SFRP2 Methylation Research Articles

DNA Methylation status of Wnt antagonist SFRP5 can predict the response to the EGFR-tyrosine kinase inhibitor therapy in non-small cell lung cancer

BackgroundIt is well known that genetic alternation of epidermal growth factor receptor (EGFR) plays critical roles in tumorgenesis of lung cancer and can predict outcome of non-small-cell lung cancer treatment, especially the EGFR tyrosine-kinase inhibitors (EGFR-TKIs) therapy. However, it is unclear whether epigenetic changes such as DNA methylation involve in the response to the EGFR-TKI therapy.MethodsTumor samples from 155 patients with stages IIIB to IV NSCLC who received EGFR-TKI therapy were analyzed for DNA methylation status of Wnt antagonist genes, including SFRP1, SFRP2, SFRP5, DKK3, WIF1, and APC, using methylation specific PCR (MSP) method. EGFR mutations detections were performed in the same tissues samples using Denaturing High Performance Liquid Chromatography (DHPLC).ResultsWe found that Wnt antagonists tend to methylate simultaneously. Methylation of sFRP1 and sFRP5 are reversely correlated with EGFR mutation (P = 0.005, P = 0.011). However, no correlations of methylations of other Wnt antagonist genes with EGFR mutation were found. The patients with methylated SFRP5 have a significant shorter progression free survival than those with unmethylated SFRP5 in response to EGFR-TKI treatment (P = 0.002), which is independent of EGFR genotype.ConclusionsPatients with unmethylated SFRP5 are more likely to benefit from EGFR-TKI therapy.

Effects of 5-Aza-2-deoxycytidine on DNA methylation of anti-oncogenes in non-small cell lung cancer cells

To observe the expression of SFRP1 gene methylation in non-small cell lung cancer (NSCLC), and study the effect of 5-Aza-2-deoxycytidine (5-Aza-CdR) on DNA methylation and expression of SFRP1, p16 and MGMT genes in the human lung cancer cell line SPC-A-1 cells. SP immunohistochemistry and methylation-specific PCR were used to detect the SFRP1 methylation in 60 NSCLC cases, and 21 cases of benign lung diseases were used as control group. SPC-A-1 cells were cultured and treated with 5-Aza-CdR. The promoter methylation status of SFRP1, p16 and MGMT genes were detected by methylation-specific polymerase (MSP) chain reaction, and mRNAs were detected by real-time PCR. The positive rate of SFRP1 gene methylation in NSCLC was significantly higher than that in normal lung tissue (58.3% vs. 14.3%; χ(2) = 12.118, P = 0.001). SFRP1 gene methylation was closely correlated with lymph node metastasis and degree of differentiation in NSCLC (P < 0.05). SFRP1 protein expression was correlated with clinical stage, degree of differentiation and lymph node metastasis in NSCLC (P < 0.05). The positive expression of SFRP1 protein in 30 cases of NSCLC tissue containing SFRP1 gene methylation was significantly higher than that in non-methylated NSCLC (68.6% vs. 24.0%; χ(2) = 9.613, P = 0.002). SFRP1 gene methylation was closely correlated with SFRP1 gene protein expression in NSCLC (P < 0.05). Negative expression of SFRP1 protein was correlated with the differentiation, clinical stage, and lymph node metastasis in NSCLC (all P < 0.05). Without 5-Aza-CdR treatment, the expressions of methylation of SFRP1, p16 and MGMT genes and their mRNA were low. After 5-Aza-CdR treatment at different concentrations, their expressions were significantly elevated (all P < 0.05). SFRP1 gene methylation is closely associated with carcinogenesis and development of NSCLC. 5-Aza-CdR may reverse the methylation of SFRP1, p16 and MGMT genes, and facilitate the re-expression of the anti-oncogenes.

Epigenetic dysregulation of secreted frizzled-related proteins in myeloproliferative neoplasms complements the JAK2V617F-mutation

