Wif-1 Promoter Methylation Research Articles

LncRNA LINC00592 mediates the promoter methylation of WIF1 to promote the development of bladder cancer.

Epigenetic alteration is a key feature that contributes to the progression of bladder cancer (BC) and long non-coding RNAs serve crucial role in the epigenetic modulation. This study was designed to explore the epigenetic regulation of LINC00592 in BC. LINC00592 expression in BC was examined. Then, LINC00592 was silenced in BC cell followed by cell behavior analyses using CCK-8, transwell, western blot, or flow cytometry. Potential downstream target of LINC00592 was explored using RNA pull-down assay and methylation of WIF1 was determined using methylated-specific PCR. In addition, WIF1 or/and LINC00592 were silenced in BC cells followed by cell behavior analyses to explore the regulation between them. Upregulation of LINC00592 was significantly detected in BC tissues and cells. In BC cells silencing LINC00592 suppressed the proliferation, migration, and epithelial-mesenchymal transitions (EMT), but enhanced apoptosis. Moreover, LINC00592 recruited DNMT1, DNMT3A, and DNMT3B to enhance WIF1 promoter methylation. In addition, WIF1 overexpression suppressed the proliferation, migration, as well as EMT, but enhanced apoptosis. Silencing WIF1 significantly attenuated the role of silencing LINC00592 in suppressing the proliferative, migratory, and EMT ability of BC cells, and increasing the apoptosis. LINC00592 promoted the growth and metastasis of BC via enhancing the promoter methylation of WIF1 and decreasing WIF1 transcription.

WIF1 promoter hypermethylation induce endometrial carcinogenesis through the Wnt/β-catenin signaling pathway.

This study explores the mechanism underlying WIF1 promoter methylation and its relationship with the pathogenesis of endometrial carcinoma. WIF1 promoter methylation was detected using methylation-specific polymerase chain reaction (MSP). WIF1 expression was examined through qRT-PCR and western blotting. Furthermore, 5-aza-2'-deoxycytidine (5-Aza) was used to demethylate the WIF1 promoter. The roles of WIF1 were investigated using in vitro loss- and gain-of-function assays. Xenograft models were used to analyze WIF1 expression and downstream genes, and results were confirmed using immunofluorescence and western blotting. WIF1 promoter methylation in endometrial cancer cells was significantly higher than that in normal cells, but the WIF1 mRNA and protein levels were reduced. The expression of WIF1 increased significantly after 5-Aza treatment (p<.05). Thus, 5-Aza treatment can inhibit the proliferation of endometrial cancer cells and induce apoptosis, while knockdown of WIF1 significantly inhibits the effects of 5-Aza. 5-Aza treatment can also inhibit Wnt pathway genes, including phosphorylation of β-catenin protein, c-Myc, and CyclinD1, inhibit downstream functional genes, and activate the tumor suppressor gene APC, which can be blocked by WIF1 knockdown in endometrial carcinoma cells. Finally, 5-Aza inhibited the proliferation of subcutaneous tumor-bearing nude mice with endometrial cancer cells, but the effect was weaker than that of WIF1 overexpression. Our research shows that WIF1 promoter hypermethylation may promote the progression of endometrial cancer by downregulating WIF1 expression, activating the Wnt/β-catenin pathway, and promoting proliferation and inhibiting apoptosis. WIF1 may be a potential biological target for gene therapy and drug development for the treatment of endometrial cancer.

