Wnt Inhibitory Factor-1 Promoter Research Articles

WIF1 was downregulated in cervical cancer due to promoter methylation.

Wnt inhibitory factor 1 (WIF1) is frequently downregulated in a variety of cancer due to promoter methylation. However, the methylation status of the WIF1 promoter in cervical cancer remains unclear. This study aimed to elucidate the mechanism by which WIF1 promoter methylation contributes to cervical cancer development. The expression of WIF1 in cervical cancer tissues was examined by immunohistochemistry. The methylation status of the WIF1 promoter in cervical cancer cells was detected by methylation specific PCR. WIF1 mRNA levels and protein levels were detected by PCR and Western blot analysis. We found that WIF1 expression was low in cervical cancer tissues compared to adjacent normal cervical tissues. The WIF1 promoter was methylated in the cervical cancer SiHa cell line but not in the normal cervical epithelial cell line Ect1. Correspondingly, WIF1 mRNA levels and protein levels were significantly lower in SiHa cells than in Ect1 cells. Treatment with 5-aza-2-deoxycytidine (AZA) led to the upregulation of WIF1 mRNA and protein levels in SiHa cells, but the effects were abrogated by treatment with WIF1 siRNA. In addition, AZA treatment induced apoptosis and inhibited the invasion of SiHa cells, and the effects were abrogated by WIF1 siRNA. The protein levels of survivin, c-myc and cyclinD1 were significantly lower in SiHa cells treated with AZA, but their levels were upregulated after treatment with WIF1 siRNA. In conclusion, the methylation of the WIF1 promoter leads to the downregulation of WIF1 and the activation of Wnt/β-catenin signaling in cervical cancer cells. WIF1 is a tumor suppressor that is inactivated in cervical cancer.

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.

Aberrant promoter methylation of Wnt inhibitory factor-1 gene is a potential target for treating psoriasis

Psoriasis is a chronic inflammatory skin disease mediated by immune and complex genetic factors. The wingless-related integration site (Wnt) signaling pathway plays a critical role in psoriasis, but how the Wnt pathway is regulated in psoriatic skin and whether it can be exploited for therapeutic benefits is unclear. By comparing biopsies from healthy and psoriatic skin, we found that Wnt inhibitory factor 1 (WIF1), an inhibitor of Wnt signaling, showed reduced expression at both mRNA and protein levels in psoriatic skin. We then quantified methylation of the WIF1 gene promoter by DNA methylation sequencing and found that the WIF1 promoter region was hypermethylated. We further showed that recombinant WIF1 injection ameliorates the imiquimod (IMQ) mouse model of psoriasis. We also revealed that treatment with the DNA methylation inhibitor, decitabine, inhibited proliferation of immortalized human keratinocytes (HaCaT) in a psoriasis-like inflammatory environment. Finally, we applied decitabine to the IMQ mouse model and demonstrated that treatment of mice with decitabine ameliorates the disease. Therefore, our study reveals that methylation of the WIF1 gene is associated with the pathogenesis of psoriasis, and suggests that pharmacological targeting of DNA methylation is a potential treatment strategy for psoriasis.

A Single Nucleotide Polymorphism in the WIF1 Promoter Region Regulates the Wool Length in Rabbits

Wool length is an important trait in the rabbit wool industry. Wnt inhibitory factor 1 (WIF1), a key gene, regulates hair follicle growth and development. This study explores the WIF1 gene relationship with wool production traits in three different rabbit breeds, Rex rabbit, New Zealand rabbit, and Angora rabbit. The rabbit WIF1 gene sequence was obtained from GenBank, and PCR and Sanger sequencing were applied to screen SNP (single nucleotide polymorphism) from the gene exons and promoter regions. Sequencing results found no SNP in the PCR products of 10 exons from the rabbit WIF1 gene, indicating that the WIF1 gene was not polymorphic. Thus, the gene was conserved in all three rabbit breeds. Only one SNP was found in the WIF1 promoter region, associated with wool-producing traits in rabbits. Luciferase activity assays showed that the AA genotype had higher transcriptional activities than the AG or GG genotype (p < 0.01). In addition, WIF1 was significantly upregulated in the Rex rabbit compared to the New Zealand rabbit and Angora rabbit (p < 0.01). In summary, a key SNP in the WIF1 promoter region was found to be associated with rabbit fiber length trait.