BackgroundSecreted frizzled-related proteins (SFRPs) are antagonists of the Wnt signaling pathway, which plays a central role in stem cell maintenance and differentiation of stem cells and hematopoietic progenitors. Epigenetic downregulation of SFRPs by promoter hypermethylation has been described to be involved in the pathogenesis of hematopoietic malignancies. There is an association between aberrant Wnt signaling and the established cancer stem cell concept. In contrast to BCR-ABL1-positive chronic myeloid leukemia CML, BCR-ABL1-negative myeloproliferative neoplasms (Ph-MPN) are characterized by the frequent occurrence of an autoactivating mutation in the JAK2 tyrosine kinase (JAK2V617F) or other mutations in the JAK-STAT pathway. However, pathogenetic mechanisms of JAK2 mutated or unmutated Ph-MPN remain not completely understood. We determined the promoter methylation status of SFRP-1, -2, -4, and -5 in 57 MPN patient samples by methylation-specific polymerase chain reaction (PCR) (MSP). JAK2V617F was assessed by allele-specific PCR.ResultsAberrant methylation among primary MPN samples was 4% for SFRP-1, 25% for SFRP-2, 2% for SFRP-4, and 0% for SFRP-5. Hypermethylation of SFRP-2, which was the most frequently hypermethylated gene in our study, could not be correlated to any specific MPN subtype. However, we detected a significant correlation between SFRP-2 methylation and presence of a JAK2V617F mutation (P = 0.008). None of the 10 CML samples showed any SFRP-methylation.ConclusionsOur data indicate that epigenetic dysregulation of the Wnt signaling pathway is a common event in MPN with aberrant methylation of at least one SFRP being detected in 25% of the primary patient samples and in 30% if only accounting for Ph-MPN. A significant correlation between SFRP-2 methylation and presence of JAK2V617F in our data supports the hypothesis that epigenetic dysregulation may be a complementary mechanism to genetic aberrations. Aberrant methylation of crucial stem cell maintenance genes seems to contribute to disease pathogenesis in Ph-MPN.

Hypermethylation of Wnt antagonist gene promoters and activation of Wnt pathway in myelodysplastic marrow cells

We observed aberrant gene methylation of Wnt antagonists: sFRP1, sFRP2, sFRP4, sFRP5 and DKK1 in marrow cells of 55 MDS cases. Methylation of Wnt antagonist genes was associated with activation of the Wnt signaling pathway, consistent with the up-regulation of the Wnt downstream genes TCF1 and LEF1. Azacitidine exposure induced demethylation of Wnt-antagonist gene promoters and reduction of the non-phosphorylated β-catenin (NPBC) which is prevalent during Wnt pathway inactivation. Presence of ≥5% of bone marrow blasts was associated with methylation of sFRP1 and DKK1 and with methylation of more than two of the five Wnt antagonist genes.

Relationship between Tumor DNA Methylation Status and Patient Characteristics in African-American and European-American Women with Breast Cancer

Aberrant DNA methylation is critical for development and progression of breast cancer. We investigated the association of CpG island methylation in candidate genes and clinicopathological features in 65 African-American (AA) and European-American (EA) breast cancer patients. Quantitative methylation analysis was carried out on bisulfite modified genomic DNA and sequencing (pyrosequencing) for promoter CpG islands of p16, ESR1, RASSF1A, RARβ2, CDH13, HIN1, SFRP1 genes and the LINE1 repetitive element using matched paired non-cancerous and breast tumor specimen (32 AA and 33 EA women). Five of the genes, all known tumor suppressor genes (RASSF1A, RARβ2, CDH13, HIN1 and SFRP1), were found to be frequently hypermethylated in breast tumor tissues but not in the adjacent non-cancerous tissues. Significant differences in the CDH13 methylation status were observed by comparing DNA methylation between AA and EA patients, with more obvious CDH13 methylation differences between the two patient groups in the ER- disease and among young patients (age<50). In addition, we observed associations between CDH13, SFRP1, and RASSF1A methylation and breast cancer subtypes and between SFRP1 methylation and patient's age. Furthermore, tumors that received neoadjuvant therapy tended to have reduced RASSF1A methylation when compared with chemotherapy naïve tumors. Finally, Kaplan Meier survival analysis showed a significant association between methylation at 3 loci (RASSF1A, RARβ2 and CDH13) and reduced overall disease survival. In conclusion, the DNA methylation status of breast tumors was found to be significantly associated with clinicopathological features and race/ethnicity of the patients.

