Wnt Inhibitory Factor-1 Methylation Research Articles

Epigenetic regulation of human WIF1 and DNA methylation situation of WIF1 and GSTM5 in urothelial carcinoma

WNT inhibitory factor 1 (WIF1) is known to function as a tumor suppressor gene; it inhibits oncogene activation by preventing WNT signaling. This study investigated the epigenetic regulation of WIF1 gene in bladder cancer. We observed a positive relationship between WIF1 mRNA expression and survival probability of bladder cancer patients. The WIF1 gene expression could be enhanced by DNA demethylation drug 5-aza-2′-deoxycytidine (5-aza-dC) and histone deacetylase inhibitor trichostatin A (TSA), suggesting that epigenetic modifications could regulate WIF1 gene expression. Overexpression of WIF1 inhibited cell proliferation and migration in 5637 cells, confirming the tumor suppressor role of WIF1. 5-Aza-dC dose dependently increased WIF1 gene expression while reducing DNA methylation level, suggesting that reversing WIF1 DNA methylation could activate its gene expression. We collected the cancer tissues and urine pellets of bladder cancer patients and only urine pellets from non-bladder cancer volunteers for DNA methylation analysis, but the methylation level of WIF1 gene −184 to +29 did not differ between patients and controls. We also analyzed glutathione S-transferase Mu 5 (GSTM5) gene methylation level because GSTM5 DNA hypermethylation was suggested to be a tumor biomarker in our previous study. It confirmed a higher GSTM5 DNA methylation in bladder cancer patients than in controls. In summary, this study suggests that the 5-aza-dC activated WIF1 gene which showed an anti-cancer effect, while WIF1 promoter −184 to +29 did not provide a suitable methylation assay region in clinical samples. In contrast, GSTM5 promoter −258 to −89 is a useful region for DNA methylation assay because it shows a higher methylation level in bladder cancer patients.

20(S)- Protopanaxadiol suppresses hepatic stellate cell activation via WIF1 demethylation-mediated inactivation of the Wnt/β-catenin pathway

Background20(S)-protopanaxadiol (PPD), one of the main components of ginseng, has anti-inflammatory, anti-estrogenic, and anti-tumor activities. It is known that activated hepatic stellate cells (HSCs) are the primary producers of extracellular matrix (ECM) in the liver, and the Wnt/β-catenin pathway participates in the activation of HSCs. We aimed to explore whether PPD inhibits liver fibrosis is associated with the Wnt/β-catenin pathway inactivation. MethodsThe anti-fibrotic roles of PPD were examined both in vitro and in vivo. We also examined the levels of Wnt inhibitory factor 1 (WIF1), DNA methyltransferase 1 (DNMT1) and WIF1 methylation. ResultsPPD obviously ameliorated liver fibrosis in carbon tetrachloride (CCl4)-treated mice and reduced collagen deposition. PPD also suppressed the activation and proliferation of primary HSCs. Notably, PPD inhibited the Wnt/β-catenin pathway, reduced TCF activity, and increased P-β-catenin and GSK-3β levels. Interestingly, WIF1 was found to mediate the inactivation of the Wnt/β-catenin pathway in PPD-treated HSCs. WIF1 silencing suppressed the inhibitory effects of PPD on HSC activation and also restored α-SMA and type I collagen levels. The downregulation of WIF1 expression was associated with the methylation of its promoter. PPD induced WIF1 demethylation and restored WIF1 expression. Further experiments confirmed that DNMT1 overexpression blocked the effects of PPD on WIF1 expression and demethylation and enhanced HSC activation. ConclusionPPD up-regulates WIF1 levels and impairs Wnt/β-catenin pathway activation via the down-regulation of DNMT1-mediated WIF1 methylation, leading to HSC inactivation. Therefore, PPD may be a promising therapeutic drug for patients with liver fibrosis.

Gene Methylation and Silencing of WIF1 Is a Frequent Genetic Abnormality in Mantle Cell Lymphoma.

