Wif-1 Methylation Research Articles

Global hypomethylation and promoter methylation in small intestinal neuroendocrine tumors

Aberrant DNA methylation is a feature of human cancer affecting gene expression and tumor phenotype. Here, we quantified promoter methylation of candidate genes and global methylation in 44 small intestinal-neuroendocrine tumors (SI-NETs) from 33 patients by pyrosequencing. Findings were compared with gene expression, patient outcome and known tumor copy number alterations. Promoter methylation was observed for WIF1, RASSF1A, CTNNB1, CXCL14, NKX2–3, P16, LAMA1, and CDH1. By contrast APC, CDH3, HIC1, P14, SMAD2, and SMAD4 only had low levels of methylation. WIF1 methylation was significantly increased (P = 0.001) and WIF1 expression was reduced in SI-NETs vs. normal references (P = 0.003). WIF1, NKX2–3, and CXCL14 expression was reduced in metastases vs. primary tumors (P < 0.02). Low expression of RASSF1A and P16 were associated with poor overall survival (P = 0.045 and P = 0.011, respectively). Global methylation determined by pyrosequencing of LINE1 repeats was reduced in tumors vs. normal references, and was associated with loss in chromosome 18. The tumors fell into three clusters with enrichment of WIF1 methylation and LINE1 hypomethylation in Cluster I and RASSF1A and CTNNB1 methylation and loss in 16q in Cluster II. In Cluster III, these alterations were low-abundant and NKX2-3 methylation was low. Similar analyses in the SI-NET cell lines HC45 and CNDT2 showed methylation for CDH1 and WIF1 and/or P16, CXCL14, NKX2-3, LAMA1, and CTNNB1. Treatment with the demethylating agent 5-azacytidine reduced DNA methylation and increased expression of these genes in vitro. In conclusion, promoter methylation of tumor suppressor genes is associated with suppressed gene expression and DNA copy number alterations in SI-NETs, and may be restored in vitro.

MP21-04 ESTABLISHMENT OF AN ORTHOTOPIC MOUSE NON-MUSCLE INVASIVE BLADDER CANCER MODEL EXPRESSING THE MAMMALIAN TARGET OF RAPAMYCIN SIGNALING PATHWAY

genome v10 using Bismark software. Methylation analysis was performed with MethylKit and IGV. RESULTS: Egg-injected mice featured major alterations in their methylome (vs control mice) after two weeks of treatment. Bases with a depth of sequencing of less than 10 were excluded from analysis, and differential methylation was defined as a different of greater than 25%. 13,333 cytosines were hypermethylated and 6,244 were hypomethylated. Mice that were fed nitrosamine were found to have 5,554 hypermethylated cytosines and 3,335 that were hypomethylated. Of these differentially methylated bases in the group that were egg-injected 1019 were found to be within 1000 base pairs of a transcription start site for a known gene. Six of these genes are part of the Wnt canonical pathway, which is related to cell proliferation. A CpG upstream of the WIF-1 gene, a gene silenced by hypermethylation in bladder tumors and other cancers, was methylated 54% of the time in egg-injected mice, 34% in nitrosamine-fed mice and 7% in control mice. CONCLUSIONS: Our data reveal that methylation of the tumor suppressor gene Wif-1 occurs early in the schistosomiasis and carcinogen exposed bladder urothelium. Future work will determine the diagnostic, prognostic, and therapeutic potential of this early methylation event.