Abstract 4565: Prediction of prognosis by the combination of mutation and methylation in colorectal cancer

Abstract Colorectal cancer (CRC) is among the most common types of cancer in both men and women and is associated with high mortality, particularly at advanced stages. Markers for defining individual risk signatures in CRC patients are of great clinical value, as they may allow for targeted therapies to improve outcomes in CRC patients. Epigenetic alteration such as promoter hypermethylation of gene, as well as genetic abnormality such as gene mutation, occurs frequently during the pathogenesis of human cancers. Recent studies exhibit the increasing observations that both epigenetic and genetic changes combine to determine the phenotype of cancer. Thus, in the present study, we examined KRAS and PIK3CA mutations and the methylation status of BNIP3, SFRP1, and Dkk1, and analyzed the correlation between these molecular alterations and the clinicopathological features of CRC. We aimed to clarify whether a combination of genetic and epigenetic alterations can be used to classify CRC patients in relation to their clinicopathological features and outcomes. Tissue samples from 122 CRC patients who underwent surgical resection were examined. The methylation status of SFRP1 and Dkk1 and the sequences of exon 1 of KRAS gene and exons 9 and 20 of the PIK3CA gene were determined by methylation specific PCR and direct sequencing, respectively, using genomic DNA extracted from paraffin-embedded blocks. Correlation between these factors and clinicopathologic findings/patients survival were examined. As a result, there was a significant correlation between KRAS mutation and BNIP3 (p=0.0004) or Dkk1 (p=0.0009) methylation but not SFRP1 (p=0.114). Although SFRP1 and Dkk1 methylation showed no associations with disease specific survival (DSS) and other clinicopathological findings in CRC patients, BNIP3 was associated with poor DSS (p=0.0149). In CRC without both KRAS and PIK3CA mutation, patients with two or three methylated genes had poorer overall survival compared to those with no or one methylated genes (p=0.0017). To contrary, in the cases of at least one or more mutations, patients with two or three methylated genes tended to have better survival than patients with no methylation. Classifying CRC based on BNIP3, SFRP1, and Dkk1 methylation status and KRAS and PIK3CA mutation may be a clinically powerful predictor of patient outcome. 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 4565. doi:1538-7445.AM2012-4565

Epigenetic silencing of sFRP1 activates the canonical Wnt pathway and contributes to increased cell growth and proliferation in hepatocellular carcinoma

The Wnt pathway is a key regulator of embryonic development and stem cells, and its aberrant activation is associated with human malignancies, most notably hepatocellular carcinoma (HCC). Epigenetic deregulation of the genes encoding the secreted frizzled-related proteins (sFRPs), the Wnt signalling antagonists, has been linked with aberrant hyperactivation of the Wnt signalling in HCC cells; however, the precise underlying mechanism remains elusive. We investigated the methylation profiles of Wnt antagonists in liver samples of different stages of HCC development and liver cancer cell lines and studied the functional impact of aberrant epigenetic silencing of sFRPs on the canonical Wnt pathway and cell viability. We found that the sFRP1 gene encoding the subunit is a frequent target of aberrant DNA hypermethylation and silencing in HCC tumours, whereas other extracellular Wnt antagonists, WIF1 and Dkk3, exhibited no methylation in tumour cells, consistent with the notion that aberrant methylation events in cancer cells are non-randomly distributed among the genes and that there is a strong preference for hypermethylation of specific genes in HCC. In addition, by comparing sFRP1 methylation status in HCC tumours with normal, cirrhotic and chronic hepatitis liver tissues, we identified sFRP1 gene as a potential early marker of HCC. The restoration of sFRP1 expression in cancer cells by ectopic expression inhibited Wnt activity accompanied with destabilization of β-catenin and downregulation of c-Myc and cyclin D1, the known downstream targets of Wnt pathway. Importantly, restoring sFRP1 levels in cancer cells inhibited cell growth and induced apoptotic cell death. This study supports the critical role for sFRP1 silencing in hepatocellular carcinoma and reinforces the importance of the Wnt antagonists in preventing oncogenic stabilization of β-catenin and chronic activation of the canonical Wnt pathway, suggesting that sFRP1 may be an attractive target for early cancer detection and therapeutic intervention.

Epigenetic Silencing of WNT Inhibitory Proteins; SFRP1 and DKK3 in Acute Leukemia