Association of ZNF331 and WIF1 methylation in peripheral blood leukocytes with the risk and prognosis of gastric cancer

BackgroundPeripheral blood leukocyte (PBL) DNA methylation may serve as a surrogate marker to evaluate the susceptibility to and prognosis of gastric cancer (GC). In this study, blood-derived DNA methylation levels of two tumour-related genes, namely, ZNF331 and WIF1, and their impacts on the risk and prognosis of GC were evaluated.MethodsIn total, 398 GC cases and 397 controls were recruited for the study. Then, all cases were followed up for 5 years. ZNF331 and WIF1 promoter methylation status in PBLs was measured using a methylation-sensitive high-resolution melting method. Logistic and Cox regression models were used to analyse the correlation between gene methylation and the risk and prognosis of GC. Confounders were balanced through propensity score (PS) matching.ResultsHigh ZNF331 methylation significantly decreased GC risk after PS adjustment (OR = 0.580, 95% CI: 0.375–0.898, P = 0.015), which also presented in males (OR = 0.577, 95% CI: 0.343–0.970, P = 0.038). However, WIF1 methylation was not associated with GC risk. Additionally, significant combined effects between ZNF331 methylation and the intake of green vegetables and garlic were observed (OR = 0.073, 95% CI: 0.027–0.196, P < 0.001 and OR = 0.138, 95% CI: 0.080–0.238, P < 0.001, respectively). Furthermore, ZNF331 and WIF1 methylation had no impact on the prognosis of GC.ConclusionZNF331 methylation in PBLs may affect GC risk in combination with the consumption of green vegetables and garlic and may act as a potential biomarker of GC.

Prognostic Role of RASSF1A, SOX17 and Wif-1 Promoter Methylation Status in Cell-Free DNA of Advanced Gastric Cancer Patients.

Epigenetic modification of several genes is a key component in the development of gastric cancer. The methylation status of RASSF1A, SOX17 and Wif-1 genes was evaluated in the cell free circulating DNA of 70 patients with advanced gastric cancer, using methylation-specific PCR. Patients with higher cell-free DNA concentration seem to have lower PFS, than patients with lower cell-free DNA concentration (p = 0.001). RASSF1A was the tumor suppressor gene, most frequently methylated in metastatic gastric cancer patients, followed by SOX17 and Wif-1 (74.3%, 60.0% and 47.1%, respectively). Patients having the SOX17 promoter methylated, had lower progression free survival and overall survival, than unmethylated ones (p < 0.001). Patients having the Wif-1 promoter methylated, had lower progression free survival and overall survival, than unmethylated ones (p = 0.001). Patients having the RASSF1A promoter methylated, had lower progression free survival and overall survival, than unmethylated ones (p = 0.004). Promoter methylation of the examined genes was significantly associated with a decrease in progression free survival and overall survival, comparing to that of patients without methylation. Simultaneous methylation of the above genes was associated with even worse progression free survival and overall survival. The methylation of RASSF1A, SOX-17 and Wif-1 and genes, is a frequent epigenetic event in patients with advanced gastric cancer.

Association between SOX17, Wif-1 and RASSF1A promoter methylation status and response to chemotherapy in patients with metastatic gastric cancer.

Article Association between SOX17, Wif-1 and RASSF1A promoter methylation status and response to chemotherapy in patients with metastatic gastric cancer was published on February 1, 2021 in the journal Clinical Chemistry and Laboratory Medicine (CCLM) (volume 59, issue 2).

Indirubin inhibits Wnt/\u03b2-catenin signal pathway via promoter demethylation of WIF-1