MP21-03 EARLY DNA METHYLATION EVENTS IN BLADDER CARCINOGENESIS AND THEIR IMPLICATIONS FOR INFLAMMATION-INDUCED BLADDER CANCER

You have accessJournal of UrologyBladder Cancer: Basic Research I1 Apr 2014MP21-03 EARLY DNA METHYLATION EVENTS IN BLADDER CARCINOGENESIS AND THEIR IMPLICATIONS FOR INFLAMMATION-INDUCED BLADDER CANCER Simon Conti, Jared Honeycutt, and Michael Hsieh Simon ContiSimon Conti More articles by this author , Jared HoneycuttJared Honeycutt More articles by this author , and Michael HsiehMichael Hsieh More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.831AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Inflammation-induced DNA methylation of tumor suppressor genes has been implicated in various forms of carcinogenesis. Urogenital schistosomiasis is closely linked to inflammation-associated bladder cancer. N-Butyl-N-(4-hydroxybutyl)nitrosamine(BBN) is a potent bladder carcinogen and an established mouse model of chemical induced bladder cancer. We hypothesized that DNA methylation changes could be detected early after induction of experimental urogenital schistosomiasis as well as administration of a known bladder carcinogen. METHODS We combined our established mouse model of urogenital schistosomiasis with reduced representation bisulfite sequencing (genome-wide methylation analysis). Mice underwent sub-epithelial bladder injections of S. haematobium eggs or vehicle. Other mice received drinking water containing BBN. After two weeks of exposure mice were sacrificed and their urothelia dissected from the detrusor and granuloma. DNA was extracted from each specimen and the restriction enzyme Msp1 used to cleave CpG islands. After bisulfite treatment samples were purified to a length of 175-225 bp and amplified by PCR. Next generation sequencing was performed with the Illumina Hi-Seq platform. The output was aligned with the UCSC M. Musculus genome v10 using Bismark software. Methylation analysis was performed with MethylKit and IGV. RESULTS Egg-injected mice featured major alterations in their methylome (vs control mice) after two weeks of treatment. Bases with a depth of sequencing of less than 10 were excluded from analysis, and differential methylation was defined as a different of greater than 25%. 13,333 cytosines were hypermethylated and 6,244 were hypomethylated. Mice that were fed nitrosamine were found to have 5,554 hypermethylated cytosines and 3,335 that were hypomethylated. Of these differentially methylated bases in the group that were egg-injected 1019 were found to be within 1000 base pairs of a transcription start site for a known gene. Six of these genes are part of the Wnt canonical pathway, which is related to cell proliferation. A CpG upstream of the WIF-1 gene, a gene silenced by hypermethylation in bladder tumors and other cancers, was methylated 54% of the time in egg-injected mice, 34% in nitrosamine-fed mice and 7% in control mice. CONCLUSIONS Our data reveal that methylation of the tumor suppressor gene Wif-1 occurs early in the schistosomiasis and carcinogen exposed bladder urothelium. Future work will determine the diagnostic, prognostic, and therapeutic potential of this early methylation event. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e225-e226 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Simon Conti More articles by this author Jared Honeycutt More articles by this author Michael Hsieh More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Wif-1 methylation and β-catenin expression in colorectal serrated lesions

To investigate methylation status of Wif-1 and β-catenin expression in colorectal serrated lesions. Various colorectal lesions were collected including 52 cases of hyperplastic polyps, 41 cases of sessile serrated adenoma, 23 cases of traditional serrated adenoma, 24 cases of colorectal cancer and 24 cases of normal mucosa. All specimens were subject to immunohistochemical staining of β-catenin.SYBR Green PCR analysis of Wif-1 promoter methylation was performed in 29 cases of hyperplastic polyps, 29 cases of sessile serrated adenoma, 19 cases of traditional serrated adenoma, 14 cases of colorectal cancer and 16 cases of normal mucosa. Abnormal expression rates of β-catenin in normal mucosa, hyperplastic polyps, sessile serrated adenoma, traditional serrated adenoma and colorectal cancer were 12.5% (3/24), 59.6% (31/52), 63.4% (26/41), 73.9% (17/23) and 100.0% (24/24), respectively. The corresponding methylation rates of Wif-1 promoter were 2/16, 10/29 (34.5%), 16/29 (55.2%), 15/19 and 13/14 (P < 0.05), respectively. Abnormal β-catenin expression was positively correlated with Wif-1 promoter methylation in traditional serrated adenomas (r = 0.536, P < 0.05). Abnormal β-catenin expression and methylation rate of Wif-1 promoter are significantly higher in colorectal serrated lesions. Methylation of Wif-1 promoter may be related to the abnormal expression of β-catenin through activation of Wnt/β-catenin signaling pathway, which may contribute to the development of colorectal serrated lesions.

