Background: The important role of Wingless-type (Wnt)/β-catenin pathway is well established in various human tumors. Previously, we have demonstrated that Wnt inhibitory factor 1 (WIF1) is significantly downregulated in human salivary gland tumors. The tumor suppressor microRNA, miR-200c, is downregulated in many cancers and is known to repress migration and invasion of cancer cells.
Aims: To determine the tumor suppressive effects of WIF1 and delineate the mechanisms by which WIF1 regulates microRNAs in human malignant salivary gland cells.
Methods: Human malignant salivary gland cells (Carcinoma ExPleomorphic adenoma, CaExPA79) were transiently transfected with either empty vector or pCI blast-WIF1 and cell proliferation was determined by MTT assay at various time points. CaExPA79 cells transfected with WIF1 for 48 h were used for total RNA isolation and gene expression studies. To determine the effect of WIF1 restoration on salivary gland cancer cells, CaExPA79 cells were treated with 50 μM of 5-aza-2′-deoxycytidine (DAC), a demethylating agent, for 4 days. After DAC treatment, the cells were either used for cell cycle analysis or total RNA isolation and real-time RT-PCR analysis. To determine the effect of WIF1 on miR-200c, CaExPA79 cells were transfected with pCI blast-WIF1 along with miR200c-Luc reporter vector and Renilla luciferase expressing plasmid pRL-TK for 24 h. Luciferase activity was determined using Dual-Luciferase Reporter Assay System.
Results: WIF1 significantly inhibited the proliferation of CaExPA79 at 24, 48 and 72 h. Treatment with DAC increased the WIF1 mRNA expression and resulted in a significant accumulation of cells in S phase. DAC treatment also resulted in a significant increase in miR-200c expression. WIF1 over-expression resulted in a significant increase in miR-200c expression and led to a significant reduction in the expression of miR-200c targeted genes as documented by luciferase reporter gene assays. Conclusions: WIF1 inhibits salivary gland cancer cell proliferation and induces cell cycle arrest. Most importantly, our findings suggest that WIF1 is a positive transcriptional regulator of the tumor suppressor microRNA miR-200c and leads to a corresponding downregulation of its downstream targets. Taken together, this study reveals a novel mechanism by which WIF1 elicits it's tumor suppressive effects on salivary gland cancer cells.
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: Ilangovan Ramachandran, David Obeso, Sripathi M. Sureban, Elangovan Thavathiru, Antonio Reis, Lurdes Queimado. microRNA-200c mediates the tumor suppressive effects of Wnt inhibitory factor 1 in human malignant salivary gland cells. [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 4298. doi:10.1158/1538-7445.AM2013-4298