Wnt Inhibitory Factor-1 Downregulation 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.

METTL3 is aberrantly expressed in endometriosis and suppresses proliferation, invasion, and migration of endometrial stromal cells.

Endometriosis (EM) is one of the leading gynecological disorders, and associated with excessive functioning of endometrial stromal cells (ESCs). The current study was conducted to determine the expression and role of methyltransferase-like 3 (METTL3) in the proliferation, invasion, and migration of ESCs in EM. The documented expression levels of METTL3, microRNA (miR)-21-5p, and WNT inhibitory factor 1 (WIF1) in eutopic (Eut) and ectopic (Ect) endometrial tissues and ESCs were determined by a combination of real-time quantitative polymerase chain reaction and Western blot assay. After transfection with pcDNA3.1-METTL3, miR-21-5p mimic, and WIF1 small interfering RNA, cell counting kit-8, colony formation, and Transwell assays were performed in the Ect ESCs (Ect-ESCs). Subsequently, the binding of miR-21-5p to METTL3 was analyzed, along with quantification of the N6-methyladenosine (m6A) level, the enrichments of METTL3 and m6A on WIF1, and the mRNA stability of WIF1. In our findings, METTL3 was downregulated in the EM tissues and cells. METTL3 overexpression intrinsically reduced the proliferation, invasion, and migration of Ect-ESCs. miR-21-5p inhibited the METTL3 expression while METTL3 enhanced the mRNA stability and expression of WIF1 via m6A modification. Additionally, a negative correlation of METTL3 was identified with miR-21-5p along with a positive correlation with the WIF1 mRNA in EM tissues. The miR-21-5p overexpression or WIF1 downregulation enhanced the proliferation, invasion, and migration of Ect-ESCs. Collectively, miR-21-5p inhibited the METTL3-mediated m6A modification and mRNA stability of WIF1, thereby facilitating the proliferation, invasion, and migration of Ect-ESCs.

Loss of CLDN5 in podocytes deregulates WIF1 to activate WNT signaling and contributes to kidney disease

Although mature podocytes lack tight junctions, tight junction integral membrane protein claudin-5 (CLDN5) is predominantly expressed on plasma membranes of podocytes under normal conditions. Using podocyte-specific Cldn5 knockout mice, we identify CLDN5 as a crucial regulator of podocyte function and reveal that Cldn5 deletion exacerbates podocyte injury and proteinuria in a diabetic nephropathy mouse model. Mechanistically, CLDN5 deletion reduces ZO1 expression and induces nuclear translocation of ZONAB, followed by transcriptional downregulation of WNT inhibitory factor-1 (WIF1) expression, which leads to activation of WNT signaling pathway. Podocyte-derived WIF1 also plays paracrine roles in tubular epithelial cells, as evidenced by the finding that animals with podocyte-specific deletion of Cldn5 or Wif1 have worse kidney fibrosis after unilateral ureteral obstruction than littermate controls. Systemic delivery of WIF1 suppresses the progression of diabetic nephropathy and ureteral obstruction-induced renal fibrosis. These findings establish a function for podocyte CLDN5 in restricting WNT signaling in kidney.

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.

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

Reduced WIF-1 Expression Stimulates Skin Hyperpigmentation in Patients with Melasma

The expression of Wnt inhibitory factor-1 (WIF-1) gene, which was detected by a microarray analysis of hyperpigmented and normally pigmented skin sets of melasma patients, was significantly reduced in the hyperpigmented skin from melasma patients, but not in healthy controls, regardless of UV irradiation. Wnt signals regulate skin pigmentation; however, WIF-1 is expressed in cultured skin keratinocytes and fibroblasts, but not in melanocytes. Therefore, we examined whether WIF-1 knockdown in neighboring keratinocytes and fibroblasts plays a role in melasma. Additionally, the effect of WIF-1 overexpression on the amelioration of hyperpigmentation was examined. WIF-1 knockdown, either in fibroblasts or in keratinocytes, significantly stimulated tyrosinase expression and melanosome transfer, whereas melanocytes with WIF-1 overexpression significantly reduced those parameters. The WIF-1 knockdown decreased glycogen synthase kinase-3β (GSK-3β), β-catenin, and NFATc2 (nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 2) phosphorylation and increased microphthalmia-associated transcription factor (MITF) expression as in melanocytes with Wnt-1 overexpression, whereas the WIF-1 overexpression reversed the results. Expression of Wnts, both canonical and noncanonical, was increased in the hyperpigmented skin of melasma patients. Collectively, WIF-1 downregulation, which may occur in epidermal keratinocytes and in dermal fibroblasts, is involved in melasma development because of the stimulation of melanogenesis and melanosome transfer through upregulation of the canonical and the noncanonical Wnt signaling pathway.