Aberrant methylation of NPY, PENK, and WIF1 as a promising marker for blood-based diagnosis of colorectal cancer

BackgroundDNA methylation is a well-known epigenetic mechanism involved in epigenetic gene regulation. Several genes were reported hypermethylated in CRC, althought no gene marker was proven to be individually of sufficient sensitivity or specificity in routine clinical practice. Here, we identified novel epigenetic markers and assessed their combined use for diagnostic accuracy.MethodsWe used methylation arrays on samples from several effluents to characterize methylation profiles in CRC samples and controls, as established by colonoscopy and pathology findings, and selected two differentially methylated candidate epigenetic genes (NPY, PENK). To this gene panel we added WIF, on the basis of being reported in literature as silenced by promoter hypermethylation in several cancers, including CRC. We measured their methylation degrees by quantitative multiplex-methylation specific PCR (QM-MSP) on 15 paired carcinomas and adjacent non-cancerous colorectal tissues and we subsequently performed a clinical validation on two different series of 266 serums, subdivided in 32 CRC, 26 polyps, 47 other cancers and 161 with normal colonoscopy. We assessed the results by receiver operating characteristic curve (ROC), using cumulative methylation index (CMI) as variable threshold.ResultsWe obtained CRC detection on tissues with both sensitivity and specificity of 100%. On serum CRC samples, we obtained sensitivity/specificity values of, e.g., 87%/80%, 78%/90% and 59%/95%, and negative predictive value/positive predictive value figures of 97%/47%, 95%/61% and 92%/70%. On serum samples from other cancers we obtained sensitivity/specificity of, e.g, 89%/25%, 43%/80% and 28%/91%.ConclusionsWe showed the potential of NPY, PENK, and WIF1 as combined epigenetic markers for CRC diagnosis, both in tissue and serum and tested their use as serum biomarkers in other cancers. We optimized a QM-MSP for simultaneously quantifying their methylation levels. Our assay can be an effective blood test for patients where CRC risk is present but difficult to assess (e.g. mild symptoms with no CRC family history) and who would therefore not necessarily choose to go for further examination. This panel of markers, if validated, can also be a cost effective screening tool for the detection of asymptomatic cancer patients for colonoscopy.

Promoter methylation of Wnt-antagonists in polypoid and nonpolypoid colorectal adenomas

BackgroundNonpolypoid adenomas are a subgroup of colorectal adenomas that have been associated with a more aggressive clinical behaviour compared to their polypoid counterparts. A substantial proportion of nonpolypoid and polypoid adenomas lack APC mutations, APC methylation or chromosomal loss of the APC locus on chromosome 5q, suggesting the involvement of other Wnt-pathway genes. The present study investigated promoter methylation of several Wnt-pathway antagonists in both nonpolypoid and polypoid adenomas.MethodsQuantitative methylation-specific PCR (qMSP) was used to evaluate methylation of four Wnt-antagonists, SFRP2, WIF-1, DKK3 and SOX17 in 18 normal colorectal mucosa samples, 9 colorectal cancer cell lines, 18 carcinomas, 44 nonpolypoid and 44 polypoid adenomas. Results were integrated with previously obtained data on APC mutation, methylation and chromosome 5q status from the same samples.ResultsIncreased methylation of all genes was found in the majority of cell lines, adenomas and carcinomas compared to normal controls. WIF-1 and DKK3 showed a significantly lower level of methylation in nonpolypoid compared to polypoid adenomas (p < 0.01). Combining both adenoma types, a positive trend between APC mutation and both WIF-1 and DKK3 methylation was observed (p < 0.05).ConclusionsMethylation of Wnt-pathway antagonists represents an additional mechanism of constitutive Wnt-pathway activation in colorectal adenomas. Current results further substantiate the existence of partially alternative Wnt-pathway disruption mechanisms in nonpolypoid compared to polypoid adenomas, in line with previous observations.

