Abstract
Abstract Background: Adenoid cystic carcinoma (ACC) is a common malignancy that arises in major and minor salivary glands. It is well characterized by relatively high rate of local recurrence, and late onset of distant metastases, which cause high morbidity and mortality. Dysregulation in the Wingless-type (Wnt)/β-catenin pathway contributes to many human tumors. Here, we report that downregulation of an important Wnt antagonist, Wnt inhibitory factor 1 (WIF1), promotes ACC cell proliferation and migration. Aims: To determine the status of WIF1 and study the effects of WIF1 on human adenoid cystic carcinoma cell growth, migration, spheroid formation and senescence. Methods: Genomic DNA isolated from human adenoid cystic carcinoma cell lines (ACC52 and ACC112) was used for methylation-specific PCR after bisulfite modification. ACC52 and ACC112 cells were treated with a demethylating agent, 5-aza-2′-deoxycytidine (DAC) [50 µM for 4 days], and used for total RNA isolation and real-time RT-PCR to determine WIF1 mRNA expression. To determine the growth suppressive effects of WIF1, ACC52 and ACC112 cells were transiently transfected with either pCI blast (vector) or pCI blast-WIF1 and assessed for cell proliferation, migration, spheroid formation and senescence. Cell proliferation was determined at different time intervals (24, 48, 72 h) by hexosaminidase assay. Cell migration and spheroid formation were assessed by scratch assay and soft agar assay, respectively. Cellular senescence was determined using senescence β-galactosidase staining kit. Results: Methylation-specific PCR demonstrate that WIF1 is silenced by promoter methylation in ACC cells. DAC treatment increased the WIF1 mRNA expression by 2.3-fold and 3.8-fold in ACC52 and ACC112 cells, respectively compared with vehicle treatment. WIF1 significantly decreased the proliferation and migration of adenoid cystic carcinoma cells. Additionally, WIF1 significantly decreased the number of spheroids formed at 7 and 17 days. Furthermore, WIF1 induced a remarkable increase in cellular senescence in both the cell lines. Conclusions: We demonstrate that WIF1 is downregulated by promoter hypermethylation in human adenoid cystic carcinoma cells. Importantly, WIF1 inhibited the cell growth, migration and cancer stemness, while inducing cellular senescence. Our findings suggest that WIF1 is a potent growth inhibitor of adenoid cystic carcinoma cells and therefore could be a potential therapeutic agent for ACC. Grant support: This work was supported by the Adenoid Cystic Carcinoma Research Foundation (LQ). LQ holds a Presbyterian Health Foundation Endowed Chair in Otorhinolaryngology. Citation Format: Ilangovan Ramachandran, Vengatesh Ganapathy, Lurdes Queimado. Wnt inhibitory factor 1 reduces the growth and migration of human adenoid cystic carcinoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5274. doi:10.1158/1538-7445.AM2014-5274
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