ESR1 Methylation Research Articles

Maternal folate supply and sex influence gene‐specific DNA methylation in the fetal gut

Epidemiological evidence supports the developmental origins of health and disease hypothesis that developmental under/over-nutrition increases adulthood disease risk. Epigenetic markings are one potential mechanism mediating these effects. Altered folate supply may influence methyl group availability for DNA methylation. We reported low folate supply in utero was associated with reduced global DNA methylation in the murine small intestine of adult offspring. We hypothesised that aberrant methylation would be observed during early development. Female C57BL/6J mice were fed diets containing 2 mg folic acid/kg or 0.4 mg folic acid/kg 4 wk before mating and during pregnancy. At 17.5 day gestation, gene methylation in fetal gut was analysed by Pyrosequencing(®) . Low folate reduced overall methylation of Slc394a by 3.4% (p=0.038) but did not affect Esr1 or Igf2 differentially methylated region (DMR) 1. There were sex-specific differences in Slc394a and Esr1 methylation (2.4% higher in females (p=0.002); 4% higher in males (p=0.0014), respectively). This is the first study reporting causal effects of maternal folate depletion on gene-specific methylation in fetal gut. These observations support reports that altered methyl donor intake during development affects DNA methylation in the offspring. The consequences of epigenetic changes for health throughout the life course remain to be investigated.

Epigenetic Control of Estrogen Receptor Expression and Tumor Suppressor Genes Is Modulated by Bioactive Food Compounds

Background: The tumor suppressor genes p15^INK4b and p16^INK4a as well as the estrogen receptor-α (ESR1) gene are abnormally methylated and expressed in colon cancer. The cancer-preventative abilities of several bioactive food components have been linked to their estrogenic and epigenetic activities. Methods: The effect of folic acid, zebularine, resveratrol, genistein and epigallocatechin-3-gallate (EGCG) on tumor cell growth, promoter methylation of ESR1, p15^INK4b and p16^INK4a and gene expression of ESR1 and ESR2 was analyzed in Caco-2 cells. Gene expression was measured using real-time PCR, and promoter CpG methylation was assessed using bisulfite conversion and methylation-specific PCR. Results: After exposure to a high concentration of folic acid (20 µmol/l), enhanced cancer cell growth and concomitant increased methylation of the ESR1 (3.6-fold), p16^INK4a and p15^INK4b promoters was observed. A lower concentration of folic acid (2 µmol/l) decreased cell growth. The phytoestrogens genistein and resveratrol enhanced expression of ESR1 (genistein 200 µmol/l: 2.1-fold; resveratrol 50 µmol/l: 6.3-fold) and ESR2 (2.6- and 3.6-fold, respectively). Genistein and resveratrol treatment increased promoter methylation of ESR1 (genistein 200 µmol/l: 2.9-fold; resveratrol 50 µmol/l: 1.4-fold). For p16^INK4a, increased methylation was found after exposure to 10 µmol/l resveratrol, but for p15^INK4b, decreased methylation was found. Both components showed growth-inhibitory activities. For EGCG, growth inhibition at 100 µmol/l and suppressed promoter methylation of tumor suppressor genes (p16^INK4a: 0.9-fold; p15^INK4b: 0.6-fold) was seen. Conclusions: Our results show that these food compounds regulate ESR and tumor suppressor gene expression by multiple mechanisms including epigenetic processes. An improved understanding of these epigenetic effects could therefore support specific dietary concepts of epigenetic cancer prevention and intervention.

Aberrant methylation in breast cancer and correlate with tumor phenotypes, prognostic factors, and patient outcome

Abstract Purpose: Posttranslational modifications are known to be altered in cancer cells, and loss of selected histone acetylation and methylation marks has recently been shown to predict patient outcome in human carcinoma. Materials: Since January 2002 to June 2005, 107 women with breast cancer and 108 control subjects were recruited. A sensitive SYBR green methylation-specific PCR quantitative technique was used to analyze the utility of circulating DNA with CpG island hypermethylation of APC, RAR-β, E-cadherin, ESR1 and 14-3-3σ gene promoter regions as breast cancer biomarkers. Sera were collected in 107 operable breast cancer patients (pts) previously surgery and in 60 of those pts after treatment. Respect controls, 34 had benign breast disease and 74 with no evidence of breast disease Results: Mean serum values of methylated ESR1 and 14-3-3σ gene promoters significantly differed between breast cancer patients and healthy controls (p = 0.0112 for ESR1 and p= 0.0047 for 14-3-3- σ). When their results were combined, it was found that hypermethylation of these two genes differentiated between breast cancer patients and healthy controls (p≤0.0001) with a sensitivity of 81% (95% confidence interval: 72—88%) and specificity of 88% (95% CI: 78—94%). Presence of methylated ESR1 in serum of breast cancer patients was associated with ER-negative phenotype (p = 0.0179); and presence of methylated 14-3-3-σ was associated with T3-4 stage (OMS) (p< 0.05) and nodal positive status (p< 0.05). We observed lower methylated ERS1 or 14-3-3-σ values after surgery, respect pretreatment levels, but without an overall statistically significant difference. With a median follow up of 6 years, we found that patients with a significant decrease of sera methylated levels of both genes after surgery had better time to progression an overall survival respect patients without this observation Conclusions: These results also suggest that this panel of genes detected in ductal lavage and blood specimens could be useful to biomarkers for early detection breast cancer. These findings cast some doubts on the utility for early cancer diagnosis of highly sensitive techniques to identify hypermethylation of specific gene promoters in DNA extracted from serum. Although numerous issues remain to be resolved, the quantitative measurement of circulating methylated DNA is a promising tool for cancer risk assessment.