Wif1 hypermethylation as unfavorable prognosis of non-small cell lung cancers with EGFR mutation.

Lung cancer is a leading cause of cancer-related mortality across the world and tobacco smoking is the major risk factor. The Wnt signaling pathway is known to be involved in smoke-induced tumorigenesis in the lung. Promoter hypermethylation of Wnt inhibitory factor 1 (Wif1) has become a common event in a number of human tumors. Using a methylation-specific PCR, hypermethylation of the Wif1 gene promoter was evaluated in 139 primary nonsmall cell lung cancers (NSCLCs) and its correlation with clinicopathological and prognostic parameters was evaluated. Methylation of Wif1 was observed in 47.5% and 20.9% of neoplastic and adjacent normal lung tissues, respectively. Its methylation rate tended to be higher in stage I than stages II-IIIA. Results of Kaplan-Meier analysis showed no significant difference in overall survival according to Wif1 methylation status. However, Wif1 methylation showed an association with unfavorable prognosis of adenocarcinoma (AC) patients with EGFR mutation. According to our current findings, Wif1 promoter methylation is an early, frequent event as an epigenetic field manner and could be considered as a useful prognostic marker for AC patients with EGFR mutation. Further investigation into the therapeutic potential of this finding is warranted.

Loss of WIF-1 and Wnt5a expression is related to aggressiveness of sporadic breast cancer in Tunisian patients

Activation of the Wnt/β-catenin signaling pathway is common in various human cancers. The aim of this study was to investigate the expression of 2 members of the Wnt family (WIF-1 and Wnt5a) in sporadic and hereditary breast cancer tissues. WIF-1, is a secreted antagonist that binds Wnt ligands, and therefore inhibits the canonical Wnt/β-catenin pathway. Wnt5a is one of the members of the noncanonical Wnt family that mainly acts through calcium signaling pathway. The expression of WIF-1 was analyzed by methylation-specific PCR and RT-PCR, and the level of Wnt5a ligand was quantified by RT-QPCR in breast cancer tissues. Methylation of WIF-1 was detected in 71.3 % and 81.8 % of sporadic and hereditary cases, respectively. Aberrant methylation of WIF-1 was associated with advanced TNM stage and triple negative cases in sporadic breast carcinoma (p=0.001 and p=0.037, respectively). In hereditary cases, methylation of WIF-1 correlated with age at diagnosis (p=0.027) and p53 status (p=0.035). Regarding patients' survival, WIF-1 methylated promoter conferred a reduced overall survival rate, and particularly in a group of patients with advanced TNM stage (p log rank=0.006). Furthermore, aberrant CpG methylation of the WIF-1 promoter was significantly associated with transcriptional silencing of this tumor suppressor gene in sporadic breast cancer tissues (p=0.036). On the other hand, in sporadic tumor tissues, the level of Wnt5a mRNA was significantly lower compared to normal tissues (p=0.031) and lower still in those showing more aggressive behavior, suggesting that Wnt5a, a ligand involved in the noncanonical Wnt/β-catenin pathway, could act as a tumor suppressor gene in breast cancer.

P-0198 Identification of Prognostic Methylation Markers in Patients With Early Stage Colorectal Cancer