Background: Over-activation of Wnt (derived from names of two genes; DrosophilaWingless and mouse Int-1) pathway is incriminated in leukemogenesis. Functionalloss of Wnt antagonists; DKK (Dikkopf) and SFRPs (secreted frizzled-relatedprotein), can contribute to Wnt hyper-activation. Silencing of Wnt antagonists byhypermethylation is reported in human malignancies as well as in hematopoieticmalignancies. Our aim was to estimate the frequency and the possible impact ofhypermethylation of the SFRP1 and DKK3 in acute leukemia. Methods: We evaluatedSFRP1 and DKK3 methylation status using methylation specific polymerase chainreaction (MS-PCR) in 50 acute myeloid leukemia (AML) and 30 B-acutelymphoblastic leukemia (B-ALL) patients and 20 age and sex matched controls.Results: The frequency of methylation in B-ALL patients was 40% for SFRP1, 40%for DKK-3, in AML patients; the frequency was 44% for SFRP1, 36% for DKK-3. Allthe control subjects had no aberrant methylation in either SFRP1 or DKK-3. B-ALLand AML groups showed no statistical significant difference in the frequency ofSFRP1 or DKK-3 methylation. B-ALL patients with M-SFRP1 had a significantlyhigher mean platelets count and a lower mean age compared to B-ALL patients withUM-SFRP1, no other significant clinical or hematological difference wasencountered between patients with M-SFRP1 and UM-SFRP1 or between M-DKK3and UM-DKK3 patients in the ALL or the AML group. Different B-ALL and AMLprognostic cytogenetic groups showed nearby frequency of SFRP1 and DKK3methylation. Conclusion: SFRP1 and DKK3 methylation is frequent in acuteleukemia. Treatment with demethylating agents may reverse the overactivated Wntsignaling in patients with methylated phenotype.

Epigenetic inactivation of the canonical wnt antagonist secreted frizzled-related protein 1 in hepatocellular carcinoma cells

Secreted Frizzled-related protein 1 (sFRP1), as one of most important Wnt antagonists, is frequently silenced by promoter hypermethylation in many types of tumor, including hepatocellular carcinoma (HCC). In this study, we aimed to investigate whether restoration of sFRP1 affected HCC metastatic behavior. sFRP1 mRNA expression and promoter methylation in HCC tissues and cell lines were examined using RT-PCR and methylation-specific PCR (MS-PCR), respectively. sFRP1 protein expression was assessed by Western Blot. We generated stable HCC cell line restoration of sFRP1 in HepG2 cells, which naturally do not express detectable sFRP1 mRNA. The effects of exogenous sFRP1 on HepG2 cell invasion were investigated using trans-well assay. Also the effects of sFRP1 re-expression on the β-catenin/T-cell factor-dependent transcription activity was measured by luciferase assay.sFRP1 promoter methylation was frequently observed in HCC tissues (60%) and cell lines (75%). All samples with sFRP1 methylation showed down-regulation of sFRP1 expression in HCC cell lines. Demethylation treatment with 5-aza-20-deoxycytidine in HCC cells restored sFRP1 expression. Restoration of sFRP1 substantially impaired the invasive potentials of HepG2 cells. Moreover, exogenous sFRP1 caused significant decrease of β-catenin/T-cell factor-dependent transcription activity.These findings demonstrate that sFRP1 silencing due to promoter hypermethylation is a major event during tumorigenesis. sFRP1 is also a negative modulator of canonical Wnt signaling, which could contribute to metastasis in HCC progression, thus providing a possible therapeutic strategy against HCC.

Secreted-frizzled related protein 1 is a transcriptional repression target of the t(8;21) fusion protein in acute myeloid leukemia

AbstractSecreted-frizzled related proteins (SFRPs) are modulators of the Wnt signaling pathway that is closely involved in normal and malignant hematopoiesis. Epigenetic deregulation of Wnt modulators leading to aberrant signaling has been reported in adult patients with acute myeloid leukemia (AML), but its occurrence in childhood patients with AML and the role of individual modulators are unclear. In this study, we examined SFRP1, SFRP2, SFRP4, and SFRP5 promoter methylation in 83 patients with AML (59 children and 24 adults) and found preferential SFRP1 methylation and mRNA down-regulation in the prognostically favorable subgroup of AML with t(8;21) translocation. Among the 4 genes, SFRP1 methylation independently predicted prolonged event-free and relapse-free survivals in childhood patients with nonacute promyelocytic leukemia with nonadverse cytogenetics. Mechanistically, we further demonstrated that RUNX1-ETO, the t(8;21) fusion product, specifically bound the SFRP1 promoter and repressed its transcription via a consensus RUNX binding site. In t(8;21)–leukemia cells, SFRP1 selectively inhibited canonical Wnt signaling and cellular proliferation that were associated with concomitant down-regulation of Wnt/β-catenin target genes, including CCND1 and MYC. Taken together, we identified SFRP1 as a transcriptional repression target of the t(8;21) fusion protein and demonstrated a novel mechanism of Wnt activation in a specific subtype of AML.