DNA methylation of ESR-1 and N-33 in colorectal mucosa of patients with ulcerative colitis (UC)

Epigenetic marking such as DNA methylation influence gene transcription and chromosomal stability and may also be affected by environmental exposures. Few studies exist on alteration in DNA methylation profiles (genomic and gene specific methylation) in patients with Ulcerative Colitis (UC) and none assessing its relationship with lifestyle exposures.

DNA hypermethylation markers of poor outcome in laryngeal cancer

This study examined molecular (DNA hypermethylation), clinical, histopathological, demographical, smoking, and alcohol variables to assess diagnosis (early versus late stage) and prognosis (survival) outcomes in a retrospective primary laryngeal squamous cell carcinoma (LSCC) cohort. The study cohort of 79 primary LSCC was drawn from a multi-ethnic (37% African American), primary care patient population, diagnosed by surgical biopsies in the Henry Ford Health System from 1991 to 2004 and followed from 5 to 18 years (through 2009). Of the 41 variables, univariate risk factors of p < 0.10 were tested in multivariate models (logistic regression (diagnosis) and Cox (survival) models (p < 0.05)). Aberrant methylation of estrogen receptor 1 (ESR1; p = 0.01), race as African American (p = 0.04), and tumor necrosis (extensive; p = 0.02) were independent predictors of late stage LSCC. Independent predictors of poor survival included presence of vascular invasion (p = 0.0009), late stage disease (p = 0.03), and methylation of the hypermethylated in cancer 1 (HIC1) gene (p = 0.0002). Aberrant methylation of ESR1 and HIC1 signified independent markers of poorer outcome. In this multi-ethnic, primary LSCC cohort, race remained a predictor of late stage disease supporting disparate diagnosis outcomes for African American patients with LSCC.

Correlation of serum ESR1 methylation levels with RE status in tumor and molecular subtypes of breast cancer.

e13648 Background: To determine whether estrogen receptor (ER) (+) and ER (-) status relates to epigenetic changes in breast cancer-related genes and to correlate with molecular breast cancer subtypes. Methods: From January 2002 to June 2005, we quantified methylation levels ERS1 gene in serum of 92 pts breast cancer. A PCR quantitative technique was used to analyze levels of methylation gene. We also examined and correlationed the expression of ESR1 AND HER2+ in tumors by immunohistochemistry with molecular phenotype. Results: Median age was 58 years (32-88); 69% were postmenopausal women. Nodal involvement (N0;63%,N1;30%,N2;7%), tumor size (T1;58%, T2;35%, T3;4%, T4;4%) and grade (G1;20%, G2;37%, G3;30%). Of the cases, 37pts (40%) were Luminal A (LA), 32pts (33%) Luminal B (LB), 14pts (15%) triple-negative (TN), and 9pts (10%) HER2+. The methylated ESR1 in serum was significantly associated with ER(-) in breast tumors >80% (p=0.0179). Methylation ESR1 was preferably associated with TN (80%) and HER2+ (60%) subtype. Nevertheless unmethylation ESR1 was found more frequently in LA (71%) and LB (59%) phenotype. With a median follow up of 5 years, we found worse overall survival (OS) with more frequent ESR1 methylation gene (p>0.05), Luminal A; ESR1 Methylation OS at 5 years 81% vs. 93% when was ESR1 Unmethylation. Luminal B; ESR1 Methylation 86% SG at 5 years vs. 92% in Unmethylation ESR1. Triple negative; ESR1 Methylation SG at 5 years 75% vs. 80% in unmethylation ESR1. HER2; ESR1 Methylation SG at 5 years was 66.7% vs. 75% in unmethylation ESR1. Conclusions: Gene promoter region hypermethylation is a significant event in primary breast cancer. However, its impact on tumor progression and potential predictive implications remain relatively unknown. Our study identifies the presence of variations in global levels of methylation promoters ESR1 genes in breast cancer with different phenotype classes and shows that these differences have clinical significance. Although numerous issues remain to be resolved, quantitative measurement of circulating methylated DNA may be of significance in the assessment and search of targeted therapy resistance related to ESR1 and HER2 status by epigenetic or transcriptional cancer therapy No significant financial relationships to disclose.