ABSTRACT Introduction Colorectal cancer (CRC) is one of the most common forms of cancer. Surgical excision at clinical stages I and II is highly effective, but in about 20-30% of patients, the disease can recur. Therefore, it is highly important to identify patients at high risk for disease recurrence, who would benefit from adjuvant chemotherapy. Aberrant promoter methylation represents a hallmark of colorectal cancer; therefore detection of promoter methylation could be a suitable marker to identify such high risk patients. The overall objective of this study was to identify prognostic methylation markers to assess prognosis in stage II CRC patients. Methods For selection of candidate methylated genes in cancer patients we performed a Methyl Profiler Assay (Qiagen) of 48 gene promoters in two different cell lines, the tumorigenic cell line SW480 and the non-tumorigenic cell line Caco-2. To assess whether promoter methylation was associated with transcriptional silencing, we analyzed mRNA expression in the presence or absence of the demethylating agent 5-Aza-2′-dCytidine (5-Aza) in 4 different colon cancer cell lines (SW480, HT29, HCT116, Caco-2). Additionally, we analyzed the methylation status using established MethyLight assays after 5-Aza treatment compared to the untreated control. Markers that showed promoter demethylation associated with an increase in expression (>10-fold) were then analyzed in a set of 57 primary tumors from stage II CRC patients. Results Of 48 gene promoters 19 showed aberrant methylation in SW480 while 16 promoters where differentially methylated in Caco-2. To verify that the methylation is leading to down-regulation of these genes, we chose 17 of the methylated markers for qPCR analysis after treatment with the demethylating 5-Aza in 4 colorectal cancer cell lines. In all cell lines expression of WIF1 could be restored after 5-Aza treatment. Three cell lines showed also a significant increase in expression of UHCL1. Caco-2 did not show an increase in UHCL1 expression which is in line with results of the Methyl Profiler and the MethyLight assay where the UCHL1 promoter did not reveal any aberrant methylation. For all other cell lines we detected a significant decrease of DNA methylation after 5-Aza treatment corresponding to the mRNA analysis. To evaluate whether this aberrant methylation is also present in clinical samples we analyzed a set of 57 primary tumors from stage II CRC patients. Of 57 tumors 13 (22.8%) could not be analyzed due to low DNA concentrations. The WIF1 promoter was aberrantly methylated in 59.6% (35 of 57) whereas UCHL1 promoter hypermethylation was seen in 29.8% of the tumors (17 of 57). Conclusion Taken together, these results indicate a direct association between WIF1 and UCHL1 promoter methylation and repression of expression. Aberrant promoter methylation of WIF1 and UCHL1 was found in 59.6% and 29.8% of primary tumors, respectively. Thus, WIF1 and UCHL1 appear to be suitable for further analysis and prognostic evaluation of progression and recurrence of CRC.

Prospective study of tumor suppressor gene (TSG) methylation as a prognostic biomarker in surgically resected non-small cell lung cancer (NSCLC).

7066 Background: In the New England Journal of Medicine, Brock et al. (2008) published a nested case-control study which tested the association between early recurrence of NSCLC after surgical resection, and TSG promoter methylation in tumor and lymph nodes detected by methylation-specific PCR (MSP). They reported that promoter methylation of the TSGs p16, CDH13, RASSF1A, and APC was significantly associated with early recurrence in 51 patients with stage I NSCLC compared to matched patients without early recurrence. We attempted to confirm these findings. Methods: In a prospective study, fresh frozen tumor tissue was acquired after surgical resection in 107 patients with stage I-IIIA NSCLC between 2003-2008. The promoter methylation status of the same 4 genes examined by Brock, et al (p16, CDH13, RASSF1A, APC), as well as 6 additional TSGs (MGMT, WIF-1, METH-2, GSTP1, SOCS3, DAPK) were assessed in the tumor tissue using quantitative MSP (MethyLight assay), with any amount of methylation scored as positive. Methylation status was correlated with clinical features, pathologic stage, disease-free survival (DFS), and overall survival (OS). Results: No significant associations were observed between promoter methylation of individual TSGs and DFS. No significant associations were observed between the number of methylated TSGs and DFS, or OS. Increased RASSF1A methylation was observed in poorly-differentiated and undifferentiated tumors compared to tumors that were well- or moderately-differentiated (p=0.031). Increased WIF-1 methylation and GSTP1 methylation were associated with increasing T (p=0.01) and N stage (p=0.028), respectively. Squamous cell carcinomas (SQCCs) were characterized by increased p16 methylation (p=0.0314) and decreased APC methylation (p=0.0146) compared to tumors of non-SQCC histologies. Conclusions: In this prospective study, we did not confirm that the promoter methylation of p16, CDH13, RASSF1A, and APC, or 6 other TSGs, was prognostic for early recurrence in surgically resected NSCLC.