BackgroundPsoriasis is a common inflammatory skin disease. Abnormal proliferation of keratinocytes is one of the psoriatic histopathological features. Indirubin has an essential effect on the proliferation and activation of keratinocytes; however, in psoriasis, the specific mechanism of action of indirubin on keratinocytes is unclear. In the present study, we revealed the effects of indirubin on DNA methyltransferase 1 (DNMT1), wnt inhibitory factor 1 (wif-1), and wnt/β-catenin signal pathway, in the meantime, we explored the effects of indirubin on proliferation, cell cycle and the apoptosis of HaCaT cells.MethodsThe expression of DNMT1, wif-1, Frizzled2, Frizzled5, and β-catenin in HaCaT cells treated with different concentrations of indirubin were detected by Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR). The expression levels of DNMT1 and wif-1 were observed after treated with different concentrations of indirubin by enzyme-linked immunosorbent assay (ELISA). The wif-1 promoter methylation status was detected by DNA methylation-specific PCR (MSP). The transcriptional activities of wif-1 and β-catenin were discovered by a luciferase reporter gene system. Cell viability was determined by Cell Counting Kit-8 (CCK8) method. The cell cycle was detected by flow cytometry. The apoptotic cells were surveyed by the apoptosis kit. The expression of Inolucrin, Loricrin, Filaggrin, Keratin 17, and transcriptional activation of transglutaminase 1(TGase1) were detected by Western blotting.ResultsIndirubin inhibited the expression of DNMT1 and the methylation of the wif-1 promoter. In the wnt signal pathway, indirubin restored the protein expression of wif-1 and inhibited expression of Frizzled2, Frizzled5, and β-catenin. Besides, indirubin inhibited the proliferation of HaCaT cells, induced apoptosis, and arrest cell cycle. We also reported that indirubin could down-regulate the expression of Involucrin, TGase 1, and keratin 17, but the expression of Filaggrin and Loricrin had no significant effect.ConclusionOur research showed that indirubin promoted the demethylation of wif-1 and suppressed the wnt/β-catenin signal pathway, thereby exerted an anti-proliferative effect. This study reveals the anti-proliferation mechanism of indirubin, which may provide an effective option for the treatment of proliferative diseases.

DNA methylation of SFRP1, SFRP2, and WIF1 and prognosis of postoperative colorectal cancer patients

BackgroundAs biomarkers, DNA methylation is used to detect colorectal cancer (CRC) and make assessment of CRC prognosis. The published findings showed the association between the methylation of SFRP1, SFRP2, and WIF1, located in the Wnt signaling pathway, and the prognosis of CRC were not consistent. Our study aimed to explore the potential possibility of SFRP1, SFRP2, and WIF1 concomitant promoter methylation as prognostic biomarkers of postoperative CRC patients.MethodsAs a total of 307 sporadic postoperative CRC patients were followed up, we detected SFRP1, SFRP2, and WIF1 methylation obtained from tumor tissues and adjacent non-tumor tissues respectively on the basis of methylation-sensitive high resolution melting analysis. Univariate and multivariate Cox regressions were carried out so as to assess the potential possibility of SFRP1, SFRP2, and WIF1 promoter methylation as predictors of prognosis. Confounders in our study were controlled by Propensity Score (PS) analysis.ResultsThe SFRP1, SFRP2, and WIF1 methylation levels in tumor tissues were significantly higher than that in adjacent non-tumor tissues (P < 0.001). SFRP2 hypermethylation was significantly associated with a favorable clinical outcome at the hazard ratio (HR) of 0.343 [95% confidence intervals (CI): 0.164–0.718, P = 0.005] and 0.410 (95% CI: 0.200–0.842, P = 0.015) in multivariate Cox regression and PS analysis, respectively. Co-hypermethylation of SFRP1 and SFRP2 was significantly associated with a favorable clinical outcome at the HR of 0.333 (95% CI: 0.159–0.694, P = 0.003) and 0.398 (95% CI: 0.192–0.821, P = 0.013) in multivariate Cox regression and PS analysis, respectively. Co-hypermethylation of SFRP1, SFRP2 and WIF1 was significantly associated with a favorable clinical outcome at the HR of 0.326 (95% CI: 0.117–0.908, P = 0.032) and 0.401 (95% CI: 0.146–1.106, P = 0.077) in multivariate Cox regression and PS analysis, respectively.ConclusionsSFRP1, SFRP2, and WIF1 were frequently hypermethylated in CRC tumor tissues. It was apparent that the promoter hypermethylation of SFRP2 and co-hypermethylation of SFRP1 and SFRP2 might be considered as independent prognostic predictors for survival advantage of postoperative CRC patients.