Abstract 5625: Wnt inhibitory factor 1 is a potent growth inhibitory agent for salivary gland tumor cells

Abstract Background: Aberrant activation of the Wingless-type (Wnt)/β-catenin pathway plays an important role in many human cancers. We have shown that Wnt inhibitory factor 1 (WIF1), a Wnt antagonist, is rearranged in salivary gland pleomorphic adenomas and down-regulated in carcinoma ex-pleomorphic adenomas. Here, we studied the mechanisms of WIF1 down-regulation and also the potential therapeutic implications of WIF1 in human salivary gland tumor cells. Aims: To characterize the mechanisms of WIF1 down-regulation in salivary gland tumor cells and to determine the effects of restoration of WIF1 expression on salivary gland tumor cell growth. Methods: Salivary gland tumor cell lines were treated with the demethylating agent 5-aza-2′-deoxycytidine (50 µM) for 4 days. Then we isolated total RNA and performed real-time RT-PCR to determine WIF1 mRNA expression. To determine the growth suppressive effects of WIF1, salivary gland pleomorphic adenoma and carcinoma ex-pleomorphic adenoma cells were stably or transiently transfected with either empty vector or pCI blast-WIF1. The growth inhibitory role of WIF1 was also assessed by exposure of salivary gland tumor cells to pure WIF1 protein. Cell proliferation was assessed at different time points by MTT assay. We performed cell cycle analysis after 72 h of WIF1 transfection by flow cytometry using FACSCalibur analyzer. Results: Treatment with DAC caused about 30-fold and 6-fold increase in WIF1 mRNA expression in salivary gland carcinoma ex-pleomorphic adenoma cells and pleomorphic adenoma cells, respectively compared with vehicle treatment. Stable transfection of salivary gland tumor cells with WIF1 vector induced dramatic nuclear fragmentation and cell death. No viable colonies were observed. Transient transfection with pCI blast-WIF1 or exposure to WIF1 protein significantly decreased the proliferation of salivary gland tumor cells. Cell cycle analysis showed a significant accumulation of cells in G1 phase suggesting that WIF1 re-expression induces G1 arrest in salivary gland tumor cells. Conclusions: Our findings demonstrate that WIF1 is epigenetically silenced by promoter hypermethylation in carcinoma ex-pleomorphic adenoma cells and pleomorphic adenoma cells. Importantly, salivary gland tumor cells stably transfected with WIF1 are non-viable, and exposure to high levels of WIF1 protein resulted in significant growth inhibition. These data show that WIF1 is a potent growth inhibitory agent for salivary gland tumor cells and may serve as a potential therapeutic drug for salivary gland cancer. Grant support: This work was supported by the Oklahoma Center for the Advancement of Science &amp; Technology (LQ) (HR08-018). LQ holds a Presbyterian Health Foundation Endowed Chair in Otorhinolaryngology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5625. doi:1538-7445.AM2012-5625

Wnt inhibitory factor 1 induces apoptosis and inhibits cervical cancer growth, invasion and angiogenesis in vivo