Simultaneous CXCL12 and ESR1 CpG island hypermethylation correlates with poor prognosis in sporadic breast cancer

BackgroundCXCL12 is a chemokine that is constitutively expressed in many organs and tissues. CXCL12 promoter hypermethylation has been detected in primary breast tumours and contributes to their metastatic potential. It has been shown that the oestrogen receptor α (ESR1) gene can also be silenced by DNA methylation. In this study, we used methylation-specific PCR (MSP) to analyse the methylation status in two regions of the CXCL12 promoter and ESR1 in tumour cell lines and in primary breast tumour samples, and correlated our results with clinicopathological data.MethodsFirst, we analysed CXCL12 expression in breast tumour cell lines by RT-PCR. We also used 5-aza-2'-deoxycytidine (5-aza-CdR) treatment and DNA bisulphite sequencing to study the promoter methylation for a specific region of CXCL12 in breast tumour cell lines. We evaluated CXCL12 and ESR1 methylation in primary tumour samples by methylation-specific PCR (MSP). Finally, promoter hypermethylation of these genes was analysed using Fisher's exact test and correlated with clinicopathological data using the Chi square test, Kaplan-Meier survival analysis and Cox regression analysis.ResultsCXCL12 promoter hypermethylation in the first region (island 2) and second region (island 4) was correlated with lack of expression of the gene in tumour cell lines. In the primary tumours, island 2 was hypermethylated in 14.5% of the samples and island 4 was hypermethylated in 54% of the samples. The ESR1 promoter was hypermethylated in 41% of breast tumour samples. In addition, the levels of ERα protein expression diminished with increased frequency of ESR1 methylation (p < 0.0001). This study also demonstrated that CXCL12 island 4 and ESR1 methylation occur simultaneously at a high frequency (p = 0.0220).ConclusionsThis is the first study showing a simultaneous involvement of epigenetic regulation for both CXCL12 and ESR1 genes in Brazilian women. The methylation status of both genes was significantly correlated with histologically advanced disease, the presence of metastases and death. Therefore, the methylation pattern of these genes could be used as a molecular marker for the prediction of breast cancer outcome.

Hormone therapy, DNA methylation and colon cancer.

Observational epidemiological studies and randomized trials have reported a protective effect of estrogen and progestin therapy (EPT) on the risk of colorectal cancer but the findings on estrogen-alone therapy (ET) are less consistent. The mechanism by which menopausal hormones influence risk of colorectal cancer has not been well studied. To further investigate the relationship between menopausal hormones and risk of colon cancer, we conducted a population-based case-control study in Los Angeles County involving 831 women with newly diagnosed colon cancer and 755 population-based control women. Risk of colon cancer decreased significantly with increasing duration of current use of ET and EPT; the adjusted relative risk was 0.83 [95% confidence interval (95% CI) = 0.76-0.99)] per 5 years of ET use and 0.88 (95% CI = 0.78-0.99) per 5 years of EPT use. Risk of colon cancer was unrelated to past ET or EPT use. We explored if current use of menopausal hormones is associated with DNA methylation of estrogen receptor (ESR1 and ESR2), progesterone receptor and other genes in the colonic tissues of a subset of colon cancer patients (n = 280) we interviewed. Our results suggest that current menopausal hormone users compared with non-current users displayed increased DNA methylation of progesterone receptor in the 'normal' colonic tissues (P = 0.055) and increased DNA methylation of ESR1 in the 'tumorous' colonic tissues (P = 0.056). These findings on DNA methylation and hormone therapy use need confirmation in larger studies.

Epigenetics Differences between Estrogen Receptor (ERS1) and Her2/Neu Status in Breast Cancer Patients.