WNT inhibitory factor 1 promoter hypermethylation is an early event during gallbladder cancer tumorigenesis that predicts poor survival

Gallbladder cancer (GBC) is the most common malignant tumor in the human biliary tract, but the lack of a marker for timely diagnosis leads to an extremely poor prognosis. In this study, we assessed CpG sites in the WIF-1 promoter using bisulfite sequencing PCR and methylation-specific PCR to detect methylation in gallbladder cancer and cholecystitis tissues. WIF-1 promoter methylation was present in 36 of 50 (72.0%) gallbladder cancers but only 5 of 20 (25.0%) cholecystitis tissues (P=0.000<0.05), suggesting that WIF-1 promoter methylation might participate in the malignant transformation of cholecystitis into gallbladder cancer. WIF-1 methylation was negatively correlated with WIF-1 protein expression by immunohistochemistry, demonstrating that WIF-1 expression is downregulated by promoter hypermethylation. We analyzed the prognosis of 50 GBC patients with 5years of follow-up. Univariate analysis revealed that patients with hypermethylated WIF-1 exhibited worse overall survival than those with hypomethylated WIF-1 (χ2=8.137, P=0.004<0.05). Furthermore, multivariate analysis revealed that WIF-1 methylation was an independent prognostic factor for 5-year overall survival (P=0.011). Therefore, WIF-1 methylation is a candidate as a marker for early gallbladder cancer diagnosis and prognosis.

Extracellular activation of Wnt signaling through epigenetic dysregulation of Wnt inhibitory factor-1 (Wif-1) is associated with pathogenesis of adrenocortical tumor.

Wnt/β-catenin signaling is considered to be an essential regulator of adrenocortical oncogenesis. Wnt inhibitory factor-1 (Wif-1), an extracellular regulator of Wnt signaling, is frequently down-regulated by hypermethylation of the promoter CpG. We investigated epigenetic regulation of Wif-1 and its association with adrenocortical (AC) tumor pathogenesis in light of Wnt activation. The AC tumors showed a high prevalence of Wif-1 promoter methylation and low prevalence of Wif-1 mRNA transcription as compared to the normal adrenal (NA) samples. Furthermore, a significant correlation was found between Wif-1 promoter methylation and mRNA transcription in the tumors. Either intracellular β-catenin accumulation or β-catenin mRNA transcription was significantly elevated in the AC tumors, which also showed an inverse correlation with Wif-1 mRNA transcription. Cyclin D1, a target gene of Wnt signaling, was also up-regulated in the AC tumors as compared with the NA samples. In addition, down-regulation of Wif-1 was correlated with increased cyclin D1 at both mRNA and protein levels. However, despite the proposed activation of Wnt signaling in AC tumors, only 2 of 20 with intracellular β-catenin accumulation showed β-catenin mutations. Thus, genetic alterations of β-catenin and epigenetics-related Wif-1 promoter hypermethylation may be important mechanisms underlying AC tumor formation though aberrant canonical Wnt/β-catenin signaling activation.

PD1-12 PATHOGENESIS OF ADRENOCORTICAL TUMOR IS ASSOCIATED WITH EXTRACELLULAR ACTIVATION OF WNT SIGNALING THROUGH EPIGENETIC DYSREGULATION OF WNT INHIBITORY FACTOR-1