DNA Methylation and the HOXC6 Paradox in Prostate Cancer

Overexpression of the classical homeobox transcription factor HOXC6 is frequent in prostate cancers and correlates with adverse clinical parameters. Since surprisingly many HOXC6 target genes are downregulated in prostate cancer, it has been posited that oncogenic effects of HOXC6 in prostate cancer may be unmasked by concurrent epigenetic downregulation of target genes exerting tumor suppressive effects. To test this hypothesis, we have studied the expression of three HOXC6 target genes, CNTN1 (encoding a cell adhesion protein), DKK3 and WIF1 (encoding WNT growth factor antagonists) as well as DNA methylation of DKK3 and WIF1. HOXC6 upregulation and association with poor prognosis were confirmed in our tissue series. The three target genes were each significantly downregulated in cancer tissues and expression of each one correlated inversely with that of HOXC6. Cases with lower WIF1 expression showed significantly earlier recurrence (p = 0.021), whereas no statistical significance was reached for CNTN1 and DKK3. Hypermethylation of DKK3 or WIF1 gene promoters was observed in a subset of cancers with downregulated expression, but was often weak. Our data support the hypothesis that HOXC6 target genes exerting tumor-suppressive effects are epigenetically downregulated in prostate cancer, but DNA methylation appears to follow or bolster rather than to cause their transcriptional inactivation.

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.

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.

Epigenetic markers for early detection of nasopharyngeal carcinoma in a high risk population

BackgroundUndifferentiated nasopharyngeal carcinoma (NPC) is strongly related to Epstein-Barr virus (EBV) infection, allowing aberrant antibodies against EBV and viral DNA load as screening tools in high risk populations. Methylation analysis in the promoter of tumor suppressor genes (TSGs) may serve as a complementary marker for identifying early cases. This study determined methylation status of multiple TSGs and evaluated whether it may improve early detection.MethodsNasopharyngeal brushings were taken from 53 NPC patients, 22 high risk subjects and 25 healthy EBV carriers. Corresponding NPC paraffin tissue was included. DNA was bisulfite-modified preceding analysis by methylation-specific PCR (MSP). Ten TSGs were studied.ResultsNPC paraffin and brushing DNA revealed an 81.8% concordance so that MSP analysis was done using either one of both specimens. NPC samples showed methylation for individual TSGs (DAPK1 79.2%, CDH13 77.4%, DLC1 76.9%, RASSF1A 75.5%, CADM1 69.8%, p16 66.0%, WIF1 61.2%, CHFR 58.5%, RIZ1 56.6% and RASSF2A 29.2%). High risk individuals, having elevated EBV IgA and viral load, showed high frequency of methylation of CDH13, DAPK1, DLC1 and CADM1, but low frequency of methylation of p16 and WIF1 and undetectable methylation of RASSF1A, CHFR, RIZ1 and RASSF2A. Healthy subjects showed similar patterns as high risk individuals. A combination of RASSF1A and p16 gave good discrimination between NPC and non-NPC, but best results were combined analysis of five methylation markers (RASSF1A, p16, WIF1, CHFR and RIZ1) with detection rate of 98%.ConclusionMultiple marker MSP is proposed as a complementary test for NPC risk assessment in combination with EBV-based markers.

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.