Abstract Background: The CpG island methylator phenotype is associated with distinct clinicophatological characteristics as Estrogen Receptor (ESR1) positive and amplification of HER2 in breast cancer.Objetive: To evaluate epigenetics differences in tumor-related genes to ERS1 and HER2/neu status in primary breast cancer.Material and Methods: We quantified methylation levels of promoter of 5 genes (ERS1, RAR- β, 14-3-3 sigma, APC, E-Cadherin) which are to confer growth adventage to cells, in 107 women with breast cancer and 108 control subjects. Real Time QMS-PCR SYBR green (methylation-specific PCR) was used to analyze the hypermethylation. Tumours were classified as phenotype basal, luminal A, Luminal B and phenotype HER2+.Results: Ours analyses revealed low or absent methylation ESR1and 14-3-3σ in healthy controls and significant differences between breast cancer patients (pts) and healthy controls in relative serum levels of methylated gene promoters ESR1 (p=0.0112) and 14-3-3s (p=0.0047). Presence of methylated ESR1 in serum of breast cancer patients was associated with ER-negative phenotype (p=0.0179). Of the available cases, 60 pts (56%) were Luminal A, 10 pts (9.3%) Luminal B, 13 pts (12%) Basal like and 9 pts (8.4%) HER2+. We observed that methylated ERS1 was preferably associated with phenotype Basal Like and worse interval progression free and survival global though p&amp;gt;0.05 and the amplification HER2+ was correlation with significant more frequent methylation gene (p&amp;lt;0.05). Thet hypermethylation of normal ERS1 and 14-3-3σ combined differentiated between breast cancer patients and healthy controls (p=0.0001) with a sensitivity of 81% (95% CI: 72–88%) and specificity of 88% (95% CI: 78–94%).Conclusions: This study identifies the presence of variations in global levels of methylation promoters genes in healthy controls and breast cancer with different phenotype classes and shows that these differences have clinical significance. These showed that frequent methylation had a strong association with molecular phenotype of breast cancer and perhaps in the future can explain therapy resistance related to RE and HER2/neu status in breast cancer patients. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1151.

DNA Hypermethylation ofESR1andPGRin Breast Cancer: Pathologic and Epidemiologic Associations

Improved understanding of the etiology of estrogen receptor-alpha (ERalpha)-negative and progesterone receptor (PR)-negative breast cancers may permit improved risk prediction. In vitro studies implicate DNA hypermethylation of the ERalpha and PR promoters in the pathogenesis of ERalpha-negative and PR-negative tumors, but results are not definitive. We evaluated 200 invasive breast cancers selected from a population-based case-control study. DNA extracted from fixed tumor tissue cores was tested using MethyLight to assess DNA methylation at four CpG islands: ESR1 promoters A and B; PGR promoters A and B; and a CpG shore, ESR1 promoter C. DNA methylation results were compared with levels of ERalpha and PR, tumor characteristics, and breast cancer risk factors. We observed mild to moderate DNA methylation levels in most tumors for ESR1 promoters A and B and PGR promoter B, and a few tumors showed mild methylation in PGR promoter A. In contrast, ESR1 promoter C showed a wide range of methylation and was weakly correlated with lower expression levels of ERalpha (beta = -0.26; P < 0.0001) and PR (beta = -0.25; P < 0.0001). The percentage of tumors with methylated PGR promoters A and B was significantly higher for tumors with low ERalpha (A, Fisher's test P = 0.0001; B, P = 0.033) and PR levels (A, P = 0.0006; B, P = 0.001). Our data suggest that the relationships between DNA methylation of ESR1 and PGR promoters and protein expression are weak and unlikely to represent a predominant mechanism of receptor silencing. In contrast to CpG islands, ESR1 promoter C showed a wider range of methylation levels and inverse associations with ERalpha and PR expression.

MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1

AbstractAberrant DNA hypermethylation contributes to myeloid leukemogenesis by silencing structurally normal genes involved in hematopoiesis. MicroRNAs (miRNAs) are noncoding RNAs that regulate gene expression by targeting protein-coding mRNAs. Recently, miRNAs have been shown to play a role as both targets and effectors in gene hypermethylation and silencing in malignant cells. In the current study, we showed that enforced expression of miR-29b in acute myeloid leukemia cells resulted in marked reduction of the expression of DNA methyltransferases DNMT1, DNMT3A, and DNMT3B at both RNA and protein levels. This in turn led to decrease in global DNA methylation and reexpression of p15INK4b and ESR1 via promoter DNA hypomethylation. Although down-regulation of DNMT3A and DNMT3B was the result of a direct interaction of miR-29b with the 3′ untranslated regions of these genes, no predicted miR-29b interaction sites were found in the DNMT1 3′ untranslated regions. Further experiments revealed that miR-29b down-regulates DNMT1 indirectly by targeting Sp1, a transactivator of the DNMT1 gene. Altogether, these data provide novel functional links between miRNAs and aberrant DNA hypermethylation in acute myeloid leukemia and suggest a potentially therapeutic use of synthetic miR-29b oligonucleotides as effective hypomethylating compounds.

Association of serum tumor-related methylated DNA with circulating tumor cells in peripheral blood of breast cancer patients.