Aberrant activation of Wingless-type (Wnt)/β-catenin signaling is widespread in human cervical cancer. However, the underlying mechanisms of Wnt activation and the therapeutic potential of Wnt inhibition remain largely unknown. Here, we demonstrate that the Wnt inhibitory factor 1 (WIF1), a secreted Wnt antagonist, is downregulated in all human primary cervical tumors and cell lines analyzed. Our data reveal that WIF1 downregulation occurs due to promoter hypermethylation and is an early event in cervical oncogenesis. WIF1 re-expression upon 5-aza-2'-deoxycytidine treatment or WIF1 gene transfer induces significant apoptosis and G(2)/M arrest, and inhibits cervical cancer cell proliferation in vitro. Consistent with this, treatment of established mice tumor xenografts with peritumoral WIF1 gene transfer results in a significant inhibition of cancer growth and invasion. WIF1 treatment causes a significant decrease in intracellular WNT1 and TCF-4 proteins revealing novel Wnt-regulatory mechanisms. Thus, WIF1 causes a major cellular re-distribution of β-catenin and a significant inhibition of the Wnt/β-catenin pathway in tumor cells, as documented by a remarkable reversion in the expression of Wnt/β-catenin transcriptional target genes (E-cadherin, c-Myc, cyclin D1, CD44 and VEGF). Consequently, multiple critical events in tumor progression and metastasis such as cell proliferation, angiogenesis and invasion were inhibited by WIF1. In addition, WIF1 modulated the expression of specific anti-apoptotic and apoptotic proteins, thereby inducing significant apoptosis in vivo. Our findings demonstrate for the first time that WIF1 downregulation by epigenetic gene silencing is an important mechanism of Wnt activation in cervical oncogenesis. Of major clinical relevance, we show that peritumoral WIF1 gene transfer reduces not only cancer growth but also invasion in well-established tumors. Therefore, our data provide novel mechanistic insights into the role of WIF1 in cervical cancer progression, and the important preclinical validation of WIF1 as a potent drug target in cervical cancer treatment.

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.

The Wnt inhibitory factor 1 (WIF1) is targeted in glioblastoma and has a tumor suppressing function potentially by induction of senescence

Gene expression-based prediction of genomic copy number aberrations in the chromosomal region 12q13 to 12q15 that is flanked by MDM2 and CDK4 identified Wnt inhibitory factor 1 (WIF1) as a candidate tumor suppressor gene in glioblastoma. WIF1 encodes a secreted Wnt antagonist and was strongly downregulated in most glioblastomas as compared with normal brain, implying deregulation of Wnt signaling, which is associated with cancer. WIF1 silencing was mediated by deletion (7/69, 10%) or epigenetic silencing by promoter hypermethylation (29/110, 26%). Co-amplification of MDM2 and CDK4 that is present in 10% of glioblastomas was associated in most cases with deletion of the whole genomic region enclosed, including the WIF1 locus. This interesting pathogenetic constellation targets the RB and p53 tumor suppressor pathways in tandem, while simultaneously activating oncogenic Wnt signaling. Ectopic expression of WIF1 in glioblastoma cell lines revealed a dose-dependent decrease of Wnt pathway activity. Furthermore, WIF1 expression inhibited cell proliferation in vitro, reduced anchorage-independent growth in soft agar, and completely abolished tumorigenicity in vivo. Interestingly, WIF1 overexpression in glioblastoma cells induced a senescence-like phenotype that was dose dependent. These results provide evidence that WIF1 has tumor suppressing properties. Downregulation of WIF1 in 75% of glioblastomas indicates frequent involvement of aberrant Wnt signaling and, hence, may render glioblastomas sensitive to inhibitors of Wnt signaling, potentially by diverting the tumor cells into a senescence-like state.

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.

The tumor suppressor Wnt inhibitory factor 1 is frequently methylated in nasopharyngeal and esophageal carcinomas

Aberrant activation of the wingless-type- (Wnt)-signaling pathway is common in many cancers including nasopharyngeal (NPC) and esophageal squamous cell (ESCC) carcinomas, both prevalent in Southern China and Southeast Asia. However, the molecular mechanism leading to this abnormality is still obscure. Wnt inhibitory factor-1 (WIF1) is a secreted antagonist of the Wnt pathway, and is recently shown to be inactivated by epigenetic mechanism in some tumors. Here, we examined whether WIF1 is also inactivated epigenetically in NPC and ESCC. With semiquantitative reverse transcription-PCR and methylation-specific PCR, we detected WIF1 downregulation or silencing in 6/6 of NPC and 12/19 of ESCC cell lines, which is well correlated with its methylation status. Methylation was further confirmed by high-resolution bisulfite genomic sequencing. Methylation was also frequently observed in a large collection of primary tumors of NPC (85%, 55/65) and ESCC (27%, 25/92), with WIF1 expressed and unmethylated in normal NPC and esophageal cell lines and normal tissues. Treatment of 5-aza-2′-deoxycytidine demethylated WIF1 and induced its expression in NPC and ESCC cell lines, highlighting a direct role of epigenetic inactivation. Ectopic expression of WIF1 in NPC and ESCC tumor cells resulted in significant inhibition of tumor cell colony formation, similar to TP53, and also significant downregulation of β-catenin protein level in NPC cells. Thus, WIF1 functions as a tumor suppressor for both NPC and ESCC through suppressing the Wnt-signaling pathway, but is frequently silenced by epigenetic mechanism in a tumor-specific way. Our study indicates that epigenetic inactivation of WIF1 contributes to the aberrant activation of Wnt pathway and is involved in the pathogenesis of both tumors. WIF1 methylation could also serve as a specific biomarker for these tumors.