We have previously shown that the Wnt canonical pathway (WCP) is constitutively active in most cases of mantle cell lymphoma (MCL). Here, we aimed to elucidate the mechanisms underlying this biochemical deregulation. We hypothesized that gene methylation/silencing of WIF1 (Wnt inhibitory factor-1), a physiologic inhibitor of WCP, contributes to the deregulation of WCP and promotes cell growth in MCL. In support of this hypothesis, we found that the expression of WIF1 was detectable in none of the 4 MCL cell lines, and in only 2 of 5 tumors (40%) examined. Using methylation-specific PCR, we found evidence of gene methylation of WIF1 in 4 of 5 cell lines (80%) and in 24 of 29 (82%) tumors. The addition of the demethylation agent 5-aza-2′-deoxycytidine to Mino and JeKo-1, two WIF1-negative cell lines, restored the expression of WIF1 mRNA in these cells. Gene transfection of WIF1 into JeKo-1 and Mino cells significantly reduced cell growth, and this finding correlated with substantial downregulations of various proteins in WCP, such as β-catenin and pGSK-3β. In conclusion, our results support the concept that gene methylation/silencing of WIF1 is a frequent event in MCL, and this abnormality contributes to the aberrant activation of WCP. These results have provided further evidence that aberrant Wnt signaling is pathogenetically important in MCL and it may represent a potential therapeutic target.

Promoter methylation of WNT inhibitory factor-1 may be associated with the pathogenesis of multiple human tumors.

We investigated the association of WNT inhibitory factor-1 (WIF-1) gene methylation with the pathogenesis of multiple human tumors, using a meta-analysis based approach. Electronic databases and manual search was additionally employed to retrieve relevant published literature. The cohort studies relating to tumor and WIF-1 were screened based on predefined selection criteria, and all extracted data from the selected studies were analyzed through STATA software. Sixteen studies were finally enrolled in our study involved 1112 tumor samples and 612 adjacent normal samples. The study result showed that WIF-1 gene methylations in tumor tissues were significantly higher compared with adjacent/normal tissues. The result of subgroup analysis on ethnicity revealed that in the Caucasians, Asians, and Africans, the methylation status of WIF-1 gene in tumor tissues was higher than adjacent/normal tissues. Further subgroup analysis on disease types revealed that WIF-1 gene methylation status is a widespread phenomenon that is, observed in tumor tissues of patients with multiple human tumors compared with that in adjacent/normal tissues. Interestingly, there was no significant difference in WIF-1 gene methylation between tumor tissues among patients with lung cancer, gastric cancer, astrocytoma, and adjacent/normal tissues, indicating the WIF-1 gene methylation not a general nonspecific phenomenon. WIF-1 gene methylation in tumor tissues was significantly more frequent as compared to that in adjacent normal tissues, indicating that WIF-1 gene methylation may be an important event in the pathogenesis of multiple human tumors.

Diagnostic value of WIF1 methylation for colorectal cancer: a meta-analysis.

As a common antagonist of Wnt/β-catenin signaling, Wnt inhibitory factor 1 (WIF1) plays an important role in the tumor progression. The aim of our meta-analysis was to summarize the diagnostic value of WIF1 methylation in colorectal cancer (CRC). Eligible studies were retrieved by a systemic search among PubMed, Embase, CNKI, and Wanfang literature databases. The diagnostic value of WIF1 methylation for CRC was assessed by the summary receiver operating characteristics (SROC) test. Our meta-analysis of 12 studies between 1420 CRC samples and 946 control samples showed that WIF1 hypermethylation was significantly associated with CRC (P < 0.001, OR = 30.10, 95% CI = 19.48-46.50). WIF1 hypermethylation, as a diagnostic biomarker for CRC, has a pooled sensitivity of 0.40 (95% CI: 0.37-0.42), a pooled specificity of 0.95 (95% CI: 0.93-0.96), a pooled positive-likelihood ratio (PLR) of 8.65 (95% CI, 4.47-16.73), and a pooled negative-likelihood ratio (NLR) of 0.41 (95% CI, 0.30-0.55), a diagnostic odds ratio (DOR) of 26.86 (95% CI: 15.73-45.89), and an area under the curve (AUC) of 0.9115. In conclusion, our study established that WIF1 hypermethylation might be a promising diagnostic biomarker for CRC.

The effect of EBV on WIF1, NLK, and APC gene methylation and expression in gastric carcinoma and nasopharyngeal cancer.