Epigenetic fingerprint in endometrial carcinogenesis: The hypothesis of a uterine field cancerization

Transcriptional silencing by CpG island hypermethylation plays a critical role in endometrial carcinogenesis. In a collection of benign, premalignant and malignant endometrial lesions, a methylation profile of a complete gene panel, such steroid receptors (ERα, PR), DNA mismatch repair (hMLH1), tumor-suppressor genes (CDKN2A/P16 and CDH1/E-CADHERIN) and WNT pathway inhibitors (SFRP1, SFRP2, SFRP4, SFRP5) was investigated in order to demonstrate their pathogenetic role in endometrial lesions. Our results indicate that gene hypermethylation may be an early event in endometrial endometrioid tumorigenesis. Particularly, ERα, PR, hMLH1, CDKN2A/P16, SFRP1, SFRP2 and SFRP5 revealed a promoter methylation status in endometrioid carcinoma, whereas SFRP4 showed demethylation in cancer. P53 immunostaining showed weak-focal protein expression level both in hyperplasic lesions and in endometrioid cancer. Non-endometrioid cancers showed very low levels of epigenetic methylations, but strong P53 protein positivity. Fisher exact test revealed a statistically significant association between hMLH1, CDKN2A/P16 and SFRP1 genes methylation and endometrioid carcinomas and between hMLH1 gene methylation and peritumoral endometrium (p < 0.05). Our data confirm that the methylation profile of the peritumoral endometrium is different from the altered molecular background of benign endometrial polyps and hyperplasias. Therefore, our findings suggest that the methylation of hMLH1, CDKN2A/P16 and SFRP1 may clearly distinguish between benign and malignant lesions. Finally, this study assessed that the use of an epigenetic fingerprint may improve the current diagnostic tools for a better clinical management of endometrial lesions.

Promoter methylation analysis of WNT/β-catenin signaling pathway regulators to detect adenocarcinoma or its precursor lesion of the cervix

Objective Cervical adenocarcinoma (AdCA) and adenocarcinoma in situ (ACIS) are frequently missed in cytology-based screening programs. Testing for high-risk human papillomavirus (hrHPV) improves their detection, but novel ACIS/AdCA specific biomarkers are needed to increase specificity for these lesions. Novel markers may be deduced from the WNT/β-catenin signaling pathway, which is aberrantly activated during cervical carcinogenesis. Methods Promoter methylation of nine WNT-antagonists (APC, AXIN2, DKK3, SFRP2, SFRP4, SFRP5, SOX17, WIF1 and WNT5A) was evaluated by methylation-specific PCR (MSP) on a small series of cervical tissue specimens, including AdCA and SCC. To estimate the diagnostic potential of the genes most frequently methylated in AdCA an extended series of ACIS, AdCA, CIN3, SCC, and normal cervical tissue specimens (n = 131) as well as 49 hrHPV-positive scrapings were analyzed by quantitative MSP (qMSP). Results The frequency of DKK3 and SFRP2 methylation was significantly higher in AdCA compared to SCC, i.e. 82% vs. 18% (p < 0.01) and 84% vs. 39% (p < 0.01), respectively, while SOX17 methylation frequency was significantly higher in SCC than AdCA, i.e. 89% vs. 62% (p < 0.05). Methylation of WIF1 was common in both AdCA (71%) and SCC (54%). Methylation frequencies ranged from 4% to 55% in precursor lesions and from 0% to 5% in normal biopsies. When tested on HPV-positive cervical scrapings, qMSP of the best ACIS/AdCA discriminator genes, i.e. DKK3 and SFRP2, detected all women with underlying ACIS/AdCA, compared to 3% of controls. Conclusions DKK3 and SFRP2 promoter methylation is highly indicative for the presence of ACIS/AdCA, thereby providing promising triage markers for HPV-positive women at risk of ACIS/AdCA.

Distinct DNA methylation epigenotypes in bladder cancer from different Chinese sub-populations and its implication in cancer detection using voided urine