A long non-coding RNA, HOTAIR, promotes cartilage degradation in osteoarthritis by inhibiting WIF-1 expression and activating Wnt pathway

BackgroundLong noncoding RNAs (lncRNAs) are recently found to be critical regulators of the epigenome. However, our knowledge of their role in osteoarthritis (OA) development is limited. This study investigates the mechanism by which HOTAIR, a key lncRNA with elevated expression in OA, affects OA disease progression.ResultsHOTAIR expression was greatly elevated in osteoarthritic compared to normal chondrocytes. Silencing and over-expression of HOTAIR in SW1353 cells respectively reduced and increased the expression of genes associated with cartilage degradation in OA. Investigation of molecular pathways revealed that HOTAIR acted directly on Wnt inhibitory factor 1 (WIF-1) by increasing histone H3K27 trimethylation in the WIF-1 promoter, leading to WIF-1 repression that favours activation of the Wnt/β-catenin pathway.ConclusionsActivation of Wnt/β-catenin signalling by HOTAIR through WIF-1 repression in osteoarthritic chondrocytes increases catabolic gene expression and promotes cartilage degradation. This is the first study to demonstrate a direct link between HOTAIR, WIF-1 and OA progression, which may be useful for future investigations into disease biomarkers or therapeutic targets.

Promoter hypermethylation of Wnt inhibitory factor-1 in patients with lung cancer: A systematic meta-analysis.

Background:Promoter hypermethylation of Wnt inhibitory factor-1 (WIF-1)—a tumor suppressor gene—has been detected in several types of human tumors. However, the association between WIF-1 promoter hypermethylation and lung cancer remains to be elucidated. Therefore, we conducted this study to evaluate the clinical significance of WIF-1 promoter hypermethylation in lung cancer.Methods:A comprehensive literature search was conducted to obtain eligible studies. The combined odds ratios (ORs) or hazard ratios and 95% confidence intervals were used to estimate the strength of associations.Results:A total of 8 eligible publications with 626 cases and 512 controls were included in our study. The combined ORs revealed that WIF-1 promoter hypermethylation was significantly higher in lung cancer than in controls (OR 10.53, P < 0.001). Moreover, WIF-1 promoter hypermethylation was significantly associated with smoking behavior (OR 1.88, P = 0.002). No significant correlation was found between WIF-1 promoter hypermethylation and sex status, age status, tumor stage, and pathological types in cancer. Multivariate analysis results indicated the absence of correlation between WIF-1 promoter hypermethylation and with relapse-free survival and overall survival. Subgroup analysis by sample type demonstrated that promoter hypermethylation of WIF-1 was significantly associated with an increased risk of lung cancer in the tissue (OR 7.89, P < 0.001), blood (OR 21.83, P = 0.034), and pleural effusion subgroups (OR 157.43, P = 0.001).Conclusions:Promoter hypermethylation of WIF-1 may play a crucial role in lung cancer carcinogenesis. It may be a noninvasive biomarker using blood or pleural effusion detection. WIF-1 promoter hypermethylation is correlated with smoking behavior, but not with sex status, age status, tumor stage, pathological types, and the prognosis of lung cancer patients in terms of relapse-free survival and overall survival. More investigations, including a larger number of subjects, are required to further confirm the findings of our analysis.

Correlations of Promoter Methylation in WIF-1, RASSF1A, and CDH13 Genes with the Risk and Prognosis of Esophageal Cancer.