Epstein-Barr virus (EBV) is an important DNA tumor virus that is associated with approximately 10% of gastric carcinomas and 99% of nasopharyngeal cancers (NPC). DNA methylation and microRNAs (miRNAs) are the most studied epigenetic mechanisms that can prompt disease susceptibility. This study aimed to detect the effect of EBV on Wnt inhibitory factor 1 (WIF1), Nemo-like kinase (NLK), and adenomatous polyposis coli (APC) gene methylation, and expression in gastric carcinoma and NPC. The WIF1, NLK, and APC gene mRNA expression levels were measured by real-time quantitative RT-PCR in four EBV-positive cell lines and four EBV-negative cell lines. Bisulfite genomic sequencing or methylation-specific PCR was used to detect the methylation status of the WIF1, NLK, and APC promoters. All cell lines were treated with 5-azacytidine (5-aza-dC), miR-BART19-3p mimics or an inhibitor, and analyzed by flow cytometry and MTT cell proliferation assays. The WIF1, NLK, and APC promoters were hypermethylated in all eight cell lines. 5-Aza-dC displayed a growth inhibitory effect on cells . After transfection with miR-BART19-3p mimics, the expression of WIF1, and APC decreased, and the cellular proliferation rate increased. After transfection with the miR-BART19-3p inhibitor, the expression levels were higher, and the cell growth was inhibited. In the NPC and GC cell lines, the promoters of WIF1, NLK, and APC are highly methylated, and the expression of these three genes is regulated by miR-BART19-3p. The activity of the Wnt pathway in EBV-associated tumors may be enhanced by miR-BART19-3p.

Wnt inhibitory factor 1 (WIF1) methylation and its association with clinical prognosis in patients with chondrosarcoma

Chondrosarcoma (CS) is a rare cancer, but it is the second most common primary malignant bone tumor and highly resistant to conventional chemotherapy and radiotherapy. Aberrant DNA methylation in the promoter CpG island of Wnt inhibitory factor 1 (WIF1) has been observed in different cancers. However, no studies have shown the relationship between WIF1 methylation and CS. In this study, we found promoter methylated WIF1 in both CS cell lines (CS-1 and SW1353) and tumor tissues. Western blot analysis confirmed loss WIF1 expression and activation of Wnt pathway proteins (Wnt5a/b, LRP6, and Dvl2). We subsequently examined the correlation between levels of WIF1 methylation and overall survival (OS) and progression-free survival (PFS) in CS patient samples with a follow-up spanning 234 months (mean: 57.6 months). Kaplan-Meier survival curves and log-rank tests revealed that high levels of WIF1 methylation were associated with lower OS and PFS rates (p < 0.05). Multivariate Cox hazard analysis suggested that detection of high level methylation of WIF1 could be an independent prognostic factor in OS and PFS. In conclusion, we found that WIF1 is epigenetically silenced via promoter DNA methylation in CS and propose that WIF1 methylation may serve as a potential prognostic marker for patients with CS.

Hypermethylation of WIF1 and its inhibitory role in the tumor growth of endometrial adenocarcinoma.

Endometrial carcinoma is the most common malignancy of the female genital tract and is the fourth most common malignancy among women worldwide. Endometrial adenocarcinoma (EAC) accounts for ~80% of endometrial carcinoma cases. Numerous critical genetic events have been determined to serve an essential role in EAC progression; however, the precise molecular mechanisms underlying EAC progression remain unclear. Pyrosequencing and methylation-specific PCR were used to detect the methylation status of Wnt inhibitory factor 1 (WIF1). Immunohistochemistry and western blot were used to detect the expression of WIF1, Wnt family member 1 and other related pathways. The anticancer role of WIF1 in EAC was investigated in vitro and in vivo. Two of the three EAC cases exhibited significantly high methylation in five CpG sites, and the WIF1 methylation rate in EAC and endometrial tissues was 43.4 and 8%, respectively (P<0.05). The kappa consistency coefficient was −0.369 between methylation and mRNA expression (P<0.05) and WIF1 expression levels were significantly downregulated in EAC tissues compared with non-tumorous tissues (P<0.01). The 5-year overall survival rates were significantly lower for patients with tumors that negatively expressed WIF1 when compared with the 77.9% exhibited by those with positive WIF1 expression. Furthermore, the proliferation rate of KLE cells was significantly reduced following 5-aza-20-deoxycytidine treatment or WIF1 overexpression in vitro and in vivo, which may be associated with downregulated c-Myc and phosphorylated-extracellular signal-regulated kinase expression. These results demonstrated the important role of WIF1 in EAC tumorigenesis, and suggested that WIF1 may be a potential drug target in EAC treatment.