Abstract Abstract #5017 Circulating tumor cells (CTC) and free tumor-related methylated DNA in blood have been separately associated with poorer disease outcome in breast cancer patients. However, no studies have looked into the relation between both molecular markers in breast cancer. In this study, we investigated the correlations between plasma total DNA, serum methylated DNA and CTC in blood from breast cancer patients.&amp;#x2028; We simultaneously obtained matched triplets of peripheral blood, plasma and serum samples from 4 patients with localized breast cancer (group A), 59 patients with metastatic disease under treatment (group B), 16 untreated patients with metastatic disease (group C) and 20 healthy controls. CTC levels in blood were measured with the CellSearch System (Veridex). Plasma total DNA levels were determined by a qPCR method. Sera were analyzed by methylation-specific qPCR for three methylated markers: APC, RASSF1A and ESR1.&amp;#x2028; Plasma total DNA levels in breast cancer patients were significantly increased when compared to healthy controls (P&amp;lt;0.001). Differences between total DNA levels in different patient groups also reached statistical significance (P=0.04). The largest differences were measurable between groups A and C (median of 7 vs 29 ng/ml) (P=0.02) and groups B and C (median of 12 vs 29 ng/ml) (P=0.06). The sensitivity and the specificity of the total DNA assay for predicting malignancy was respectively 72.5% and 85%. Total DNA levels correlated with CTC (r=0.418, P&amp;lt;0.001) and patient age (r=0.298, P=0.01), but not with other clinical variables. Hypermethylation of one or more genes was detected in 42 (53%) serum samples from breast cancer patients and in 3 (16%) serum samples of healthy controls (P=0.003). APC was hypermethylated in 23 (29%), RASSF1A in 28 (35%) and ESR1 in 16 (20%) breast cancer cases. CTC were detected in 19 (70%) patients with serum methylated DNA and in 8 (30%) patients without methylated DNA (P=0.03). The presence of methylated DNA markers was only associated with ER status. On univariate analysis, the detection of CTC, high levels of circulating DNA, methylation of RASSF1A and the combinations of APC or RASSF1A methylation with ESR1 methylation were significantly associated with progressive disease. Multivariate analysis indicated that only RASSF1A methylation, high total DNA levels, HER2 and PR status were significantly associated with disease status.&amp;#x2028; The number of CTC was significantly increased in patients with high levels of total DNA and in patients with at least one methylated DNA marker in serum, suggesting that CTC might be a potential source of circulating tumor-related DNA. Only RASSF1A methylation and high DNA levels were independently associated with progressive disease. The combination assay of CTC and serum methylated DNA did not enable a more informative assessment of disease status. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5017.

TGFβR2 Methylation Assessed by Quantitative-MSP in Multiple Myeloma Patients: An Independent Prognostic Marker

Introduction: Multiple myeloma (MM) is a B-cell neoplasm characterized by multiorgan dysfunction as a result of bone marrow infiltration by malignant cells and systemic damage of monoclonal circulating protein. Molecular studies have largely focused on acquired genetic aberrations in MM. The accumulation of genetic events is thought to be crucial for the malignant transformation of plasma cells. DNA methylation is associated with several changes in chromatin structure, including the regulation of histone methylation and acetylation and the recruitment of proteins to the methylated sites. This leads to the obstruction of the promoter, and subsequent gene silencing. Aberrant promoter methylation of genes has been described for several genes in MM. This epigenetic event acts as an alternative to mutations and deletions to disrupt tumor suppressor gene function.Objectives: We determined the aberrant DNA methylation status of 20 genes (AIM1, CCNA1, CCND2, CDH1, CDKN2A, CDKN2B, DCC, ESR1, GSTP1, HIC1, MGMT, MINT31, p14ARF, PTGS2, RARβ, RB1, SFN, SOCS1, TGFβR2, and THBS1) in 51 samples of MM and compared the methylation profile with clinicopathological characteristics of the patients.Methods: DNA was isolated using the TRIzol reagent (Invitrogen), from bone marrow aspirates of MM patients. The promoter methylation pattern was determined by quantitative methylation specific PCR (QMSP).Results: The QMSP analysis showed that PTGS2 (100.0%), SFN (100.0%), CDKN2B (90.2%), CDH1 (88.2%), ESR1 (72.5%), HIC1 (70.5%), CCND2 (62.7%), DCC (45.1%), and TGFβR2 (39.2%) were frequently methylated in MM at diagnosis while hypermethylation of RARβ (16.6%), MGMT (12.5%), AIM1 (12.5%), CDKN2A (8.3%), SOCS1 (8.3%), CCNA1 (8.3%), and THBS1 (4.1%) were rare events. There was no methylation of GSTP1, MINT31, p14ARF and RB1 in the samples tested. The median age of the 51 MM patients was 65 years (range, 27–80 years) and 56.8% were male. According the monoclonal component isotype, the patients were classified as IgG isotype (56.6%), IgA isotype (24.5%) and others (18.8%). The kappa light chain monoclonal protein was present in 64.7% of the patients, while the lambda protein was detected in 27.4% of the cases. Based on Durie Salmon staging system, 5.9% were IA, 3.9% were IIA, 52.9% were IIIA and 33.4% were IIIB, confirming that most of our patients were diagnosed at advanced stage disease. According to ISS system, 13.7% of cases were ISS 1, 31.4% ISS 2, 49% ISS 3. More than 86% of the cases have > 50% of monoclonal plasma cells in their bone marrow assessed by biopsies. Methylation of ESR1 was correlated positively with isotype IgA (p = 0.016), while methylation of THBS1 correlated negatively with isotype IgG (p = 0.031). The 3-year overall survival was 31.5%. The clinicopathological parameters such as Durie Salmon Stage III (p = 0.015), ISS 3 (p = 0.007) and non-transplanted cases (p = 0.019) were significantly associated with reduced overall survival. The aberrant DNA methylation analyses showed that hypermethylation of DCC and TGFβR2 were also correlated with poor survival (p = 0.0034 and p = 0.0016, respectively). The multivariate analysis showed ISS (95% CI, 1.24 – 5.86, p = 0.012) and methylated TGFβR2 (95% CI, 1.02 – 4.62, p = 0.044) strongly correlated with poor outcome.Conclusion: The current study represents the first reported quantitative evaluation of MM methylation profile and our results demonstrated that aberrant promoter methylation is a frequent event in this disease. Furthermore, our data provide evidence that TGFβR2 methylation may be useful as prognostic indicator in MM.

