR and HER-2 negative status, and then conversely triple negative (ER, PR and HER-2 negative) profile. Twenty nine percent of the triple negative tumors presented and hypermethylated status, comparing to 5 and 2% for HR+/HER-2- and HER-2+ tumors respectively. No statistical association was found between a hypermethylation status and the PARP cytosolic activity. Conclusion: Cytosolic PARP activity is equally represented in TN, HER2+ and SBR II and III HR+ breast cancers. The only parameter clearly associated with the cytosolic PARP activity was the SBR Mitotic count score. In contrast, hypermethylation of the BRCA1 gene promoter appears restricted to a subgroup of TN tumors expressing an aggressive phenotype. No statistical association was found between a hypermethylation status and the PARP cytosolic activity. These results could allow a more accurate stratification of patients without BRCA1 famillial mutation in the future PARP inhibitors clinical trials. 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 679. doi:1538-7445.AM2012-679

Oxidative Damage Targets Complexes Containing DNA Methyltransferases, SIRT1, and Polycomb Members to Promoter CpG Islands

Cancer cells simultaneously harbor global losses and gains in DNA methylation. We demonstrate that inducing cellular oxidative stress by hydrogen peroxide treatment recruits DNA methyltransferase 1 (DNMT1) to damaged chromatin. DNMT1 becomes part of a complex(es) containing DNMT3B and members of the polycomb repressive complex 4. Hydrogen peroxide treatment causes relocalization of these proteins from non-GC-rich to GC-rich areas. Key components are similarly enriched at gene promoters in an invivo colitis model. Although high-expression genes enriched for members of the complex have histone mark and nascent transcription changes, CpG island-containing low-expression genes gain promoter DNA methylation. Thus, oxidative damage induces formation and relocalization of a silencing complex that may explain cancer-specific aberrant DNA methylation and transcriptional silencing.

Systematic CpG islands methylation profiling of genes in the wnt pathway in epithelial ovarian cancer identifies biomarkers of progression-free survival.

Wnt pathways control key biological processes that potentially impact on tumor progression and patient survival. We aimed to evaluate DNA methylation at promoter CpG islands (CGI) of Wnt pathway genes in ovarian tumors at presentation and identify biomarkers of patient progression-free survival (PFS). Epithelial ovarian tumors (screening study n = 120, validation study n = 61), prospectively collected through a cohort study, were analyzed by differential methylation hybridization at 302 loci spanning 189 promoter CGIs at 137 genes in Wnt pathways. The association of methylation and PFS was examined by Cox proportional hazards model. DNA methylation is associated with PFS at 20 of 302 loci (P < 0.05, n = 111), with 5 loci significant at false discovery rate (FDR) less than 10%. A total of 11 of 20 loci retain significance in an independent validation cohort (n = 48, P ≤ 0.05, FDR ≤ 10%), and 7 of these loci, at FZD4, DVL1, NFATC3, ROCK1, LRP5, AXIN1, and NKD1 genes, are independent from clinical parameters (adjusted P < 0.05). Increased methylation at these loci associates with increased hazard of disease progression. A multivariate Cox model incorporates only NKD1 and DVL1, identifying two groups differing in PFS [HR = 2.09; 95% CI (1.39-3.15); permutation test P < 0.005]. Methylation at DVL1 and NFATC3 show significant association with response. Consistent with their epigenetic regulation, reduced expression of FZD4, DVL1, and ROCK1 is an indicator of early-disease relapse in an independent ovarian tumor cohort (n = 311, adjusted P < 0.05). The data highlight the importance of epigenetic regulation of multiple promoter CGIs of Wnt pathway genes in ovarian cancer and identify methylation at NKD1 and DVL1 as independent predictors of PFS.

Abstract 63: Methylation status of histone H3 Lysine 4 in normal melanocytes is a major determinant of DNA hypermethylation in melanoma