Retracted Article] Methylation of the Wnt Signaling Antagonist, Wnt Inhibitory Factor 1 and Dickkopf-1 Genes in Acute Myeloid Leukemia at the Time of Diagnosis

Background: In acute myeloid leukemia (AML), a large number of tumor suppressor genes are silenced through DNA methylation such as CDKN2B and p73. Wnt inhibitory factor 1 (WIF1) and Dickkopf-1 (DKK-1) are negative regulator of the Wnt signaling pathway. Objectives: In the present study, we studied the methylation status of WIF1 and DKK-1 genes in AML patients. Patients and Methods: In this case-control study, blood samples from 120 AML patients and 25 healthy control subjects collected, isolated DNA was treated with sodium bisulphite and examined by methylation specific PCR (MS-PCR) with primers specific for methylated and unmethylated sequences of the WIF1 and DKK-1 genes. Results: The frequency of aberrant hypermethylation of WIF1 and DKK-1 genes in AML patients determined 35% (42/120) and 28.3% (34/120), respectively. In addition, for all subjects in control group, methylation of WIF1 and DKK-1 genes were negative. Patients with M0 subtype of French-American-British (FAB)-AML had the highest incidence of hypermethylation of WIF1 (P = 0.003) and DKK-1 (P = 0.005) genes. Conclusions: The present study showed that, like many solid tumors, WIF1 and DKK-1 genes methylation also occurs in AML. The study of other antagonists of Wnt signaling pathway are recommended.

Expression and promoter methylation of Wnt inhibitory factor-1 in the development of oral submucous fibrosis.

Oral squamous cell carcinoma (OSCC) is a type of head and neck malignancy with a high mortality rate. Oral submucous fibrosis (OSF) is the pre-cancerous lesion of OSCC, whose molecular mechanisms in OSCC tumorigenesis remain largely unclear. Activation of the Wnt/β-catenin signaling pathway plays an important role in oral mucous carcinogenesis, although rare mutations of Wnt signaling molecules are found in OSCC, suggesting an epigenetic mechanism mediating aberrant Wnt/β‑catenin signaling in OSCC. Wnt inhibitory factor-1 (WIF1) is an Wnt antagonist, and its downregulation and methylation have been reported in a number of malignancies. However, the expression and methylation of WIF1 in the development of OSF have yet to be reported. In the present study, we investigated the WIF1 expression level by immuno-histochemical staining and semi‑quantitative RT-PCR in normal oral, OSF and OSCC tissues, as well as the methylation status by methylation-specific PCR and bisulfite genomic sequencing. The results showed that WIF1 was readily expressed in normal oral mucous tissues, but decreased gradually in OSF early, moderately advanced and advanced tissues, and was less expressed in OSCC tissues. Moreover, WIF1 was able to translocate from the nuclear to cytoplasm in OSF and OSCC tissues. Furthermore, WIF1 was frequently methylated in OSCC cases with betel quid chewing habit, but not in normal oral mucous and different stages of OSF tissues, suggesting WIF1 methylation is tumor-specific in the development of OSF. Thus, the results demonstrated that WIF1 is frequently downregulated or silenced by promoter methylation in the carcinogenesis of OSF, which serves as a potential epigenetic biomarker for the early detection of OSCC.

Promoter methylation of WNT inhibitory factor-1 and expression pattern of WNT/β-catenin pathway in human astrocytoma: pathologic and prognostic correlations

WNT inhibitory factor-1 (WIF1) is an antagonist of the WNT signaling pathway. We investigated the relationship between WIF1 promoter methylation and regulation of the WNT/β-catenin signaling pathway, tumor grade, and survival in patients with astrocytoma. This study included 86 cases of astrocytoma, comprising 20 diffuse astrocytomas and 66 glioblastomas. In addition, 17 temporal lobectomy specimens from patients with epilepsy were included as controls. The ratio of methylated DNA to total methylated and unmethylated DNA (% methylation) was measured by methylation- and unmethylation-specific PCR. Representative tumor tissue was immunostained for WIF1, β-catenin, cyclin D1, c-myc, and isocitrate dehydrogenase 1. Levels of WIF1 promoter methylation, mRNA expression, and protein expression in a glioblastoma cell line were compared before and after demethylation treatment. The mean percent methylation of the WIF1 promoter in astrocytomas was higher than that in control brain tissue. WIF1 protein expression was lower in the tumor group with >5% methylation than in the group with <5% methylation. Cytoplasmic β-catenin staining was more frequently observed in tumors with a low WIF1 protein expression level. Demethylation treatment of a glioblastoma cell line increased WIF1 mRNA and protein expression. Increased WIF1 promoter methylation and decreased WIF1 protein expression were not related to patient survival. In conclusion, WIF1 expression is downregulated by promoter methylation and is an important mechanism of aberrant WNT/β-catenin pathway activation in astrocytoma pathogenesis.