BackgroundThis study was designed to explore the correlations of promoter methylation in Wnt inhibitory factor-1 (WIF-1), ras-association domain family member 1A (RASSF1A), and Cadherin 13 (CDH13) genes with the risk and prognosis of esophageal cancer (EC).Material/MethodsA total of 71 EC tissues from resection and 35 adjacent normal tissues were collected. Methylation status in the promoter region was detected by methylation- and non-methylation-specific primers. Corresponding mRNA levels were detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Correlations between the methylations of these 3 genes and clinicopathologic characteristics were analyzed. Kaplan-Meier method and Cox regression model were used to investigate the relationships between WIF-1, RASSF1A, and CDH13 promoter methylations and the prognosis of EC.ResultsCompared with adjacent normal tissues, the methylation frequencies of WIF-1, RASSF1A, and CDH13 genes were significantly higher but the mRNA levels of these 3 genes were significantly lower in EC tissues (all P<0.05). WIF-1 and CDH13 promoter methylations were associated with the degree of tumor differentiation and WIF-1 and RASSF1A promoter methylations were associated with age (all P<0.05). The survival rates of patients with WIF-1, RASSF1A, and CDH13 methylations were significantly lower than those of patients without methylation (all P<0.05). WIF-1, RASSF1A, and CDH13 promoter methylations were independent risk factors affecting the prognosis of EC (all P<0.05).ConclusionsWIF-1, RASSF1A, and CDH13 promoter methylations are associated with EC. The methylation levels are negatively related with the prognosis in EC.

Role of bacterial infection in the epigenetic regulation of Wnt antagonist WIF1 by PRC2 protein EZH2.

The Enhancer of Zeste Homolog-2 (EZH2) represses gene transcription through histone H3 lysine-27-trimethylation (H3K27me3). Citrobacter rodentium (CR) promotes crypt hyperplasia and tumorigenesis by aberrantly regulating Wnt/β-catenin signaling. We aimed at investigating EZH2’s role in epigenetically regulating Wnt/β-catenin signaling following bacterial infection. NIH:Swiss outbred and ApcMin/+ mice were infected with CR (108cfu); BLT1−/−ApcMin/+ mice, AOM/DSS-treated mice and de-identified human adenocarcinoma samples were models of colon cancer. Following infection with wild type but not mutant CR, elevated EZH2 levels in the crypt at days-6 and 12 (peak hyperplasia) coincided with increases in H3K27me3 and β-catenin levels, respectively. Chromatin immunoprecipitation revealed EZH2 and H3K27me3’s occupancy on WIF1 (Wnt Inhibitory Factor-1) promoter resulting in reduced WIF1 mRNA and protein expression. Following EZH2 knockdown via siRNA or EZH2-inhibitor DZNep either alone or in combination with HDAC inhibitor SAHA, WIF1 promoter activity increased significantly while overexpression of EZH2 attenuated WIF1-reporter activity. Ectopic overexpression of SET domain mutant (F681Y) almost completely rescued WIF1 reporter activity and partially rescued WIF1 protein levels while H3K27me3 levels were significantly attenuated suggesting that an intact methyltransferases activity is required for EZH2-dependent effects. Interestingly, while β-catenin levels were lower in EZH2-knocked-down cells, F681Y mutants exhibited only partial reduction in β-catenin levels. Besides EZH2, increases in miR-203 expression in the crypts at days-6 and 12 post-infection correlated with reduced levels of its target WIF1; overexpression of miR-203 in primary colonocytes decreased WIF1 mRNA and protein levels. Elevated levels of EZH2 and β-catenin with concomitant decrease in WIF1 expression in the polyps of CR-infected ApcMin/+ mice paralleled changes recorded in BLT1−/−ApcMin/+, AOM/DSS and human adenocarcinomas. Thus, EZH2-induced downregulation of WIF1 expression may partially regulate Wnt/β-catenin-dependent crypt hyperplasia in response to CR infection.

Downregulation of WIF-1 and Wnt5a in patients with colorectal carcinoma: clinical significance.