Epigenetic Signatures of Familial Cancer Are Characteristic of Tumor Type and Family Category

Tumor suppressor genes (TSG) may be inactivated by methylation of critical CpG sites in their promoter regions, providing targets for early detection and prevention. Although sporadic cancers, especially colorectal carcinoma (CRC), have been characterized for epigenetic changes extensively, such information in familial/hereditary cancer is limited. We studied 108 CRCs and 63 endometrial carcinomas (EC) occurring as part of hereditary nonpolyposis CRC, as separate familial site-specific entities or sporadically, for promoter methylation of 24 TSGs. Eleven genes in CRC and 6 in EC were methylated in at least 15% of tumors and together accounted for 89% and 82% of promoter methylation events in CRC and EC, respectively. Some genes (e.g., CDH13, APC, GSTP1, and TIMP3) showed frequent methylation in both cancers, whereas promoter methylation of ESR1, CHFR, and RARB was typical of CRC and that of RASSF1(A) characterized EC. Among CRCs, sets of genes with methylation characteristic of familial versus sporadic tumors appeared. A TSG methylator phenotype (methylation of at least 5 of 24 genes) occurred in 37% of CRC and 18% of EC (P = 0.013), and the presence versus absence of MLH1 methylation divided the tumors into high versus low methylation groups. In conclusion, inactivation of TSGs by promoter methylation followed patterns characteristic of tumor type (CRC versus EC) and family category and was strongly influenced by MLH1 promoter methylation status in all categories. Paired normal tissues or blood displayed negligible methylation arguing against a constitutional methylation abnormality in familial cases.

Profiling CpG island field methylation in both morphologically normal and neoplastic human colonic mucosa

Aberrant CpG island (CGI) methylation occurs early in colorectal neoplasia. Quantitative methylation-specific PCR profiling applied to biopsies was used to quantify low levels of CGI methylation of 18 genes in the morphologically normal colonic mucosa of neoplasia-free subjects, adenomatous polyp patients, cancer patients and their tumours. Multivariate statistical analyses distinguished tumour from mucosa with a sensitivity of 78.9% and a specificity of 100% (P=3 × 10−7). In morphologically normal mucosa, age-dependent CGI methylation was observed for APC, AXIN2, DKK1, HPP1, N33, p16, SFRP1, SFRP2 and SFRP4 genes, and significant differences in CGI methylation levels were detected between groups. Multinomial logistic regression models based on the CGI methylation profiles from normal mucosa correctly identified 78.9% of cancer patients and 87.9% of non-cancer (neoplasia-free+polyp) patients (P=4.93 × 10−7) using APC, HPP1, p16, SFRP4, WIF1 and ESR1 methylation as the most informative variables. Similarly, CGI methylation of SFRP4, SFRP5 and WIF1 correctly identified 61.5% of polyp patients and 78.9% of neoplasia-free subjects (P=0.0167). The apparently normal mucosal field of patients presenting with neoplasia has evidently undergone significant epigenetic modification. Methylation of the genes selected by the models may play a role in the earliest stages of the development of colorectal neoplasia.