Prognostic value of Wnt inhibitory factor-1 expression in hepatocellular carcinoma that is independent of gene methylation

Recently, Wnt inhibitory factor-1 (WIF-1) was found to be epigenetically inactivated in several solid tumors, but the biological and clinical relevance of WIF-1 methylation and expression status in hepatocellular carcinoma (HCC) are still unclear. In the present study, reverse transcription polymerase chain reaction (PCR) and methylation-specific PCR were used to examine the WIF-1 expression and methylation in HCC cell lines. In addition, methylation and expression status of WIF-1 in 105 HCC cases were correlated with clinicopathological parameters and prognosis after tumor resection. WIF-1 was expressed in one HCC cell line and L02, both of which were not methylated in promoter region. DNA hypermethylation of WIF-1 promoter was identified in the other four HCC cell lines without WIF-1 expression. In neoplastic and non-neoplastic tissue samples, the rates of WIF-1 methylation were 61.9% and 37.1% (P = 0.001), respectively. WIF-1 was significantly downregulated in neoplastic tissues at messenger ribonucleic acid (mRNA) level, as compared to adjacent non-neoplastic tissues (P = 0.006). A significant inverse association was observed between WIF-1 methylation of and WIF-1 expression (P 0.017, R = -0.232). Methylation of WIF-1 was not associated with patient survival. In contrast, patients whose tumors exhibited negative WIF-1 mRNA expression had lower rates of overall survival. These findings suggested that aberrant methylation of WIF-1 is a common event in hepatocarcinogenesis. In addition, expression, but not methylation, of WIF-1 is a predictor of good outcome in patients undergoing resection of HCC.

Epigenetic Alteration of the Wnt Inhibitory Factor-1 Promoter Is Common and Occurs in Advanced Stage of Tunisian Nasopharyngeal Carcinoma

ABSTRACTActivation of the wingless-type (Wnt) signaling pathway is common in cancers. The Wnt inhibitory factor-1 (WIF-1) is a secreted antagonist that acts by binding to Wnt ligands. We examined by methylation-specific PCR (MSP), whether WIF-1 is inactivated in 68 nasopharyngeal carcinomas (NPC), and 10 normal mucosa. We showed that the WIF-1 promoter was methylated in 89.7% of tumors, whereas all normal mucosa were unmethylated. The WIF-1 methylation was associated with the tumor, node, and metastasis (TNM) (p = .003) and the age (p = .014). The Wnt-5a mRNA was higher in tumors and correlated with TNM (p = .012). The methylation of WIF-1 contributes to the activation of the Wnt pathway in NPC.

Downregulation of WIF-1 by hypermethylation in astrocytomas

Wnt inhibitory factor-1 (WIF-1) acts as a Wnt antagonist and tumor suppressor, but hypermethylation of WIF-1 gene promoter and low expression of WIF-1 activate Wnt signaling aberrantly and induce the development of several human tumors. By using RT-PCR, immunohistochemistry and methylation-specific PCR, we analyzed the expression and methylation of WIF-1 in 4 normal brain tissues, 35 freshly resected astrocytoma tissues and 4 glioblastoma-derived cell lines. Significant downregulation of WIF-1 mRNA and protein expression levels was observed in astrocytoma tissues compared with normal brain tissues. Significant association between WIF-1 downregulation and pathological grade of astrocytomas was found. WIF-1 gene aberrant methylation was observed in 19 of 35 (54.29%) tumor samples. The promoter methylation tumors showed low WIF-1 protein and mRNA expression, whereas the promoter unmethylation tumors displayed high protein and mRNA expression levels. Moreover, complete absence of WIF-1 mRNA expression was observed in four cell lines, whereas treatment with demethylating agent, 5-aza-2'-deoxycytidine, restored WIF-1 expression. Our results suggested that the WIF-1 gene is frequently silenced in astrocytoma by aberrant promoter methylation. This may be an important mechanism in astrocytoma carcinogenesis.