BackgroundBladder cancer is the sixth most common cancer in the world and the incidence is particularly high in southwestern Taiwan. Previous studies have identified several tumor-related genes that are hypermethylated in bladder cancer; however the DNA methylation profile of bladder cancer in Taiwan is not fully understood.MethodsIn this study, we compared the DNA methylation profile of multiple tumor suppressor genes (APC, DAPK, E-cadherin, hMLH1, IRF8, p14, p15, RASSF1A, SFRP1 and SOCS-1) in bladder cancer patients from different Chinese sub-populations including Taiwan (104 cases), Hong Kong (82 cases) and China (24 cases) by MSP. Two normal human urothelium were also included as control. To investigate the diagnostic potential of using DNA methylation in non-invasive detection of bladder cancer, degree of methylation of DAPK, IRF8, p14, RASSF1A and SFRP1 was also accessed by quantitative MSP in urine samples from thirty bladder cancer patients and nineteen non-cancer controls.ResultsThere were distinct DNA methylation epigenotypes among the different sub-populations. Further, samples from Taiwan and China demonstrated a bimodal distribution suggesting that CpG island methylator phentotype (CIMP) is presented in bladder cancer. Moreover, the number of methylated genes in samples from Taiwan and Hong Kong were significantly correlated with histological grade (P < 0.01) and pathological stage (P < 0.01). Regarding the samples from Taiwan, methylation of SFRP1, IRF8, APC and RASSF1A were significantly associated with increased tumor grade, stage. Methylation of RASSF1A was associated with tumor recurrence. Patients with methylation of APC or RASSF1A were also significantly associated with shorter recurrence-free survival. For methylation detection in voided urine samples of cancer patients, the sensitivity and specificity of using any of the methylated genes (IRF8, p14 or sFRP1) by qMSP was 86.7% and 94.7%.ConclusionsOur results indicate that there are distinct methylation epigenotypes among different Chinese sub-populations. These profiles demonstrate gradual increases with cancer progression. Finally, detection of gene methylation in voided urine with these distinct DNA methylation markers is more sensitive than urine cytology.

Effect of wnt aberrant methylation and epidermal growth factor receptor pathway mutation on responsiveness of non-small cell lung cancer to gefitinib and erlotinib.

e18011 Background: Wnt or Epidermal growth factor receptor (EGFR) signaling pathways play critical roles in predicting outcome of non-small-cell lung cancer treatment. Little is known about the synchronous effect on EGFR tyrosine-kinase inhibitors (EGFR-TKIs) treatment in NSCLC. Methods: Tumor samples from 138 patients with stages IIIB to IV NSCLC taking EGFR-TKIs therapy were analyzed for EGFR mutations in exons 19 and 21 by using denaturing high-performance liquid chromatography. Wnt antagonist methylation,including sFRP1, sFRP2, sFRP5, DKK-3, WIF-1, APC, were also analyzed from these samples through methylation specific PCR method. Results: We found that EGFR mutations were related to smoking status and gender, while methylation of wnt antagonists did not have these trends. The wnt antagonists tend to methylate simultaneously. Methylation of sFRP1 and sFRP5 are reversely correlated with EGFR mutation (P=0.037 and P=0.048). Patients with EGFR mutations have a significant longer progression-free survival (PFS) than those without mutations (7.8 ms vs 2.0 ms, P=0.009), while patients with methylation of sFRP5 and WIF-1 have a significant shorter PFS than those without methylation in TKI treatment (1.2 ms Vs 5.3 ms, P=0.002; 1.1 ms Vs 4.9 ms, P=0.006). Methylation of sFRP5 is an independent factor affecting PFS in multivariate analysis. Patients with sFRP1 methylation have no a significant shorter PFS than patients without its methylation in TKI therapy. Patients with WIF-1 methylation have a significant shorter overall survival (OS) than patients without its methylation (P=0.006 by log-rank test). Conclusions: Patients without sFRP5 or WIF-1 methylation are more likely to benefit from EGFR tyrosine-kinase inhibitor (TKIs) therapy. Methylation of sFRP5 and WIF-1 could be predictive markers related to poor PFS in TKIs treatment.

Abstract 816: Anti-inflammatory effects of black raspberries in ulcerative colitis are associated with demethylation of genes in the Wnt signaling and protective modulation of toll-like receptor pathway