Activation of the wingless-type (Wnt) signaling pathway is common in various human cancers including colorectal cancer (CRC). Wnt inhibitory factor-1 (WIF-1) is a secreted antagonist that can bind Wnt ligands and therefore inhibits the Wnt signaling pathway. In this study, we aimed to analyze the expression of two members of Wnt signaling (WIF-1 and Wnt5a) in Tunisian patients with sporadic CRC. WIF-1 was frequently methylated in tumor tissues (87.95 %) compared to normal mucosa (39.54 %) and correlated with distant metastasis and vascular invasion (P = 0.001 and 0.037, respectively). The unmethylated profile of the WIF-1 promoter conferred a benefit to patients in terms of overall survival (P log rank = 0.024). In addition, in the group of patients with methylated WIF-1 promoter, the overall survival rate was significantly prolonged for those with small tumor size (<5 cm) and absence of distant metastasis (P log rank = 0.007 and 0.036, respectively). Aberrant CpG methylation of the WIF-1 promoter leads to transcriptional silencing of this tumor suppressor gene in tumor tissues (P = 0.001). Furthermore, we showed that the level of Wnt5a mRNA was significantly lower in tumor compared to normal tissues (P = 0.031) and lower still in those showing more aggressive behavior (presence of lymph nodes and advanced TNM stage). Our finding supports that WIF-1 is frequently methylated and that Wnt5a acts as a tumor suppressor gene in CRC. Loss of WIF-1 and Wnt5a functions results in more aggressive behavior of the disease.

Wnt inhibitory factor 1 suppresses cancer stemness and induces cellular senescence.

Hyperactivation of the Wingless-type (Wnt)/β-catenin pathway promotes tumor initiation, tumor growth and metastasis in various tissues. Although there is evidence for the involvement of Wnt/β-catenin pathway activation in salivary gland tumors, the precise mechanisms are unknown. Here we report for the first time that downregulation of the Wnt inhibitory factor 1 (WIF1) is a widespread event in salivary gland carcinoma ex-pleomorphic adenoma (CaExPA). We also show that WIF1 downregulation occurs in the CaExPA precursor lesion pleomorphic adenoma (PA) and indicates a higher risk of progression from benign to malignant tumor. Our results demonstrate that diverse mechanisms including WIF1 promoter hypermethylation and loss of heterozygosity contribute to WIF1 downregulation in human salivary gland tumors. In accordance with a crucial role in suppressing salivary gland tumor progression, WIF1 re-expression in salivary gland tumor cells inhibited cell proliferation, induced more differentiated phenotype and promoted cellular senescence, possibly through upregulation of tumor-suppressor genes, such as p53 and p21. Most importantly, WIF1 significantly diminished the number of salivary gland cancer stem cells and the anchorage-independent cell growth. Consistent with this observation, WIF1 caused a reduction in the expression of pluripotency and stemness markers (OCT4 and c-MYC), as well as adult stem cell self-renewal and multi-lineage differentiation markers, such as WNT3A, TCF4, c-KIT and MYB. Furthermore, WIF1 significantly increased the expression of microRNAs pri-let-7a and pri-miR-200c, negative regulators of stemness and cancer progression. In addition, we show that WIF1 functions as a positive regulator of miR-200c, leading to downregulation of BMI1, ZEB1 and ZEB2, with a consequent increase in downstream targets such as E-cadherin. Our study emphasizes the prognostic and therapeutic potential of WIF1 in human salivary gland CaExPA. Moreover, our findings demonstrate a novel mechanism by which WIF1 regulates cancer stemness and senescence, which might have major implications in the field of cancer biology.