Aberrant promoter methylation of WIF-1 and SFRP1, 2, 4 genes in mesothelioma

WIF-1 is a negative regulator of the Wnt-signaling pathway that may have important implications for tumorigenesis. Microarray analysis of whole genome expression in mesothelioma tissue revealed down-regulation of 491 genes and up-regulation of 167 genes involved mainly in Jak-STAT signaling (8 genes), MAPK signaling (16 genes) and Wnt signaling (13 genes) pathways. Of these, WIF-1 gene was down-regulated in mesothelioma 72-fold compared to normal tissue. We also analyzed WIF-1 and SFRPs promoter methylations in 46 mesothelioma tissues, 8 mesothelioma cell lines by methylation-specific polymerase chain reaction (MSP). WIF-1 promoter methylation was observed in 34 of 46 mesothelioma tissues (73.9%) and in all 8 mesothelioma cell lines. SFRP1, 2 and 4 promoter methylation was observed in 21 of 37 (56.8%), 26 of 42 (61.9%) and 17 of 36 (47.2%) mesothelioma tissues, respectively. Promoter methylation of any WIF-1 and/or SFRP genes was observed in 44 of 46 (95.6%) mesothelioma tissues. The treatment of mesothelioma cell lines with 5-aza-2'-deoxycytidine (5-aza-2dC) showed WIF-1 promoter methylation recovery followed by restoration of WIF-1 expression in 6 of 8 mesothelioma cell lines. The cytoplasmic expression of beta-catenin was observed in 38 of 43 cases of mesothelioma without any nuclear reactivity. The eight mesothelioma cell lines and 27 cases of mesothelioma examined showed no mutation in exon 3 of beta-catenin suggesting no alteration of canonical Wnt signaling pathway. Our data suggest that WIF-1 promoter methylation is a common event in mesothelioma.

Molecular Characterization of Chronic Obstructive Pulmonary Disease-Related Non-Small Cell Lung Cancer Through Aberrant Methylation and Alterations of EGFR Signaling

The aim of this study was to evaluate the molecular influence of chronic obstructive pulmonary diseases (COPD) on the pathogenesis of non-small cell lung cancer (NSCLC). The methylation profiles of 12 genes, and the epidermal growth factor receptor (EGFR) and KRAS mutations were determined for samples from 229 NSCLC patients. In addition, protein expression of EGFR and HER2 in 116 NSCLCs was analyzed based on the presence or absence of COPD. IL-12Rbeta2 and Wif-1 methylation and HER2 overexpression were more frequent events in the COPD group. Eighty nonmalignant lung tissues had no correlation with any molecular changes between the COPD and the non-COPD group. EGFR mutation was significantly higher in the non-COPD group, while EGFR expression was inversely correlated with %FEV1.0. In the COPD group, unmethylated SPARC and sFRP-2 genes or a negative CpG island methylator phenotype (CIMP) was a negative prognostic factor, while methylation of p16(INK4A) and WNT antagonist genes was a negative prognostic factor in the non-COPD group. Novel characteristics of COPD-related NSCLC were identified by examination of methylation profiles and alterations of EGFR signaling. In consideration of the high sensitivity to smoking in patients with COPD, NSCLC with COPD might be a distinct population of smoke-related NSCLC, the genetic profile of which is quite different from non-COPD NSCLC.

Promoter hypermethylation of the p16 and Wif-1 genes as an independent prognostic marker in stage IA non-small cell lung cancers

Hypermethylation of promoter CpG islands is a major inactivation mechanism of tumor suppressor genes, some of which are thought to be related to the prognosis of patients with non-small cell lung cancer (NSCLC). Therefore, hypermethylation of the specific genes may be expected to serve as a prognostic biomarker for NSCLC. In this study, the methylation status of 14 genes was analyzed in 44 stage IA NSCLC cases using methylation-specific PCR. Hypermethylation was detected in PTGER2 (70% of cases), DRM/Gremlin (66%), sFRP-2 (57%), IL-12Rbeta2 (48%), Reprimo (41%), APC (39%), CXCL12 (39%), HPP1 (30%), SPARC (30%), sFRP-5 (30%), p16 (25%), RUNX3 (20%), sFRP-1 (20%) and Wif-1 (16%). Patients with p16, sFRP-5, Wif-1 or CXCL12 methylation had a significantly shorter duration of relapse-free survival than their counterparts with an unmethylated gene (p16, P=0.011; sFRP-5, P=0.030, Wif-1, P=0.036; CXCL12, P=0.026). Also, those with methylated HPP1, p16 or Wif-1 had a significantly shorter duration of overall survival (HPP1, P=0.031; p16, P=0.026; Wif-1, P=0.008). Multivariate analysis revealed that p16 methylation in relapse-free survival and Wif-1 methylation in overall survival were the strongest independent prognostic factors (p16, P=0.036; Wif-1, P=0.035). In conclusion, the hypermethylation of the p16 and Wif-1 genes has potential as biomarkers that may be used to predict the prognosis of stage IA NSCLC.

