Abstract 4299: Down-regulation of Wnt inhibitory factor 1 is an early event in oropharyngeal cancer associated with tobacco-use.
Authors: Queimado Lurdes VF , Raju Ryan , Cheng Liu , Ramachandran Ilangovan , Brabcova Eva , Naifeh Matt , Krempl Greg 
Published: August 2014

Background: Tobacco use is the primary risk factor for head and neck squamous cell carcinoma (HNSCC), the sixth most common malignancy worldwide. Aberrant activation of the Wnt pathway is a key event in tumorigenesis, but the precise mechanisms leading to up-regulation of Wnt signaling in HNSCC have not been defined. Wnt inhibitory factor 1 (WIF1) is significantly down-regulated in many tumors but its expression status in oropharyngeal HNSCC is unknown.

Aims: To characterize WIF1 expression in oropharyngeal normal and tumor samples and delineate the mechanisms leading to alterations in WIF1 expression.

Methods: Paraffin-embedded tissue samples from fifty patients with HNSCC identified from the annotated tumor registry at the University of Oklahoma were obtained. Additionally, fifty paraffin-embedded tissues were obtained from smoker and non-smoker individuals undergoing tonsillectomy at the University of Oklahoma for non-cancerous diagnosis. Histological diagnoses were reviewed and samples from non-cancer individuals were matched for age and sex. Smoking habits were registered in pack-years. WIF1 protein expression was determined by immunohistochemistry. Genomic DNA isolated from normal and tumor samples were treated with bisulfite, and methylation-specific PCR and bisulfite sequence analysis were performed.

Results: WIF1 expression was very high in normal tonsil epithelium obtained from non-smokers. However, WIF1 expression was reduced in smokers compared to non-smokers (p=0.01). WIF1 expression was frequently and significantly reduced in tumors compared to normal adjacent epithelium (p&lt;0.001). Methylation-specific PCR and bisulfite sequence analysis revealed that WIF1 promoter methylation is common in oropharyngeal HNSCC and occurs in some normal epithelium samples obtained from smokers.

Conclusions: WIF1 promoter hypermethylation is frequent in oropharyngeal cancer and contributes to WIF1 down-regulation. Importantly, by studying samples obtained from non-cancer patients, we show clearly and for the first time that WIF1 promoter methylation is an early event associated with tobacco-use. Taken together, this study establishes an important role for WIF1 in oropharyngeal HNSCC and suggests that WIF1 promoter methylation in normal epithelium is a biomarker of risk for tobacco-associated cancer.

Grant support: This work was supported by the Oklahoma Center for the Advancement of Science & Technology. LQ holds a Presbyterian Health Foundation Endowed Chair in Otorhinolaryngology.

Citation Format: Lurdes VF Queimado, Ryan Raju, Liu Cheng, Ilangovan Ramachandran, Eva Brabcova, Matt Naifeh, Greg Krempl. Down-regulation of Wnt inhibitory factor 1 is an early event in oropharyngeal cancer associated with tobacco-use. [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 4299. doi:10.1158/1538-7445.AM2013-4299