Effects of the estrogen receptor antagonist fulvestrant on F344 rat prolactinoma models

The relationship between estrogen and prolactinoma is well documented. But the anti-tumor effects of a pure estrogen receptor antagonist fulvestrant on prolactinomas, especially in vivo, and the possible mechanisms are still unclear. Therefore, the aim of this study was to evaluate the effects of fulvestrant and the involvement of the Wnt signaling pathway on rat prolactinoma models. Forty female F344 rat prolactinoma models were established by subcutaneous administration of 10mg 17β-estradiol for 6weeks. Rats were intramuscularly injected with fulvestrant (0, 0.5, 3, 20, 40mg/kg), and tumor size, weight and serum prolactin (PRL) levels were evaluated before and after fulvestrant treatment at 3, 7 and 14days. Expression of estrogen receptor α (ERα), β-catenin and Wnt inhibitory factor-1 (WIF-1) in prolactinomas was measured using quantitative PCR and western blotting, and methylation of the WIF-1 promoter was investigated using pyrosequencing. Tumor size, weight and serum PRL levels were inhibited in dose-dependent and time-dependent manners after fulvestrant treatments. β-catenin expression was downregulated but WIF-1 expression was upregulated following fulvestrant treatment. The methylation of the CpG site of the WIF-1 promoter was negatively correlated to the expression of WIF-1. In addition, the anti-cell proliferation of fulvestrant on GH3 cells was partly disrupted by Wnt signaling pathway agonist SB 216763. In conclusion, fulvestrant inhibited tumor proliferation and PRL secretion of prolactinomas via ERα, and the Wnt signaling pathway was involved in this anti-tumor effect. Therefore, fulvestrant may be a potential new drug for prolactinomas.

Abstract A30: Epigenetic regulation of Wnt/β-catenin signaling in vivo by PRC2 protein EZH2

Abstract Introduction: Colon cancer results from the accumulation of both acquired genetic and epigenetic changes that transform normal glandular epithelium into invasive adenocarcinomas. The Enhancer of Zeste Homolog 2 (EZH2) protein is a core component of the Polycomb Repressive Complex 2 (PRC2) that represses gene transcription through histone H3 lysine 27 trimethylation (H3K27me3). EZH2 is overexpressed in several malignancies including colon cancer; EZH2's role in tumor initiation however, is less clear. Citrobacter rodentium (CR)-induced transmissible murine colonic hyperplasia (TMCH) that utilizes a type-three secretion system (T3SS) to activate Wnt/β-catenin signaling, represents the earliest molecular and functional changes associated with colon carcinogenesis; the increases in β-catenin recorded during TMCH however, are neither due to mutations in Adenomatous polyposis coli (Apc) or β-catenin (CTNNB1) gene. Aim: To investigate if EZH2 is critical in epigenetically regulating Wnt/β-catenin signaling in response to bacterial infection. Experimental Procedures: TMCH or tumorigenesis were induced by CR infection (108cfu) in wild type and ApcMin/+ mice, respectively. AOM (@10mg/Kg body wt.)/DSS (2.5%; 3 cycles) model of colitis-associated cancer and de-identified human adenoma and adenocarcinoma samples were used to further examine evidence of epigenetically-regulated Wnt/β-catenin signaling. Results: Following CR infection, EZH2 protein increased significantly in the crypt at day 6 and particularly at day 12 (peak hyperplasia) and coincided with expression of EZH2 target protein H3K27me3. The changes accrued were specific to CR as T3SS mutant escV failed to elicit alterations in either EZH2, H3K27me3 or crypt hyperplasia. Mechanistically, siRNA-mediated knockdown of EZH2 led to: i) attenuated cell migration, ii) reduced colonosphere formation and, iii) decreased β-catenin/TCF4-dependent TOPflash reporter activity due to loss of β-catenin. Chromatin immunoprecipitation revealed EZH2's occupancy on WIF1 (Wnt Inhibitory Factor 1) promoter resulting in reduced WIF1 mRNA and protein expression. Following EZH2 knockdown either via siRNA or EZH2-inhibitor DZNep (5μM), relative levels of H3K27me3 decreased which led to the transcriptional upregulation of WIF1 as was evidenced by WIF1 promoter activity. Besides EZH2, we also discovered increases in miR-203 expression in the crypts at days-6 and 12 post-infection that correlated with reduced levels of its target WIF1, at these time points; overexpression of miR-203 in Young Adult Mouse Colon primary cells resulted in decreased WIF1 transcription. Tumors in the distal colons of ApcMin/+ mice following CR infection expressed high levels of EZH2 and β-catenin and a concomitant decrease in WIF1. In response to AOM/DSS, colonic tumors at 24 weeks expressed high levels of EZH2 and β-catenin and a concomitant decrease in WIF1. Pharmacological intervention with honokiol (HNK; @5mg/Kg body wt. p.o.) a week prior to and during AOM/DSS-induced colon carcinogenesis reduced both tumor burden and tumor size. Immunohistochemical analysis of HNK-treated tissues revealed significant reduction in EZH2 and β-catenin staining while WIF1 expression was dramatically upregulated. In human adenomas/adenocarcinomas, simultaneous increases in EZH2 and β-catenin staining coincided with a corresponding decrease in WIF1 positivity. Conclusions: 1. CR infection induces EZH2 overexpression that correlates with aberrant Wnt/β-catenin signaling in the colonic crypts.2. EZH2 downregulates WIF1 expression which may correlate with earliest stages of colon tumorigenesis. 3. Honokiol-induced upregulation of WIF1 to block aberrant Wnt/β-catenin signaling may be a novel strategy to target epigenetic abnormalities for colon cancer prevention and therapeutics. Citation Format: Badal C. Roy, Dharmalingam Subramaniam, Ishfaq Ahmed, Shrikant Anant, Shahid Umar. Epigenetic regulation of Wnt/β-catenin signaling in vivo by PRC2 protein EZH2. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr A30.