WIF1, an inhibitor of the Wnt pathway, is rearranged in salivary gland tumors

Chromosome rearrangements involving 12q13-15 are frequent among several tumors, including pleomorphic adenomas. The common molecular target for these aberrations is the HMGA2 gene, but various fusion partners of HMGA2 have been reported in tumors. Here we report the identification of the WNT inhibitory factor 1 (WIF1) gene as a novel HMGA2 fusion partner in a salivary gland pleomorphic adenoma. In normal salivary gland tissue WIF1 is expressed at a high level and HMGA2 is not expressed. However, in the pleomorphic adenoma expressing the HMGA2/WIF1 fusion transcript, we observed re-expression of HMGA2 wild-type transcripts and very low levels of WIF1 expression. These data suggest a possible synergistic effect between upregulation of HMGA2 and downregulation of WIF1. We screened 13 additional benign and malignant salivary gland tumors and detected WIF1 rearrangement in one out of two carcinomas ex-pleomorphic adenoma analyzed. In this malignant tumor, the rearrangement of one WIF1 allele coexists with loss of the other allele, a classic signature of a tumor suppressor gene. WIF1 is an antagonist of the Wnt signaling pathway, which plays a critical role in human cancer. In transgenic mouse models, Wnt activation leads to a high frequency of benign and malignant salivary gland tumors. To our knowledge, this is the first report suggesting that WIF1 is a recurrent target in human salivary gland oncogenesis and that downregulation of WIF1 plays a role in the development and/or progression of pleomorphic adenomas.

Frequent epigenetic inactivation of Wnt inhibitory factor-1 in human gastrointestinal cancers

Aberrant activation and upregulation of the Wnt pathway is a key feature of many cancers. Wnt antagonists have recently attracted wide attention. Wnt inhibitory factor-1 (WIF-1) is a secreted antagonist that can bind to Wnt proteins directly and inhibit Wnt signaling pathway. It has been reported that WIF-1 expression is down regulated in several solid tumors and that WIF-1 is silenced by promoter hypermethylation in lung and colorectal cancer. By using RT-PCR, bisulfite sequence analysis, and methylation-specific PCR, we analysed expression and methylation of WIF-1 in cancer cell lines and freshly resected cancer tissues of the esophagus, stomach, colorectum, and pancreas. Downregulation of WIF-1 mRNA expression was observed in 61 (91.0%) of 67 cancer cell lines, 16 (80.0%) of 20 esophageal, 23 (74.2%) of 31 gastric, 41 (82.0%) of 50 colorectal, and six (75.0%) of eight pancreatic cancer tissues. Downregulation of WIF-1 expression was also observed at protein level. No significant association between WIF-1 downregulation and clinicopathological characteristics was found, suggesting that downregulation of WIF-1 expression is an early event in carcinogenesis of these cancers. Indeed, downregulation of WIF-1 expression was observed in 32 (72.7%) of 44 colorectal adenoma tissues and 18 (78.2%) of 23 early mucosal or submucosal colorectal carcinoma tissues. CpG island hypermethylation in the WIF-1 promoter region correlated with downregulation of WIF-1 expression in cancer cell lines and tissues. Treatment with demethylating agent, 5-aza-2'-deoxycytidine (5-aza-dC), restored WIF-1 expression in cancer cell lines. A combined treatment of 5-aza-dC and a histone deacetylase inhibitor, trichostatinA, restored WIF-1 expression synergistically, indicating the role of cytosine methylation and histone deacetylation in the silencing of the WIF-1 gene. Transfection of the WIF-1 gene construct into TE-1 esophageal cancer cell lines or SW48 colon cancer cell lines lacking WIF-1 expression resulted in a significant inhibition on colony formation, cell proliferation, anchorage-independent growth in soft agar. TOPflash assay showed WIF-1 inhibits Wnt canonical signaling in these cell lines. These results suggest tumor suppressive function of WIF-1, due to its ability to inhibit Wnt signaling. Our results suggest that WIF-1 silencing due to promoter hypermethylation is an important mechanism underlying aberrant activation of the Wnt signaling pathway in carcinogenesis of the digestive organs. Modulation of the Wnt pathway, through reversal of WIF-1 silencing by demethylating agents, is a potential target for treatment and/or prevention of gastrointestinal cancers.