Abstract 4932: Genistein Can Induce Demethylation Of Wnt Negative Regulatory Genes In Prostate Cancer Cells

Abstract Among American men, prostate cancer is a prevalent malignancy that accounts for an estimated 192,280 new cases and 27,360 deaths in 2009. Previous work has determined that the adenomatous polyposis coli (APC) gene that negatively regulates the Wnt pathway is hypermethylated in a high proportion of prostate cancers. We hypothesized that methylation of additional Wnt negative regulatory genes may also play a role in prostate cancer progression such as SRY box 7 (SOX7), Wnt inhibitory factor 1 (WIF1), secreted frizzled-related protein 1 (SFRP1) and dickkopf-related protein 3 (DKK3). To test this hypothesis, the basal level methylation status was tested in several prostate cancer patient tumor and adjacent normal samples by methylation specific PCR. When the methylation status of some of these genes was explored, there was variation in the methylation of some of these genes, particularly DKK3 among different patients. This preliminary finding may suggest that the methylation status of some of these genes may play a role in the progression of prostate cancer. In this study, we propose that demethylation of these Wnt negative regulatory genes may allow for a decrease activity of the Wnt signaling pathway. Genistein is a soy isoflavone that has many potentially therapeutic activities, including demethylation of DNA. We hypothesized that treatment of prostate cancer cells could lead to demethylation and re-expression of these genes. To test our hypothesis, prostate cancer cell lines LNCAP and DU145 were treated with for 6 days with concentrations of 5, 10, and 20 µM genistein. We observed a dose-dependent reduction in methylation of SOX7, DKK3, WIF1, and SFRP1. Taken together, these data indicate that genistein may have therapeutic value for patients with prostate cancer. As a therapeutic agent, genistein treatment has potential solely or in combination with other compounds to demethylate Wnt negative regulatory genes and possibly impact patient outcomes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4932.

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.

Procaine and procainamide inhibit the Wnt canonical pathway by promoter demethylation of WIF-1 in lung cancer cells

Secreted Wingless type (Wnt) ligands have previously been shown to be involved in tumor developmental processes and oncogenesis. Aberrant promoter methylation of Wnt inhibitory factor-1 (WIF-1) is a fundamental mechanism of epigenetic silencing in human cancers. Procaine, a local anesthetic drug, and procainamide, a drug for the treatment of cardiac arrhythmias, have been reported as inhibitors of DNA methylation, causing demethylation and reactivation of methylation-silenced genes such as RARbeta and GSTP1. The promoter demethylation of WIF-1 has not previously been reported on. We demonstrated previously that WIF-1 is silenced due to promoter hypermethylation in lung cancer cell lines. In this study, we demonstrate promoter demethylation of WIF-1; restoration of WIF-1 expression, and underexpression of cytosolic beta-catenin protein and TCF reporter activity, after procaine and procainamide treatment in H460 and A549 cell lines. Our results provide the first evidence that procaine and procainamide reactivate WIF-1 in these cancer cells and downregulate the Wnt canonical pathway. These results further suggest that procaine and procainamide may have a potential use for preventing the development of lung cancer.

Prognostic relevance of Wnt-inhibitory factor-1 (WIF1) and Dickkopf-3 (DKK3) promoter methylation in human breast cancer