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.

Abstract 4299: Down-regulation of Wnt inhibitory factor 1 is an early event in oropharyngeal cancer associated with tobacco-use.

Abstract Background: Tobacco use is the primary risk factor for head and neck squamous cell carcinoma (HNSCC), the sixth most common malignancy worldwide. Aberrant activation of the Wnt pathway is a key event in tumorigenesis, but the precise mechanisms leading to up-regulation of Wnt signaling in HNSCC have not been defined. Wnt inhibitory factor 1 (WIF1) is significantly down-regulated in many tumors but its expression status in oropharyngeal HNSCC is unknown. Aims: To characterize WIF1 expression in oropharyngeal normal and tumor samples and delineate the mechanisms leading to alterations in WIF1 expression. Methods: Paraffin-embedded tissue samples from fifty patients with HNSCC identified from the annotated tumor registry at the University of Oklahoma were obtained. Additionally, fifty paraffin-embedded tissues were obtained from smoker and non-smoker individuals undergoing tonsillectomy at the University of Oklahoma for non-cancerous diagnosis. Histological diagnoses were reviewed and samples from non-cancer individuals were matched for age and sex. Smoking habits were registered in pack-years. WIF1 protein expression was determined by immunohistochemistry. Genomic DNA isolated from normal and tumor samples were treated with bisulfite, and methylation-specific PCR and bisulfite sequence analysis were performed. Results: WIF1 expression was very high in normal tonsil epithelium obtained from non-smokers. However, WIF1 expression was reduced in smokers compared to non-smokers (p=0.01). WIF1 expression was frequently and significantly reduced in tumors compared to normal adjacent epithelium (p&amp;lt;0.001). Methylation-specific PCR and bisulfite sequence analysis revealed that WIF1 promoter methylation is common in oropharyngeal HNSCC and occurs in some normal epithelium samples obtained from smokers. Conclusions: WIF1 promoter hypermethylation is frequent in oropharyngeal cancer and contributes to WIF1 down-regulation. Importantly, by studying samples obtained from non-cancer patients, we show clearly and for the first time that WIF1 promoter methylation is an early event associated with tobacco-use. Taken together, this study establishes an important role for WIF1 in oropharyngeal HNSCC and suggests that WIF1 promoter methylation in normal epithelium is a biomarker of risk for tobacco-associated cancer. Grant support: This work was supported by the Oklahoma Center for the Advancement of Science &amp; Technology. LQ holds a Presbyterian Health Foundation Endowed Chair in Otorhinolaryngology. Citation Format: Lurdes VF Queimado, Ryan Raju, Liu Cheng, Ilangovan Ramachandran, Eva Brabcova, Matt Naifeh, Greg Krempl. Down-regulation of Wnt inhibitory factor 1 is an early event in oropharyngeal cancer associated with tobacco-use. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4299. doi:10.1158/1538-7445.AM2013-4299