Wnt inhibitory factor-1 is silenced by promoter hypermethylation in human lung cancer.

Aberrant activation of the Wingless-type (Wnt) signaling pathway is associated with a variety of human cancers, and we recently reported the importance of aberrant Wnt signaling in lung cancer. On the other hand, inhibition of Wnt signaling suppresses growth in numerous cell types. Wnt inhibitory factor-1 (WIF-1) is a secreted antagonist that can bind Wnt in the extracellular space and inhibit Wnt signaling. Recently, down-regulation of WIF-1 has been reported in several human cancers. To discover the mechanism of WIF-1 silencing in lung cancer, we first identified the human WIF-1 promoter and subsequently examined the methylation status in the CpG islands. By using methylation-specific PCR and sequence analysis after bisulfite treatment, we demonstrate here frequent CpG island hypermethylation in the functional WIF-1 promoter region. This hypermethylation correlates with its transcriptional silencing in human lung cancer cell lines. Moreover, treatment with 5-aza-2'-deoxycytidine restores WIF-1 expression. We then studied WIF-1 expression in 18 freshly resected lung cancers, and we show a down-regulation in 15 of them (83%). This silencing also correlates with WIF-1 promoter methylation. Our results suggest that methylation silencing of WIF-1 is a common and likely important mechanism of aberrant activation of the Wnt signaling pathway in lung cancer pathogenesis, raising its therapeutic interest.

WIF1, a component of the Wnt pathway, is down-regulated in prostate, breast, lung, and bladder cancer.

To detect novel Wnt-pathway genes involved in tumourigenesis, this study analysed the RNA expression levels of 40 genes of the Wnt pathway by chip hybridization of microdissected matched pairs of 54 primary prostate carcinomas. Eleven genes showed greater than two-fold differential expression in at least 10% of prostate cancers. Three of these genes encode extracellular components of the Wnt pathway (WNT2, WIF1, SFRP4); two are receptors (FZD4, FZD6); two belong to the intracellular signal cascade (DVL1, PPP2CB); one regulates transcription (TCF4); and three represent genes regulated by this pathway (CCND2, CD44, MYC). While SFRP4, FZD4, FZD6, DVL1, TCF4, and MYC are up-regulated, WIF1, WNT2, PPP2CB, CCND2, and CD44 are down-regulated in certain prostate cancer patients. Wnt inhibitory factor 1 (WIF1) and secreted frizzled related protein (SFRP4) showed the most significant aberrant expression at the RNA level. WIF1 was down-regulated in 64% of primary prostate cancers, while SFRP4 was up-regulated in 81% of the patients. Immunohistochemical analysis using a polyclonal antibody revealed strong cytoplasmic perinuclear WIF1 expression in normal epithelial cells of the prostate, breast, lung, and urinary bladder. Strong reduction of WIF1 protein expression was found in 23% of prostate carcinomas, but also in 60% of breast, 75% of non-small cell lung (NSCLC), and 26% of bladder cancers analysed. No significant association between WIF1 down-regulation and tumour stage or grade was observed for prostate, breast or non-small cell lung carcinomas, indicating that loss of WIF1 expression may be an early event in tumourigenesis in these tissues. However, down-regulation of WIF1 correlated with higher tumour stage in urinary bladder tumours (pTa versus pT1-pT4; p = 0.038).