You have accessJournal of UrologyAdrenal1 Apr 2014PD1-12 PATHOGENESIS OF ADRENOCORTICAL TUMOR IS ASSOCIATED WITH EXTRACELLULAR ACTIVATION OF WNT SIGNALING THROUGH EPIGENETIC DYSREGULATION OF WNT INHIBITORY FACTOR-1 Yozo Mitsui, Miho Hiraki, Kohei Ogawa, Taichi Nagami, Haruki Anjiki, Chiaki Koike, Naoko Arichi, Shigenobu Nakamura, Takeo Hiraoka, Masahiro Sumura, Hiroaki Yasumoto, and Hiroaki Shiina Yozo MitsuiYozo Mitsui More articles by this author , Miho HirakiMiho Hiraki More articles by this author , Kohei OgawaKohei Ogawa More articles by this author , Taichi NagamiTaichi Nagami More articles by this author , Haruki AnjikiHaruki Anjiki More articles by this author , Chiaki KoikeChiaki Koike More articles by this author , Naoko ArichiNaoko Arichi More articles by this author , Shigenobu NakamuraShigenobu Nakamura More articles by this author , Takeo HiraokaTakeo Hiraoka More articles by this author , Masahiro SumuraMasahiro Sumura More articles by this author , Hiroaki YasumotoHiroaki Yasumoto More articles by this author , and Hiroaki ShiinaHiroaki Shiina More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.131AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Wingless-type (Wnt)/β-catenin signaling is a major regulator of and/or contributor to the pathogenesis of adrenocortical (AC) tumor development. Activation of β-catenin mutations appears to be common in some types of malignancy including these tumors. However, a large number of AC tumors show intracellular β-catenin accumulation in the absence of β-catenin mutations. Extracellular activation of Wnt signaling is controlled by Wnt antagonists such as Wnt inhibitory factor-1 (Wif-1) and low levels of Wif-1 expression caused by promoter CpG hypermethylation have been found in various human tumors. We speculate that Wif-1 plays an important role in adrenocortical pathogenesis. METHODS Promoter CpG methylation of the Wif-1 gene and the effects on Wnt signaling were analyzed in 42 AC tumors and 23 normal adrenal (NA) samples. Promoter CpG methylation was analyzed by methylation specific PCR using bisulfite-modified DNA as template. Wif-1 expression was measured by quantitative RT-PCR. RESULTS The AC tumors showed a high prevalence of Wif-1 promoter methylation and low prevalence of Wif-1 mRNA transcription as compared to the NA samples. Furthermore, a significant correlation was found between Wif-1 promoter methylation and mRNA transcription in the tumors. Either intracellular β-catenin accumulation or β-catenin mRNA transcription was significantly elevated in the AC tumors, which also showed an inverse correlation with Wif-1 mRNA transcription. Cyclin D1, a target gene of Wnt signaling, was also up-regulated in the AC tumors as compared with the NA samples. In addition, down-regulation of Wif-1was correlated with increased cyclin D1 at both mRNA and protein levels. However, despite the proposed activation of Wnt signaling in AC tumors, only 2 of 20 (10%) with intracellular β-catenin accumulation showed β-catenin mutations. CONCLUSIONS This is the first report inactivation of the Wif-1 gene via epigenetic pathways in AC tumors. Genetic alterations of β-catenin and epigenetics-related Wif-1 promoter hypermethylation may be important mechanisms underlying AC tumor formation though aberrant canonical Wnt/β-catenin signaling activation. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e14 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Yozo Mitsui More articles by this author Miho Hiraki More articles by this author Kohei Ogawa More articles by this author Taichi Nagami More articles by this author Haruki Anjiki More articles by this author Chiaki Koike More articles by this author Naoko Arichi More articles by this author Shigenobu Nakamura More articles by this author Takeo Hiraoka More articles by this author Masahiro Sumura More articles by this author Hiroaki Yasumoto More articles by this author Hiroaki Shiina 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.

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.

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.

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.

Expression and aberrant promoter methylation of Wnt inhibitory factor-1 in human astrocytomas