Abstract DNA hypermethylation in gene promoter CpG islands plays a crucial role in tumorigenesis. The determinants of the switch from unmethylated in normal to methylated in cancer remain poorly understood. It has been proposed that a bivalent chromatin domain (histone H3 Lysine 4 trimethylation (H3K4me3) and H3K27me3) in ES cells predisposes to aberrant DNA methylation, but this only accounts for a small fraction of the actual DNA hypermethylation. Here, we asked whether H3K4me3 alone affects DNA methylation changes in the tumorigenic process. To answer this question, we directly compared the epigenomes of melanoma cell lines LOXIMVI and SK-MEL-28 to normal epidermal melanocytes isolated from adults (HEMa) and neonates (HEMn). Briefly, we applied Digital Restriction Enzyme Analysis of Methylation (DREAM) to quantitatively map DNA methylation on a genome-wide scale, and ChIP-seq analysis to map the H3K4me3 pattern. Among the 5000∼5500 genes analyzed, promoter CpG island methylation (&amp;gt;50%) was seen in 1% in both HEMa and HEMn, compared to 17% and 6% in LOXIMVI and SK-MEL-28, respectively. H3K4me3 peaks were present in 67% and 75% of the genes in HEMa and HEMn, compared to 58% and 41% in LOXIMVI and SK-MEL-28. Defining a DNA methylation switch of a gene as going from below 20% methylation in normal to over 50% methylation in cancer cells, we found that the presence of H3K4me3 in normal cells was highly protective against such a switch when we compare the melanoma cell lines to normal cells. Taking the changes from HEMa to LOXIMVI as an example, 30% of the 1892 H3K4me3 negative genes in HEMa gained methylation in LOXIMVI, compared to only 8% of the 3603 H3K4me3-enriched genes (P&amp;lt;0.01). Strikingly, among these H3K4me3-enriched genes that gain DNA methylation, 87% lost H3K4me3 marks. Conversely, H3K4me3-enriched genes who lost the mark in cancer had higher chance of gaining DNA methylation than those who maintained it (72% vs. 0.4%). Thus, genes without H3K4me3 in normal cells are more likely to gain methylation in melanoma than genes with H3K4me3. To characterize further the relationship between normal and neoplastic DNA methylation, we took advantage of the highly quantitative nature of DREAM and divided genes into four groups with different DNA methylation levels in HEMa: 0-1%, 1-5%, 5-10% and 10-20%. The DNA methylation switch (to &amp;gt;50%) was seen in 13%, 43%, 40% and 50% of each of these groups. Consistent with the previous findings, in the normal cells 70% of the 0-1% methylated genes were H3K4me3 enriched, compared to 41%, 31%, and 40% of the other three groups, respectively. Taken together, we conclude that H3K4me3-enriched genes are relatively protected from DNA methylation. The presence of H3K4me3 is associated with lower levels of DNA methylation in normal cells and less chance of gaining methylation in cancer. Thus, the chromatin status in normal cells is a major determinant of aberrant DNA methylation in cancer. 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 63. doi:10.1158/1538-7445.AM2011-63

The expression of CDH1 gene methylation in patients with esophagus and stomach double primary carcinoma and its significance

Objective To study the changes of CDH1 gene promoter CpG island methylation and its clinical significance in patients with esophagus and stomach double primary carcinoma(ESDC).Methods The expression of CDH1 gene methylation in cancerous tissues and adjacent cancerous tissues in 18 cases of ESDC were detected using methylation-specific PCR method. Results Eighteen patients were endoscopically diagnosed as ESDC between Jan. 2007 and Sep. 2009 in the 4th Hospital Affiliated to Hebei Medical University. The positive methylation of CDH1 gene in tissues of esophageal squamous cell carcinoma (ESCC)and adjacent cancer were 66.7% and 33. 3%, respectively, with significant difference (χ2= 4. 167, P = 0. 031). Whereas the positive methylation of CDH1 gene in tissues of gastric carcinoma (GA) and adjacent cancer were 77.8% and 44.4%, respectively, without statistical difference (χ2=1.786, P= 0. 180). There was no significant difference (P=0. 500) in positive rate of CDH1 gene methylation between ESCC tissues and GA tissues in same individual with ESDC. For 18 patients with ESDC, consistent change of CDH1 methylation in tissues of two kinds of cancers was found in 16 patients with a total agreement of 88.9 % (positive agreement of 66.7 % and negative agreement of 22. 2%). Statistical analysis showed a significant correlation between two groups (P = 0. 005). Conclusion In patients with ESDC, there is a high consistency of CDH1 methylation change, between ESCC and GA,which suggests that two kinds of cancer may have similar risk factors and molecular mechanisms. Key words: Stomach neoplasms; Esophasus neoplas double primary carcinoma; Esophageal squmaous cell carcinoma; CDH1; Methylatlion

Genomic and epigenetic profiles of gastric cancer: Potential diagnostic and therapeutic applications

Knowledge about the molecular profile of tumor tissues is crucial to effectively target cancer cells, because cancer is a genetic disease that involves multiple genetic and epigenetic alterations. Prominent aberrations include gene mutation, amplification, loss or deletion, as well as epigenetic alterations of the promoter DNA CpG islands. All of these aberrations can lead to dynamic changes in cancer cells, as demonstrated using resected tumor samples. There are two distinct pathological types of gastric cancer: the diffuse type and the intestinal type of gastric cancer. Diffuse type gastric cancer harbors aberrations in the FGFR2/ErbB3/PI3 kinase pathway, while intestinal type gastric cancer has an activated ErbB2 oncogenic pathway. On the other hand, the prometastatic oncogene PRL-3 is commonly activated in both types of advanced gastric cancer, and might represent a relevant therapeutic target for gastric cancer with lymph node metastasis or peritoneal dissemination. Numerous tumor suppressor genes can inhibit such oncogenic pathways, and DNA methylation in CpG islands of gene promoters is frequently found to suppress the expression of such genes in gastric cancer. Helicobacter pylori infection in normal gastric mucosa may cause p53 mutations through activation of activation-induced cytidine deaminase (AID) and/or promoter DNA methylation of E-cadherin, an initiator of gastric cancer, and such abnormalities are found even in the precancerous stage of gastric carcinogenesis. In addition, it has been demonstrated that there are highly relevant methylation genes involved in cancer (HRMGs) that exhibit very frequent cancer-specific methylation in gastric cancer. Such genes are potential targets for cancer treatment, and might also serve as biomarkers of gastric cancer for either the diagnosis or for determining the prognosis or the response to treatment.