Promoter methylation and mRNA expression of DKK-3 and WIF-1 in hepatocellular carcinoma

To investigate the promoter methylation status and mRNA expression of DKK-3 and WIF-1 gene in hepatocellular carcinoma (HCC). DKK-3 and WIF-1 acted as Wnt-antagonists and tumor suppressors, but hypermethylation of the gene promoter and low mRNA expression activated Wnt signaling aberrantly and induced the development of HCC. Methylation status of the DKK-3 and WIF-1 gene promoter was investigated using methylation specific polymerase chain reaction (PCR) in tumor and adjacent non-cancerous tissues from 33 HCC patients and 20 normal liver tissues served as control. The expression of DKK-3 and WIF-1 mRNA was also determined by real-time quantitative reverse transcriptase PCR. The relationship between methylation, mRNA expression, and clinical data, as well as methylation and mRNA expression of the two genes were analyzed. The methylation of DKK-3 and WIF-1 genes in HCC increased significantly compared with adjacent non-cancerous tissues and normal control tissues (chi(2) =7.79, P < 0.05; chi(2) = 4.89, P < 0.05), and no significant difference in methylation between adjacent non-cancerous tissues and normal control tissues was observed. In HCC tissues, significant differences in the DKK-3 promoter methylation were observed in age and cirrhosis, and significant differences of the WIF-1 promoter methylation were observed in HBsAg and cirrhosis. The average expression of DKK-3 mRNA in HCC and adjacent non-cancerous tissues was increased significantly compared with normal control tissues. The average expression of WIF-1 mRNA showed no significant difference among the three tissues. The mRNA expression of DKK-3 gene in HCC was decreased as the pathological grade increased. The aberrant promoter methylation and decreased expression of DKK-3 and WIF-1 may be an important mechanism in HCC, and may be a far-reaching significance in early diagnosis and therapy of HCC.

CpG island hypermethylation of E‐cadherin (CDH1) and integrin α4 is associated with recurrence of early stage esophageal squamous cell carcinoma

The prognosis of esophageal squamous cell carcinoma (ESCC) patients remains very poor, which is partially due to a high rate of recurrence. This study was aimed at identifying a recurrence-associated epigenetic prognostic marker in patients with ESCC. We retrospectively analyzed the CpG island hypermethylation of the p16, Wif-1, sFRP1, integrin alpha4, CDH1, DAP kinase and RARbeta2 genes in 251 ESCCs. The methylation status was determined by methylation-specific PCR. Hypermethylation was detected in 52% for p16, 25% for RARbeta2, 43% for CDH1, 21% for integrin alpha4, 57% for sFRP1, 38% for DAP kinase and 35% for Wif-1. Recurrence was observed in 131 (52%) of the 251 cases. For stage I cancers, CDH1 methylation was associated with a high risk of recurrence (OR = 5.26, 95% CI = 1.48-18.67; p = 0.01) and a poor recurrence-free survival after surgery (HR = 3.13, 95% CI = 1.21-8.09; p = 0.02). The hazard of failure after recurrence was about 13.17 (95% CI = 2.46-70.41; p = 0.003) times higher in patients with Wif-1 methylation than in those without. For stage II cancers, integrin alpha4 methylation was associated with an increased risk of recurrence (OR = 3.03, 95% CI = 1.09-8.37; p = 0.03) and a poor recurrence-free survival (HR = 2.12, 95% CI = 1.13-3.98; p = 0.03). In conclusion, the present study suggests that hypermethylation of CDH1 and integrin alpha4 genes may be used as recurrence-associated prognostic indicators in stage I and stage II ESCCs, respectively.