Quantitative detection of methylated ESR1 and 14-3-3-σ gene promoters in serum as candidate biomarkers for diagnosis of breast cancer and evaluation of treatment efficacy

The aim of the present study was to investigate the association between gene hypermethylation and main clinicopathological features of breast cancer, including diagnosis and treatment response. A sensitive SYBR green methylation-specific PCR technique was used to analyze the utility of circulating DNA with CpG island hypermethylation of ESR1, APC, RARB, 14-3-3-σ, and E-cad gene promoter regions as breast cancer biomarkers. Analyses were conducted of preoperative sera from 106 women with breast cancer, 34 with benign breast disease and 74 with no evidence of breast disease and of post-treatment sera from 60 of the breast cancer patients. Mean serum values of methylated ESR1 and 14-3-3-σ gene promoters significantly differed between breast cancer patients and healthy controls (P = 0.0112 for ESR1 and P = 0.0047 for 14-3-3-σ). When their results were combined, it was found that hypermethylation of these two genes differentiated between breast cancer patients and healthy controls (P < 0.0001) with a sensitivity of 81% (95% confidence interval: 72-88%) and specificity of 88% (95% CI: 78-94%). Presence of methylated ESR1 in serum of breast cancer patients was associated with the ER negative phenotype (P = 0.0179). Serum hypermethylation at ESR1 and 14-3-3-σ loci was observed in cancer patients, in situ carcinoma and benign breast disease. No significant differences in methylated ERS1 or 14-3-3-σ values were observed between pre-surgery and post-treatment measurements. Preliminary clinical applications of this approach have revealed several shortcomings, including a frequent presence of methylated 14-3-3-σ in sera from women with breast benign disease. These findings cast some doubts on the utility for early cancer diagnosis of highly sensitive techniques to identify hypermethylation of specific gene promoters in DNA extracted from serum. Although numerous issues remain to be resolved, the quantitative measurement of circulating methylated DNA remains a promising tool for cancer risk assessment.

Concurrent methylation of promoters from tumor associated genes predicts outcome in acute myeloid leukemia

By assessment of the methylation status of 25 candidate tumor suppressor genes (TSGs) in 119 acute myeloid leukemia (AML) patients and 5 controls, we aimed to determine whether simultaneous methylation of multiple TSGs exerts prognostic impact. Methylation-specific multiplex ligation probe amplification (MS-MLPA) revealed methylation of at least one TSG in 59/119 patients, while no methylation was found in controls. Methylation of different TSGs within patients was substantially correlated (intra-class correlation; 0.38). ESR1 methylation (34/119) strongly predicted concurrent methylation of other genes, OR 7.33 (95%CI 4.13–12.99). A Cox regression model that included the three most frequently methylated TSGs ESR1, CDKN2B/p15 and IGSF4, showed ESR1 to have opposite effects on overall survival (OS) compared with the other two, HR 0.22 (95% CI 0.09–0.53) and HR 1.66 (95% CI 0.73–3.79), HR 1.61 (95%CI 0.66–3.93). By assessment of CDKN2B/p15 and IGSF4 methylation, patients with methylation at multiple loci can be identified. Accumulation of methylation aberrancies is much more pronounced in ESR1 methylated patients. When combined, the methylation status of ESR1, CDKN2B/p15 and IGSF4 enable identification of patient subgroups with large differences in OS (p <0.0001). This study shows that methylation profiling allows risk stratification in AML. In addition, ESR1 methylation may reflect a biological pathway that leads to hypermethylation of multiple genes, which is reflected by methylation of IGSF4 and/or CDKN2B/p15.

Methylation Status of Tumor Suppressor Genes BEX2, IGSF4, RARB and TIMP3 Is Indicative for AML with Translocations of the MLL Gene.