BackgroundWnt inhibitory factor-1(WIF-1) acts as a Wnt-antagonists and tumor suppressor, but hypermethylation of WIF-1 gene promoter and low expression activate Wnt signaling aberrantly and induce the development of various human tumors. With this work we intended to investigate the expression and promoter methylation status of WIF-1 gene in human astrocytomas.MethodsThe tissue samples consisted of 53 astrocytomas and 6 normal brain tissues. The expression levels of WIF-1 were determined by immunohistochemistry and semiquantitative RT-PCR. The results were analyzed in correlation with clinicopathological data. Methylation status of WIF-1 gene promoter was investigated using methylation specific PCR. The relationship between methylation and expression of the genes was analyzed.ResultsThe average expression levels of WIF-1 protein and mRNA in astrocytomas were decreased significantly compared with normal control tissues. The protein and mRNA expression of WIF-1 gene in astrocytomas was decreased with the increase of pathological grade. Furthermore, WIF-1 promoter methylation was observed by MS-PCR in astrocytomas which showed significant reduction of WIF-1 expression. The WIF-1 promoter hypermethylation was associated with reduced expression of WIF-1 expression.ConclusionOur results demonstrate that the WIF-1 gene is frequently down-regulated or silenced in astrocytomas by aberrant promoter methylation. This may be an important mechanism in astrocytoma carcinogenesis.

Epigenetic silencing of WIF-1 in hepatocellular carcinomas

To examine the expression profile and promoter methylation status of WIF-1 in hepatocellular carcinoma (HCC) and identify the possible relationship between the WIF-1 expression pattern and promoter methylation status. Quantitative real-time PCR was performed to detect mRNA level of WIF-1 in 4 HCC cell lines, 15 paired HCC clinical samples and 3 normal liver tissues. Methylation-specific PCR and bisulfite DNA sequencing were used in methylation analysis. In vitro assays for HCC cells, colony formation and cell proliferation assay were carried out to observe the effect of WIF-1 on cell growth; TOP-flash luciferase analysis was employed to determine its role in the Wnt pathway. Quantitative real-time PCR analysis showed the extensive low expression of WIF-1 mRNA in HCC, and this down-regulation was generally dependent on the degree of methylation at its promoter region. In vitro assays indicated WIF-1 can inhibit cell growth by blocking Wnt signaling in HCC cells. WIF-1 silencing as a result of its promoter hypermethylation may be a frequent event in HCC.

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.

Epigenetic Inactivation of Wnt Inhibitory Factor-1 Plays an Important Role in Bladder Cancer through Aberrant Canonical Wnt/β-Catenin Signaling Pathway

Aberrant activation of the Wingless-type (Wnt) pathway plays a significant role in the pathogenesis of several human cancers. Wnt inhibitory factor-1 (Wif-1) was identified as one of the secreted antagonists that can bind Wnt protein. We hypothesize that Wif-1 plays an important role in bladder cancer pathogenesis. To test this hypothesis, epigenetic and genetic pathways involved in the Wif-1 gene modulation and expression of Wnt/beta-catenin-related genes were analyzed in 4 bladder tumor cell lines and 54 bladder tumor and matched normal bladder mucosa. Wif-1 mRNA expression was significantly enhanced after 5-aza-2'-deoxycytidine treatment in bladder tumor cell lines. Wif-1 promoter methylation level was significantly higher and Wif-1 mRNA expression was significantly lower in bladder tumor samples than in bladder mucosa samples. In the total bladder tumor and bladder mucosa samples, an inverse correlation was found between promoter methylation and Wif-1 mRNA transcript levels. However, loss-of-heterozygosity at chromosome 12q14.3 close to the Wif-1 gene loci was a rare event (3.7%). Nuclear accumulation of beta-catenin was significantly more frequent in bladder tumor than in bladder mucosa and inversely correlated with Wif-1 expression. In addition, known targets of the canonical Wnt/beta-catenin signaling pathway, such as c-myc and cyclin D1, were up-regulated in bladder tumor compared with bladder mucosa, and this up-regulation was associated with reduced Wif-1 expression at both mRNA and protein levels. Furthermore, transfection of Wif-1 small interfering RNA into bladder tumor cells expressing Wif-1 mRNA transcripts had increased levels of c-myc and cyclin D1 and accelerated cell growth. This is the first report showing that CpG hypermethylation of the Wif-1 promoter is a frequent event in bladder tumor and may contribute to pathogenesis of bladder cancer through aberrant canonical Wnt/beta-catenin signaling pathway. The present study elucidates novel pathways that are involved in the pathogenesis of bladder cancer.