Epigenetic Inactivation of Wnt Inhibitory Factor-1 in Human Esophageal Squamous Cell Carcinoma

Wnt inhibitory factor-1 (WIF1), as one of most important Wnt antagonists, has been detected frequently silenced by promoter hypermethylation in various types of cancer. In this study, we aimed to investigate the promoter methylation profiles of WIF1 in human esophageal squamous cell carcinoma (ESCC) tissues and cell lines, as well as the functional roles of WIF1 in the human ESCC metastatic behavior. WIF1 mRNA levels and promoter methylation status in ESCC tissues and cell lines were detected using RT-PCR and methylation-specific PCR (MS-PCR), respectively. WIF1 protein levels were assessed by Western blot. Stable ESCC cell line with restoration of WIF1 was generated in EC109 cells, which naturally do not express detectable WIF1 mRNA. The effects of reexpressed WIF1 on EC109 cell proliferation and migration were investigated using crystal violet and wound healing assay, respectively. Also the effects of WIF1 reexpression on the beta-catenin/T-cell factor-dependent transcription activity was measured by luciferase assay. WIF1 promoter methylation was frequently observed in ESCC tissues (46%, 23/50) and cell lines (50%, 2/4). Treatment with demethylating agent, 5-aza-2'-deoxycytidine (5-aza-dC), increased or restored WIF1 expression in these ESCC cell lines. Restoration of the WIF1 in EC109 cells resulted in a significant inhibition on both cell proliferation and migration. Moreover, reexpression of WIF1 caused significant decrease of beta-catenin/T-cell factor-dependent transcription activity. These findings demonstrated that WIF1 silencing due to promoter hypermethylation is a major mechanism during carcinogenesis of ESCC. This would be an opportunity to prevent the development and progression of HCC through modulation of WIF1.

The Hypomethylation Agent Bisdemethoxycurcumin Acts on the WIF-1 Promoter, Inhibits the Canonical Wnt Pathway and Induces Apoptosis in Human Non-Small-Cell Lung Cancer

Hypermethylation of the Wnt inhibitory factor-1 (WIF-1) promoter has been implicated in the overactivation of the Wnt pathway in human lung cancer. Curcuminoids exert anti-cancer effects and have been reported to act as hypomethylating agents. Previously, we have investigated and compared the demethylation effects of three curcuminoids and observed that bisdemethoxycurcumin exhibited the strongest demethylation potency. In this study, we used lung cancer cell lines with WIF-1 promoter hypermethylated as a model to study the demethylating effect of bisdemethoxycurcumin on WIF-1 restoration, Wnt signaling activity and cell death. Bisdemethoxycurcumin directly suppressed the activity of DNA methyltransferase-1 (DNMT1) but did not influence DNMT1 expression. In addition, it induced WIF-1 promoter demethylation and protein re-expression. WIF-1 restoration in lung cancer cells down-regulated nuclear β-catenin and the canonical Wnt cascade. Furthermore, we also showed that down-regulation of Wnt signaling by WIF-1 was required for bisdemethoxycurcumin-induced apoptosis in certain lung cancer cell types. This report is the first to show that bisdemethoxycurcumin induces apoptosis by reactivating WIF-1 from a silenced state. Our results provide new insights into the anti-cancer actions of bisdemethoxycurcumin.

