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

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.