The methylation of CpG islands leads to the abnormal silencing of tumor suppressor genes and thus supposedly contributes to tumorigenesis. Recently, brain expressed X-linked-2 (BEX2) was described as candidate tumor suppressor gene in malignant glioma. With tissue expression array analyses, we could show that BEX2 was highly expressed in various brain-derived tissues, but not in hematopoetic cells of healthy donors. Also acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) cell lines usually did not show high levels of BEX2. However, we found BEX2 highly expressed in those AML cell lines that carried aberrations of the mixed lineage leukemia gene (MLLmu). While MLL wild-type (MLLwt) cell lines showed hypermethylation of a CpG rich area within the BEX2 promoter, the BEX2 expressing, MLLmu cell lines did not show methylation of the BEX2 promoter. Confirming that BEX2 is an epigenetically regulated gene, aza-2′deoxycytidine increased the expression of BEX2 mRNA in MLLwt cells. Hypermethylation profiles of tumor suppressor genes may be used to identify subtypes of leukemia/lymphoma. To find out whether silencing and activation patterns of BEX2 in leukemia correlate with those of other tumor suppressor genes, we performed a polymerase-chain reaction (PCR) based assay (MLPA) that allowed us to simultaneously verify methylation- and ploidy status of 24 tumor suppressor genes. Twenty-eight human leukemia cell lines were tested, fourteen AML- and ALL-derived cell lines, half of them with MLL translocations. In summary, 7/24 tumor suppressor genes (plus BEX2) appear to be promising markers allowing for the distinction between ALL and AML, and between MLLmu and MLLwt AML. All AML and ALL cell lines were affected by methylation or deletion of CKDN2B and/or ESR1. Analysis of the methylation status of DAPK1 and FHIT allowed to distinguish between ALL and AML. The promoters of both genes, DAPK1 and FHIT were methylated in the majority of ALL cell lines: 11/14 ALL vs. 1/14 AML cell lines showed methylation of DAPK1, 13/14 ALL vs. 2/14 AML showed methylation of FHIT. There was no correlation between the methylation status of tumor suppressor genes and the MLL status in ALL cell lines. In contrast, MLLmu and wt AML cell lines showed differential promoter methylation patterns of IGSF4, RARB and TIMP3. At least two of these genes were methylated in every MLLwt AML cell line, but not in MLLmu AML. Quantitative real-time PCR demonstrated that expression of BEX2 and IGSF4 was silenced in cells carrying the methylated promoters. For RARB and TIMP3, additional mechanisms besides promoter methylation appear to be involved in gene regulation. These results show that methylation status of as few as four tumor suppressor genes (BEX2, IGSF4, RARB and TIMP3) may allow conclusions about the MLLmu/wt status of AML cells. Our results raise the question whether MLLmu proteins are directly or indirectly responsible for the hypomethylation of BEX2, IGSF4, RARB and TIMP3. If this is the case, tissue-specific effectors must play important additional roles as MLLmu ALL cells do not show this phenomenon.

Sequential gene promoter methylation during HPV-induced cervical carcinogenesis

We aimed to link DNA methylation events occurring in cervical carcinomas to distinct stages of HPV-induced transformation. Methylation specific-multiplex ligation-dependent probe amplification (MS-MLPA) analysis of cervical carcinomas revealed promoter methylation of 12 out of 29 tumour suppressor genes analysed, with MGMT being most frequently methylated (92%). Subsequently, consecutive stages of HPV16/18-transfected keratinocytes (n=11), ranging from pre-immortal to anchorage-independent phenotypes, were analysed by MS-MLPA. Whereas no methylation was evident in pre-immortal cells, progression to anchorage independence was associated with an accumulation of frequent methylation events involving five genes, all of which were also methylated in cervical carcinomas. TP73 and ESR1 methylation became manifest in early immortal cells followed by RARβ and DAPK1 methylation in late immortal passages. Complementary methylation of MGMT was related to anchorage independence. Analysis of nine cervical cancer cell lines, representing the tumorigenic phenotype, revealed in addition to these five genes frequent methylation of CADM1, CDH13 and CHFR. In conclusion, eight recurrent methylation events in cervical carcinomas could be assigned to different stages of HPV-induced transformation. Hence, our in vitro model system provides a valuable tool to further functionally address the epigenetic alterations that are common in cervical carcinomas.

Aberrant DNA methyltransferase 3b activity accounts for the hypermethylator phenotype in human breast cancer cell lines

Breast cancer cell lines of the hypermethylator phenotype exhibit gene silencing related to promoter methylation of CEACAM6, CDH1, ESR1, SCNN1A, and CST6, and concurrent hypermethylation of other methylation-sensitive genes, whereas cell lines of the normal methylator phenotype express the majority of methylation-sensitive genes. To determine if aberrant DNA methyltransferase activities account for the hypermethylator phenotype in breast cancer cell lines, we conducted a DNMT analysis on representative hypermethylator cell lines, normal methylator cell lines, and the non-neoplastic MCF12A cell line. Hypermethylator cell lines exhibit increases in both total DNMT activity and individual DNMT protein levels compared to normal methylator cells. For example, hypermethylator MDA-MB-453 cells contain 5.4-fold more total DNMT activity and two-fold more DNMT3b protein compared to MCF12A cells, but DNMT1 and DNMT3a were unchanged. In contrast, DNMT activity and protein levels (including DNMT3b) of normal methylator cell line BT-20 were comparable to MCF12A cells. These findings strongly suggest that elevated expression of DNMT3b may account for the increased methylation capacity and aberrant hypermethylation events that characterize the hypermethylator phenotype of human breast cancer cells. Support: NIH CA78343 and Komen Foundation BCTR0100575.