Wnt Inhibitory Factor-1 Protein Research Articles

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.

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.

Adiponectin stimulates Wnt inhibitory factor-1 expression through epigenetic regulations involving the transcription factor specificity protein 1

Adiponectin (ADN) is an adipokine possessing growth inhibitory activities against various types of cancer cells. Our previous results demonstrated that ADN could impede Wnt/beta-catenin-signaling pathways in MDA-MB-231 human breast carcinoma cells [Wang,Y. et al. (2006) Adiponectin modulates the glycogen synthase kinase-3 beta/beta-catenin signaling pathway and attenuates mammary tumorigenesis of MDA-MB-231 cells in nude mice. Cancer Res., 66, 11462-11470]. Here, we extended our studies to elucidate the effects of ADN on regulating the expressions of Wnt inhibitory factor-1 (WIF1), a Wnt antagonist frequently silenced in human breast tumors. Our results showed that ADN time dependently stimulated WIF1 gene and protein expressions in MDA-MB-231 cells. Overexpression of WIF1 exerted similar inhibitory effects to those of ADN on cell proliferations, nuclear beta-catenin activities, cyclin D1 expressions and serum-induced phosphorylations of Akt and glycogen synthase kinase-3 beta. Blockage of WIF1 activities significantly attenuated the suppressive effects of ADN on MDA-MB-231 cell growth. Furthermore, our in vivo studies showed that both supplementation of recombinant ADN and adenovirus-mediated overexpression of this adipokine substantially enhanced WIF1 expressions in MDA-MB-231 tumors implanted in nude mice. More interestingly, we found that ADN could alleviate methylation of CpG islands located within the proximal promoter region of WIF1, possibly involving the specificity protein 1 (Sp1) transcription factor and its downstream target DNA methyltransferase 1 (DNMT1). Upon ADN treatment, the protein levels of both Sp1 and DNMT1 were significantly decreased. Using silencing RNA approaches, we confirmed that downregulation of Sp1 resulted in an increased expression of WIF1 and decreased methylation of WIF1 promoter. Taken together, these data suggest that ADN might elicit its antitumor activities at least partially through promoting WIF1 expressions.

The extracellular matrix component WIF-1 is expressed during, and can modulate, retinal development

We have shown previously that components of the extracellular matrix (ECM) modulate neuronal development. Here, we searched for additional ECM elements that might play roles in retinal histogenesis and identified a secreted glycoprotein that is heavily expressed in the retina. This molecule, named by others Wnt Inhibitory Factor-1 (WIF-1), is expressed during and after the period of rod photoreceptor morphogenesis in the mouse. We show that a potential WIF-1 ligand, Wnt4, as well as a potential Wnt4 receptor, fzd4, and a potential Wnt4 coreceptor, LRP6, are expressed in the region of, and at the time of, rod photoreceptor genesis. WIF-1 and Wnt4 are coexpressed during retinal development and bind to each other; therefore, they are likely to interact during rod production. WIF-1 protein inhibits rod production, and anti-WIF-1 antibodies increase rod production; in contrast, Wnt4 promotes rod production. Together, these data suggest that WIF-1 and Wnt4, both components of the ECM, regulate mammalian photoreceptor development.

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).