Abstract C21: Wnt inhibitory factor 1 is silenced by promoter hypermethylation in cervical cancer and its restoration suppresses tumor growth

Abstract Background and Purpose: Cervical cancer is the leading cause of morbidity and mortality among women worldwide. It is also the second most common cause of cancer deaths in women. Recent studies suggest that aberrant activation of the Wingless-type (Wnt) pathway plays an important role in cervical cancer. However, the mechanisms and implications of Wnt activation in human cervical cancer are yet to be determined. We hypothesized that the Wnt inhibitory factor 1 (WIF1), a secreted Wnt antagonist, might be silenced in human cervical cancer. Therefore, we characterized the methylation status of WIF1 gene, its mRNA and protein expression in human cervical cancer samples. We also determined the effects of WIF1 treatment on tumor growth in a xenograft mouse model. Methods: To study WIF1 promoter methylation, genomic DNA was isolated from human normal and cancerous cervical samples, processed for bisulfite modification using EZ DNA methylation kit and WIF1 methylation-specific PCR was performed. WIF1 mRNA and protein expression in human cervical normal epithelium and tumor samples were assessed by real-time RTPCR and immunohistochemistry, respectively. To determine the tumor suppressive effects of WIF1, tumor xenograft studies were performed by injecting HeLa cells subcutaneously into the flanks of nude mice. Palpable tumors were treated for 7 weeks with peritumoral injection of pCI-blast-WIF1 expression vector that expresses the full WIF1 protein. Results: Our study demonstrates that WIF1 promoter hypermethylation is a frequent mechanism leading to WIF1 down-regulation as observed by decreased WIF1 mRNA and protein expression in cervical cancer compared with normal tissue. Furthermore, treatment of human cervical cancer in a xenograft mouse model by WIF1 gene transfer significantly inhibited the tumor growth by decreasing the expression of TCF-4, β-catenin, c-myc, cyclin D1 and CD44. WIF1 elicited its tumor suppressive effects by acting at multiple levels in Wnt/β-catenin pathway, in addition to modulating the expression of antiapoptotic (Bcl-2) and apoptotic proteins (p53, p21 and caspase-3), leading to a significant reduction in tumor cell proliferation and induction of massive apoptosis. Of major clinical relevance, while vector treated tumors were highly infiltrative, WIF1 treated tumors presented mainly with pushing borders, a characteristic feature of benign tumors. In addition, WIF1 treatment decreased the expression of angiogenic factors, VEGF and CD31, and significantly reduced the tumor vascular irrigation. Conclusions: Our findings for the first time demonstrate that WIF1 is silenced by promoter hypermethylation and identify WIF1 down-regulation as an important mechanism of Wnt activation in cervical cancer. Remarkably, our in vivo study emphasizes the anti-invasive, anti-angiogenic and tumor suppressive effects of WIF1 and therefore its potential therapeutic value in the treatment of cervical cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C21.

Hypomethylation effects of curcumin, demethoxycurcumin and bisdemethoxycurcumin on WIF-1 promoter in non-small cell lung cancer cell lines

The tumor suppressor gene Wnt inhibitory factor-1 (WIF-1) has been found to be promoter hypermethylated and silenced in lung cancer cell lines and tissues. Curcuminoids are major active components of the spice turmeric, and have recently been reported to be potential hypomethylation agents. In the present study, the hypomethylation effects of three major curcuminoids, curcumin, demethoxycurcumin and bisdemethoxycurcumin, were compared in vitro using ELISA, and their demethylation potential was confirmed by methylation-specific PCR. It was found that bisdemethoxycurcumin possesses the strongest demethylation function invitro compared to the other two curcuminoids, exerting its effect at a minimal demethylation concentration of 0.5-1µM. The WIF-1 promoter region was demethylated after treatment with 20µM demethoxycurcumin and bisdemethoxycurcumin, but failed to respond to 20µM curcumin. In the A549 cell line, RT-PCR and Western blotting were used to confirm that WIF-1 expression was restored after curcuminoid-induced promoter hypermethylation. Since the results regarding the demethylation potential of the three major curcuminoids to restore WIF-1 expression indicated that bisdemethoxycurcumin has the strongest hypomethylation effect, this curcuminoid may have therapeutic use in the restoration of WIF-1 expression in NSCLC.

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.