BackgroundSecreted Wnt signaling antagonists have recently been described as frequent targets of epigenetic inactivation in human tumor entities. Since gene silencing of certain Wnt antagonists was found to be correlated with adverse patient survival in cancer, we aimed at investigating a potential prognostic impact of the two Wnt antagonizing molecules WIF1 and DKK3 in breast cancer, which are frequently silenced by promoter methylation in this disease.MethodsWIF1 and DKK3 promoter methylation were assessed by methylation-specific PCR with bisulfite-converted DNA from 19 normal breast tissues and 150 primary breast carcinomas. Promoter methylation was interpreted in a qualitative, binary fashion. Statistical evaluations included two-sided Fisher's exact tests, univariate log-rank tests of Kaplan-Meier curves as well as multivariate Cox regression analyses.ResultsWIF1 and DKK3 promoter methylation were detected in 63.3% (95/150) and 61.3% (92/150) of breast carcinoma samples, respectively. In normal breast tissues, WIF1 methylation was present in 0% (0/19) and DKK3 methylation in 5.3% (1/19) of samples. In breast carcinomas, WIF1 methylation was significantly associated with methylation of DKK3 (p = 0.009). Methylation of either gene was not associated with clinicopathological parameters, except for DKK3 methylation being associated with patient age (p = 0.007). In univariate analysis, WIF1 methylation was not associated with clinical patient outcome. In contrast, DKK3 methylation was a prognostic factor in patient overall survival (OS) and disease-free survival (DFS). Estimated OS rates after 10 years were 54% for patients with DKK3-methylated tumors, in contrast to patients without DKK3 methylation in the tumor, who had a favorable 97% OS after 10 years (p < 0.001). Likewise, DFS at 10 years for patients harboring DKK3 methylation in the tumor was 58%, compared with 78% for patients with unmethylated DKK3 (p = 0.037). Multivariate analyses revealed that DKK3 methylation was an independent prognostic factor predicting poor OS (hazard ratio (HR): 14.4; 95% confidence interval (CI): 1.9–111.6; p = 0.011), and short DFS (HR: 2.5; 95% CI: 1.0–6.0; p = 0.047) in breast cancer.ConclusionAlthough the Wnt antagonist genes WIF1 and DKK3 show a very similar frequency of promoter methylation in human breast cancer, only DKK3 methylation proves as a novel prognostic marker potentially useful in the clinical management of this disease.

Preferential Hypermethylation of the DKK-1 Promoter in Core-Binding Factor Leukaemia.

The Dickkopf-1 (DKK-1) gene product is an extracellular Wnt inhibitor. Hypermethylation of the DKK-1 promoter results in transcriptional silencing and may play an important role in cancer development. Here, we investigated hypermethylation of the DKK-1 promoter in patients with acute myeloid leukaemia (AML), especially core-binding factor (CBF) leukaemia. The methylation status of DKK-1 was analyzed by methylation-specific polymerase chain reaction in 47 patients with AML. DKK-1 methylation was found in 14 (29.8%) of the patients, and more frequently in those with CBF leukaemia (6 of 12 patients), than in those with acute promyelocytic leukaemia (0 of 6 patients) (P = .03). In contrast, Wnt inhibitory factor-1 methylation was found in acute promyelocytic leukaemia (4 of 6 patients) but not in CBF leukaemia (0 of 12 patients) (P = .001). Multivariate analyses suggested that DKK-1 methylation was a risk factor for poorer overall survival. Sequential analysis using 4 paired samples obtained at diagnosis and relapse suggested that DKK-1 methylation was involved in the progression of leukaemia. DKK-1 methylation may therefore be involved in leukaemogenesis, especially in CBF leukaemia, and may be a useful prognostic marker in AML.

Epigenetic alteration of the Wnt inhibitory factor‐1 promoter occurs early in the carcinogenesis of Barrett's esophagus

The role of Wnt antagonists in the carcinogenesis of esophageal adenocarcinoma (EAC) remains unclear. We hypothesized that downregulation of the Wnt inhibitory factor-1 (WIF-1) might be involved in the neoplastic progression of Barrett's esophagus (BE). We analyzed the DNA methylation status of the WIF-1 promoter in normal, preneoplastic, and neoplastic samples from BE patients and in EAC cell lines. We investigated the role of WIF-1 on EAC cell growth and the chemosensitization of the cells to cisplatin. We found that silencing of WIF-1 correlated with promoter hypermethylation. EAC tissue samples showed higher levels of WIF-1 methylation compared to the matched normal epithelium. In addition, we found that WIF-1 hypermethylation was more frequent in BE samples from patients with EAC than in BE samples from patients who had not progressed to EAC. Restoration of WIF-1 in cell lines where WIF-1 was methylation-silenced resulted in growth suppression. Restoration of WIF-1 could sensitize the EAC cells to the chemotherapy drug cisplatin. Our results suggest that silencing of WIF-1 through promoter hypermethylation is an early and common event in the carcinogenesis of BE. Restoring functional WIF-1 might be used as a new targeted therapy for the treatment of this malignancy.