Abstract Ulcerative Colitis (UC) causes pervasive, chronic inflammation of the colon and is a potential precursor to colon cancer. Reduced Interleukin 10 (IL10), an anti-inflammatory cytokine, is associated with an increased risk of developing UC in rodents and humans. IL10 knockout (KO) mice develop UC spontaneously with subsequent development of colon tumors. Toll-like receptor and Wnt pathways are deregulated in UC patients and IL10 KO mice. Toll-like receptor pathway plays critical roles in the inflammatory response partly by mediating the Wnt pathway. We previously showed that 5-10% dietary freeze-dried black raspberries (BRBs) reduced chronic inflammation and tumor development in IL10 KO mice; aberrant promoter methylation of negative Wnt regulators, e.g., dkk2, dkk3, and sfrp1, were observed in colon from these animals. The goals of the current study were to determine if the anti-inflammatory effects of BRBs on IL10 KO mice are associated with modulation of the Wnt pathway through DNA demethylation, with alteration of enzymes regulating DNA methylation, and with modulation of toll-like receptor pathway. IL10 KO mice were fed control or 5% BRB diet for 4 wks. Cells from bone marrow and spleen as well as colon tissue were collected. Promoter methylation of dkk2, dkk3, and sfrp1 was quantified by Pyrosequencing. Protein expression of enzymes regulating DNA methylation, e.g., histone deacetylase 1, 2 (HDAC1, 2), methyl binding domain 2 (MBD2), and DNA methyltransferase 3B (DNMT3B), and β-catenin were assessed using quantitative immunohistochemistry. mRNA expression of genes associated with the Wnt and toll-like receptor pathways were quantified using real-time PCR based arrays, each of which contained 84 genes. Increased promoter methylation of dkk2, dkk3, and sfrp1 was observed in colon from KO mice when compared with colon from wild-type mice. The methylation levels of these genes in bone marrow and spleen were lower than those in colon. BRBs significantly decreased promoter methylation of dkk2 in colon and dkk3 in spleen. This was associated with decreased protein expression of HDAC1, HDAC2, MBD2, and DNMT3B. The expression of 84 genes in both pathways was different in colon of IL10 KO mice when compared to colon of wild-type mice. BRBs modulated 80% (67/84) and 95% (80/84) of differentially expressed genes toward normal levels of expression in the Wnt and toll-like receptor pathways, respectively. Protein expression of nuclear β-catenin was decreased by BRBs. In summary, berries are capable of reversing aberrant promoter methylation of genes in the Wnt pathway presumably through inhibition of proteins regulating DNA methylation. These changes could result in protective modulation of the Wnt pathway and/or its interaction with the toll-like receptor pathway which, in turn, reduce inflammation in ulcerative colitis. 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 816. doi:10.1158/1538-7445.AM2011-816

Expansion of CpG methylation in the SFRP2 promoter region during colorectal tumorigenesis.

Secreted frizzled-related protein 2, (SFRP2) is a Wnt inhibitor whose promoter CpGs were recently found to be methylated at high frequency in colorectal cancers (CRCs). We hypothesized that the pattern of SFRP2 methylation may differ throughout the promoter during progressive tumorigenesis. Using combined bisulfite restriction analysis (COBRA), two methylation-sensitive regions (Regions A and B) of the SFRP2 promoter were investigated in 569 specimens of colorectal tissue:222 CRCs, 103 adenomatous polyps (APs), 208 normal colonic mucosa from CRC patients (N-Cs), and 36 normal colonic mucosa from subjects with no evidence of colorectal neoplasia at colonoscopy (N-Ns). Extensive (including both Regions A and B) and partial (either Region A or B) SFRP2 methylation levels were found in 61.7% and 24.8% of CRCs, 8.7% and 37.9% of APs, 3.9% and 39.9% of N-Cs, and 0% and 30.6% of N-Ns, respectively. Extensive methylation of the SFRP2 promoter was present primarily in CRCs, while partial methylation was common in APs. Whereas APs with the KRAS mutant showed no correlation to any pattern of SFRP2 methylation, extensive methylation of the SFRP2 promoter was significantly associated with KRAS mutant CRCs (p＜.0001), suggesting that genetic alteration in the RAS-RAF pathway might precede the spread of CpG methylation through the SFRP2 promoter, which is observed in over 60% of advanced colorectal tumors.

Aberrant Gene Methylation Is a Biomarker for the Detection of Cancer Cells in Peritoneal Wash Samples from Advanced Gastric Cancer Patients

To assess whether gene methylation in peritoneal fluid (PF) is clinically feasible for determining micrometastasis to the peritoneum in gastric cancer. The gene methylation of BNIP3, CHFR, CYP1B1, MINT25, SFRP2, and RASSF2 were analyzed in 107 specimens of PF by quantitative methylation-specific polymerase chain reaction. All patients were placed into one of 3 groups: group A (n = 42), patients with depth of cancer invasion at muscularis propria (MP) or less than MP; group B (n = 45), depth of cancer invasion beyond the MP; and group C (n = 20), histologically diagnosed peritoneal metastasis or cancer cells cytologically defined in the peritoneal cavity. Patients in both groups A and B were diagnosed as having no cancer cells by peritoneal cytology and histology. The methylation status of the 6 genes was found to be significantly different among the 3 groups (group A, 0-5%; group B, 0-15%; group C, 15-45%; P < 0.01). Furthermore, the rate of positive methylation in any of the 6 genes was significantly different in each group (group A, 7%; group B, 20%; group C, 75%; P < 0.001). Three of 9 patients in group B with positive methylation in any of 6 genes experienced peritoneal recurrence. On the other hand, only 1 of 36 patients without gene methylation experienced peritoneal recurrence (P < 0.05). DNA methylation in PFs is a possible marker detecting occult neoplastic cells on the peritoneum. Methylation analysis along with a cytological examination might therefore improve the positive detection of cancer cells in PF of gastric cancer.

