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

Abstract

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 & 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