Methylation status and overexpression of COX-2 in Tunisian patients with ductal invasive breast carcinoma

Inflammation and hormonal signalling induce the cyclooxygenase-2 (COX-2) expression in solid tumours including breast cancer, which in turn affects cell proliferation, apoptosis and metastasis. The aim of this study was to investigate the expression of COX-2 and its association with clinical parameters, patient's survival, hormones receptors (oestrogen, progesterone), ERBB2 and TP53 expression in 83 cases of infiltrating ductal breast carcinomas. Moreover, the methylation status at the CpG islands of the COX-2 gene promoter was also explored in 70 specimens. We showed that tumours exhibiting moderate to intense COX-2 immunostaining were significantly more frequent in patients over 45 years old (p = 0.027). Moreover, a high level of COX-2 expression correlated with a shorter survival time (p log-rank = 0.04) and was an independent prognostic factor (p = 0.022; HR 6.4; 95% CI = 1.3-31.4). On the other hand, hypermethylation of the COX-2 gene promoter was observed in 27% of cases and strongly associated with smaller tumours (<5 cm, p = 0.011). Furthermore, patients with methylated COX-2 pattern have a better 4-year disease-free survival (p = 0.022) as well as a prolonged overall survival (p log-rank test = 0.034). In conclusion, we showed that high COX-2 expression was associated with reduced survival and was an independent prognostic factor. However, hypermethylation of the COX-2 promoter correlated with a better overall survival in Tunisian patients with breast carcinoma.

Impact of MTHFR gene C677T polymorphism on Bcl-2 gene methylation and protein expression in colorectal cancer

Objective. To investigate the impact of MTHFR C677T polymorphism on Bcl-2 gene promoter CpG island (CGI) methylation and Bcl-2 protein expression. Material and methods. MTHFR polymorphisms of 86 sporadic colorectal cancer (CRC) patients and 100 healthy volunteers were analyzed by PCR-based restriction fragment length polymorphism, and Bcl-2 promoter CGI methylation in 86 CRC tissues and 86 paired nonneoplastic adjacent tissues was determined by methylation-specific PCR. Bcl-2 oncoprotein expression in 70 CRC tissues and paired nonneoplastic adjacent tissues was detected by immunohistochemistry. Results. The frequency of MTHFR 677 T allele and combined variant genotypes (677CT + TT) in CRC patients was significantly higher than that in healthy controls (p = 0.023 and p = 0.035, respectively), and there is a significant association between 677TT or 677(CT + TT) genotypes and CRC (OR = 2.534, p = 0.045 and OR = 1.888, p = 0.035, respectively). The frequency of methylated Bcl-2 promoter CGI in tumor tissues was significantly lower than that in nonneoplastic adjacent tissues (p = 0.014). The frequency of methylated Bcl-2 promoter CGI in CRC tissues of the individuals with CC genotype was significantly higher than that of those with CT/TT genotypes (p = 0.018), there was significant distribution difference of C and T alleles between individuals with methylated and unmethylated Bcl-2 promoter CGI in colorectal cancer tissues (p = 0.023). Bcl-2 promoter hypomethylation was significantly correlated with Bcl-2 oncoprotein expression in colorectal cancer tissues (r = 0.558, p < 0.001). Conclusion. Bcl-2 promoter is hypomethylated in colorectal cancer tissue, and there is a significant correlation between MTHFR 677 TT or CT/TT genotypes and CRC or Bcl-2 promoter CGI methylation/oncoprotein expression in CRC.

Methylation of p16 gene promoter in human pheochromocytomas and paragangliomas

Objective To elucidate gene mutation and promoter methylation changes of p16 gene in pheochromocytomas （PHEO） and paragangliomas （PGL） and to assess its relation with tumor clinical characters. Methods A total of 34 tumors （20 PHEO, 14 PG L, 15 benign, 19 malignant） were collected.Direct sequencing of p16 gene after PCR was performed to analyze genetic alterations. Hypermethylation of p16 gene promoter CpG island was analyzed by methylation specific PCR（MSP）. In addition, mRNA expression was detected by RT-PCR. Results Homozygous deletion and gene mutation were not observed in 34 PHEO and PGL. Aberrant methylation of p16 gene promoter CpG island was found in 35.3% （12 of 34 tumors, 3 PHEO, 9 PGL）. The p16 promoter hypermethylation in PGL was significantly higher than PHEO （P=0. 005）. The higher p16 promoter hypermethylation was associated with malignant behavior, tumor number, and younger age at presentation, but no statistical significance, due to the limited number of cases. The p16 mRNA expression in malignant cases was lower than in benign tumors（0.83±0.65 vs 1.12±0.81 ,P=0.278）. Conclusion p16 gene homozygous deletion and mutation were not frequent in PHEO and PGL. The promoter hypermethylation is mainly attributed to inactivation of the p16 gene. Key words: p16 gene; Methylation; Pheochromocytoma; Gene mutation; Gene expression