Hypermethylation and aberrant expression of Wnt-antagonist family genes in gastric cardia adenocarcinoma

The canonical Wnt signalling pathway plays a key role during embryogenesis and pathogenesis of various types of tumors. Recently, several studies have shown that the promoter hypermethylation of Wnt-antagonist genes, including sFRP-1, sFRP-2, sFRP-4, sFRP-5, Wif-1 and Dkk-3, have been certified to contribute to the tumorigenesis of several cancers. The aim of this study was to investigate the promoter methylation of Wnt-antagonist genes in gastric cardia adenocarcinoma (GCA) and corresponding adjacent non-cancerous tissues, and to establish the possible relationship between DNA methylation status and the pathogenesis of GCA. MSP, RT-PCR methods were applied respectively to examine the CpG methylation of the Wnt-antagonist genes and its mRNA expression in tumors and corresponding non-cancerous tissues, and immunohistochemistry method was used to determine protein expression of β-catenin(the key factor of the Wnt signalling pathway) and cyclin D1(the target gene of this pathway). The frequency of promoter methylation of sFRP-1, sFRP-2, sFRP-4, sFRP-5, Wif-1 and Dkk-3 genes in GCA tumor tissues were 78.7%(74/94), 76.6%(72/94), 70.2%(66/94), 77.1%(73/94), 61.7%(58/94) and 21.3%(20/94), respectively, which were significantly higher than those in adjacent non-cancerous tissues. Furthermore, the frequencies of silenced mRNA expression of these six genes in GCA tumor tissues were significantly higher than those in adjacent non-cancerous tissues. Methylation levels of these six genes were all correlated with loss of mRNA expression. The ectopic expression of β-catenin and cyclin D1 was significantly more frequent in GCA tumor tissues than that in adjacent non-cancerous tissues and correlated with each Wnt-antagonist genes hypermethylation status. Epigenetic silencing of Wnt-antagonist genes expression by promoter hypermethylation may play an important role in gastric cardia adenocarcinoma.

Sequential DNA methylation changes are associated with DNMT3B overexpression in colorectal neoplastic progression

Background and aimsAlthough aberrant methylation of key genes in the progression of colorectal neoplasia has been reported, no model-based analysis of the incremental changes through the intermediate adenoma stage has...

Acute myeloid leukemia is characterized by Wnt pathway inhibitor promoter hypermethylation

Nuclear localization of non-phosphorylated, active β-catenin is a measure of Wnt pathway activation and is associated with adverse outcome in patients with acute myeloid leukemia (AML). While genetic alterations of the Wnt pathway are infrequent in AML, inhibitors of this pathway are silenced by promoter methylation in other malignanices. Leukemia cell lines were examined for Wnt pathway inhibitor methylation and total β-catenin levels, and had frequent methylation of Wnt inhibitors and upregulated β-catenin by Western blot and immunofluorescence. One hundred sixty-nine AML samples were examined for methylation of Wnt inhibitor genes. Diagnostic samples from 72 patients with normal cytogenetics who received standard high-dose induction chemotherapy were evaluated for associations between methylation and event-free or overall survival. Extensive methylation of Wnt pathway inhibitor genes was observed in cell lines, and 89% of primary AML samples had at least one methylated gene: DKK1 (16%), DKK3 (8%), RUNX3 (27%), sFRP1 (34%), sFRP2 (66%), sFRP4 (9%), sFRP5 (54%), SOX17 (29%), and WIF1 (32%). In contrast to epithelial tumors, methylation of APC (2%) and RASSF1A (0%) was rare. In patients with AML with normal cytogenetics, sFRP2 and sFRP5 methylation at the time of diagnosis was associated with an increased risk of relapse, and sFRP2 methylation was associated with an increased risk for death. In patients with AML: (a) there is a high frequency of Wnt pathway inhibitor methylation; (b) Wnt pathway inhibitor methylation is distinct from that observed in epithelial malignancies; and (c) methylation of sFRP2 and sFRP5 may predict adverse clinical outcome in patients with normal karyotype AML.